© 2017 paula tríbulo
TRANSCRIPT
WNT SIGNALING IN THE PREIMPLANTATION BOVINE EMBRYO
By
PAULA TRIBULO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2017
© 2017 Paula Tríbulo
To my husband Marcos for betting on our ambitious dreams, working hard as a team, and for making the dreams come true
4
ACKNOWLEDGMENTS
I would like to express my deep esteem and thanks to my advisor, Dr. Peter J.
Hansen, for his guidance and mentoring. He helped make my PhD a real milestone in
my career. I truly appreciate the effort and time he has devoted to my education, and
thank him for demanding more from me than what I thought I could do. I also express
my admiration for Dr. Hansen’s skills as a team leader by keeping the lab not only
productive but making it a nice place to work in. I feel fortunate for having been
educated by such an outstanding scientist and now I have the legacy of making him
proud of me as one of his scientific descendants. I would also like to extend my
gratitude to the members of my advisory committee, Dr. Charles Wood, Dr. Geoffrey
Dahl and Dr. Paul Cooke, for their critical contributions to my research and for their
positive support.
I am grateful to the University of Florida, Department of Animal Sciences and the
Animal Molecular and Cellular Biology Graduate Program for the opportunity to pursue
my doctorate. I thank Joann Fischer (in memorium) and Renee Parks, our graduate
student advisors, for their assistance and patience.
I would like to state my appreciation to my lab mates for their collaboration and
friendship during these years; Kyle Dobbs, Anna Denicol, Sofia Ortega, Luiz Siqueira,
Jasmine Khannampuzha-Francis, Antonio Ruiz, Adriana Zolini, Liz Jannaman, Gulnur
Jumatayeva, Eliab Estrada and William Ortiz. I express my deep gratitude towards
Veronica Negrón-Perez for being always there for me during these years; without her
company, I am sure my doctoral education would have been much more difficult. I will
never forget those hours of study at night, gathering to work side by side just to support
5
each other and even go swimming when possible. I truly appreciate Vero’s wiliness to fit
everything around my son’s schedule, which was not even her problem, just to help me.
I also thank Jim Moss, who really understands how hard grad school is. He has
always been willing to help us in those unpredicted situations so that, even when only
small things were involved, he made the difference. I also thank Jim and Gail Moss for
opening their house to us, and for having the fun traditional pool parties.
I am very grateful to Luiz Siqueira, Lilian Oliveira, Beatriz Caetano da Silva Leão,
and Khoboso C. Lehloenya for helping me with my research. Without your help I would
not had been as productive! I also thank Dr. Tracy Scheffler for helping me with
Western blots.
Thanks also to William Rembert and Eddie Cummings, for ovary collection, and
owners and employees of Central Beef Packing Co., Adena Meat Products L.P., and
Florida Beef Inc. for providing ovaries. The majority of my research would not have
been possible without your hard work and generosity.
Thanks to the wonderful people I have met in Gainesville who have been
extremely important to me: Dolo Cenoz, Alberto Gochez, Guada Vera, Juanca Giugni,
Vale Zoilo, Esteban Rios, Lucas Ibarbia, Susana Braylan, Horacio Aloe, Euge Cadario,
Eduardo Ribeiro, Annette Fahrenkrog, Fito Daetz, Andres Buffoni, Karen D’Agostino,
Pili Buteler, Alvaro Gonzalez, Jime Laporta, Pancho Peñagaricano, Maca Urrets and
Lautaro Rostol.
I would like to express my most sincere gratitude to my parents, Ricardo Tríbulo
and Maria Manuela del Prado. My Dad taught me, with few words and lots of daily
examples, that success takes passion, humility and hard work. My Mom is an exemplar
6
of unconditional love that allowed me to see the importance of family. Both of them are
imprinted in me and have made me who I am today. I am thankful for the unconditional
love and support I always had from my parents. It took me only a phone call to have my
Mom come up for six month, leaving everything behind, to help me with my newborn in
the last stretch of the program. I will never forget that! Thanks mami!
Last but not least, thanks to my husband, Marcos, and my sons, Lautaro and
Octavio, for putting up with everything that Mom’s PhD implied.
7
TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES .......................................................................................................... 11
LIST OF FIGURES ........................................................................................................ 13
LIST OF ABBREVIATIONS ........................................................................................... 15
ABSTRACT ................................................................................................................... 17
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 19
Introduction ............................................................................................................. 19
Preimplantation Embryonic Development in the Cow ............................................. 21
Overview .......................................................................................................... 21
Embryonic Genome Activation ......................................................................... 22
Compaction ...................................................................................................... 25
First Lineage Commitment: Trophectoderm Differentiation .............................. 28
Origin of ICM and TE ................................................................................. 29
Transcription factors driving ICM-TE segregation ...................................... 30
Role of Hippo signaling in regulation of TE differentiation .......................... 33
Second Lineage Commitment: Hypoblast Differentiation ................................. 34
Blastulation ....................................................................................................... 37
WNT Signaling ........................................................................................................ 38
Overview .......................................................................................................... 38
Canonical Wnt signaling ............................................................................ 40
WNT/planar cell polarity pathway ............................................................... 42
Calcium signaling ....................................................................................... 43
WNT Signaling in Embryonic Stem Cells .......................................................... 43
WNT Signaling during Preimplantation Development ....................................... 46
Evidence for Association of Endometrial Expression of DKK1 and Fertility ...... 50
Objectives of the Present Investigations ................................................................. 55
2 WNT REGULATION OF EMBRYONIC DEVELOPMENT LIKELY INVOLVES PATHWAYS INDEPENDENT OF NUCLEAR β-CATENIN ..................................... 57
Introduction ............................................................................................................. 57
Materials and Methods ............................................................................................ 59
Embryo Production ........................................................................................... 59
Developmental Changes in Expression of Selected Genes Involved in WNT Signaling for Embryos Produced in Vitro (Experiment 1) .............................. 60
8
Characteristics of the WNT Signaling System in the Morula and ICM and TE of in-Vitro Produced Embryos as Revealed by RNA-Seq (Experiment 2) ..... 62
Localization of Total and Active β-catenin in Bovine Preimplantation Embryos as Determined by Immunofluorescence (Experiments 3 and 4) ..... 63
Changes in Immunoreactive Active β-catenin in Embryos Following Activation of Canonical WNT Signaling (Experiments 5-8) ........................... 65
Localization of Active β-catenin in Mouse and Bovine Embryos as Evaluated by Confocal Microscopy (Experiment 9 and 10) ........................... 66
Nuclear Localization of β-catenin in Bovine Embryonic Fibroblast Cells Following Activation of Canonical WNT Signaling (Experiment 11) .............. 67
Non-Canonical WNT Signaling Mediated by Phosphorylation of JNK (i.e., MAPK8) by WNT11 in Bovine Blastocysts (Experiment 12- 14) .................... 68
Results .................................................................................................................... 70
Developmental Changes in Expression of Selected Genes Related to WNT Signaling for Embryos Produced In Vitro (Experiment 1) .............................. 70
Characteristics of the WNT Signaling System in the Morula and ICM and TE of in Vitro Produced Embryos as Revealed by RNA-Seq (Experiment 2) ..... 71
Localization of Total and Active β-catenin in Bovine Preimplantation Embryos as Determined by Immunofluorescence (Experiments 3 and 4) ..... 73
Failure of Canonical WNT Activators to Induce Localization of Nuclear Active β-catenin (Experiments 5 to 8) ........................................................... 74
Localization of Active β-catenin in Mouse and Bovine Embryos Evaluated by Confocal Microscopy (Experiment 9 and 10) ............................................ 75
Nuclear Localization of β-catenin in Bovine Embryonic Fibroblast Cells Following Activation of Canonical WNT Signaling (Experiment 11) .............. 75
Discussion .............................................................................................................. 76
3 CONSEQUENCES OF ENDOGENOUS AND EXOGENOUS WNT SIGNALING FOR DEVELOPMENT IN THE PREIMPLANTATION BOVINE EMBRYO .............. 94
Introduction ............................................................................................................. 94
Materials and Methods ............................................................................................ 96
Embryo Production Using Non-Sex Sorted Sperm ........................................... 96
Embryo Production Using Sex-Sorted Sperm .................................................. 97
Immunolabeling of Protein in Bovine Embryos ................................................. 98
Experiment 1: Effect of Activation of Canonical WNT Signaling by Inhibition of GSK3 on Development ........................................................................... 100
Experiment 2: Effect of Activation of Canonical WNT Signaling by the Agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) in the Presence or Absence of DKK1 on Development and β-catenin Labeling........................................... 101
Experiment 3: Effects of Inhibition of Endogenous WNT Signaling with Wnt-C59 or DKK1 on Ability of Embryos to Develop to the Blastocyst Stage and Blastocyst Cell Number ........................................................................ 101
Experiments 4-5: Effects of DKK1 on Development and Blastocyst Cell Number ....................................................................................................... 102
9
Experiment 6: Effects of DKK1 on Developmental Changes in YAP1 and CDX2 Localization in Morulae and Blastocysts ........................................... 103
Experiment 7: Effects of DKK1 on Activation of JNK ...................................... 103
Experiments 8 - 11: Embryo Responses to WNT7A ....................................... 104
Statistical Analysis .......................................................................................... 105
Results .................................................................................................................. 106
Effect of Activation of Canonical WNT Signaling on Development (Experiments 1 and 2) ................................................................................. 106
Effects of Inhibition of Endogenous WNT Signaling with Wnt-C59 or DKK1 on Ability of Embryos to Develop to the Blastocyst Stage and Blastocyst Cell Number (Experiment 3) ........................................................................ 107
Effects of DKK1 on Development and Blastocyst Cell Number (Experiments 4-5) .............................................................................................................. 107
Effects of DKK1 on Developmental Changes in Immunoreactive YAP1 and CDX2 in Morulae and Blastocysts (Experiment 6) ...................................... 107
DKK1 Does not Activate pJNK (Experiment 7) ............................................... 108
Regulation of WNT Signaling by WNT7A (Experiments 8 and 11) ................. 108
Discussion ............................................................................................................ 109
4 CONSEQUENCES OF EXPOSURE OF EMBRYOS TO DICKKOPF-RELATED PROTEIN 1 AND COLONY STIMULATING FACTOR 2 ON BLASTOCYST YIELD, PREGNANCY RATE, AND BIRTH WEIGHT OF THE CALF ................... 126
Introduction ........................................................................................................... 126
Materials and Methods .......................................................................................... 127
Animals and Experimental Design .................................................................. 127
Oocyte Retrieval ............................................................................................. 128
Oocyte Classification, Transport and Maturation ............................................ 129
Embryo Production ......................................................................................... 130
Embryo Transfer and Pregnancy Diagnosis ................................................... 131
Birthweights of the Offspring .......................................................................... 132
Statistical Analyses ........................................................................................ 132
Results .................................................................................................................. 133
In Vitro Production of Embryos ....................................................................... 133
Pregnancy Rate .............................................................................................. 133
Postnatal Characteristics ................................................................................ 133
Discussion ............................................................................................................ 133
5 IDENTIFICATION OF POTENTIAL EMBRYOKINES IN THE BOVINE REPRODUCTIVE TRACT .................................................................................... 139
Introduction ........................................................................................................... 139
Materials and Methods .......................................................................................... 141
Synchronization of the Estrous Cycle ............................................................. 141
Collection of Oviductal and Endometrial Tissues and Uterine Flushings ........ 141
RNA Extraction and Gene Expression ........................................................... 143
Immunofluorescence ...................................................................................... 145
10
Western Blotting for CSF2 .............................................................................. 147
Results .................................................................................................................. 148
Expression of Putative Embryokines Expressed in Oviduct ........................... 148
Expression of Putative Embryokines Expressed in Endometrium .................. 149
Immunolocalization of Selected Embryokines within Endometrium ................ 150
Accumulation of CSF2 in Uterine Flushings ................................................... 151
Discussion ............................................................................................................ 151
6 GENERAL DISCUSSION ..................................................................................... 173
LIST OF REFERENCES ............................................................................................. 179
BIOGRAPHICAL SKETCH .......................................................................................... 207
11
LIST OF TABLES
Table page 1-1 Phenotypes generated by mutation of Wnts in mouse ....................................... 52
2-1 Primer sequences used for real-time PCR. ........................................................ 82
2-2 Effect of stage of development and cell lineage on expression of genes involved in WNT signaling .................................................................................. 83
2-3 Effect of WNT11 from day 5 to day 7 after insemination on development of embryos to the blastocyst stage at day 7 ........................................................... 85
3-1 Effect of exposure of embryos to GSK3 inhibitor from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stage ....... 115
3-2 Effect of treatment of embryos with the WNT agonist AMBMP and the endogenous regulator of WNT signaling, DKK1 from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stage ....... 116
3-3 Effects of inhibition of endogenous WNT signaling from day 5 to day 7 of development with either Wnt-C59 or DKK1, on ability of embryos to develop to the blastocyst stage, and cell number of blastocysts .................................... 117
3-4 Effect of exposure of embryos to DKK1 from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stage and cell number of day 7 blastocysts .......................................................................................... 118
3-5 Effect of treatment of embryos with DKK1 from day 5 to day of development on the ability of male and female embryos to develop to the blastocyst stage and cell number of day 7 blastocysts ................................................................ 119
3-6 Effect of treatment of embryos with recombinant WNT7A from day 1 to day 7 of development or from day 5 to 7 of development on the ability of embryos to develop ......................................................................................................... 120
3-7 Effect of treatment of embryos with recombinant WNT7A from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stage and cell number of day 7 blastocysts. ............................................................... 121
4-1 Effect of exposure of embryos to the CSF2, DKK1 or the combination on embryonic development and pregnancy rate of cows receiving an embryo ..... 137
5-1 Least-squares means for expression of 93 genes in oviduct ipsilateral to the side of ovulation during the first seven days of the estrous cycle ..................... 157
12
5-2 Genes whose expression in the oviduct ipsilateral to the side of ovulation was affected by day of the estrous cycle within the first 7 days after ovulation 160
5-3 Genes whose expression in the oviduct was differentially expressed between sides ipsilateral and contralateral to the side of ovulation within the first three days after ovulation .......................................................................................... 161
5-4 Genes differentially expressed in oviduct ipsi and contralateral to the side of ovulation that vary during the first 3 days after ovulation .................................. 162
5-5 Least-squares means for expression of 93 genes in endometrium during the first seven days of the estrous cycle averaged from both sides of the reproductive tract. ............................................................................................. 163
5-6 Genes whose expression in the endometrium was affected by day of the estrous cycle within the first 7 days after ovulation ........................................... 166
5-7 Genes whose expression was affected by the interaction between day of the estrous cycle and side of the reproductive tract relative to ovulation ................ 167
13
LIST OF FIGURES
Figure page 1-1 Position-dependent Hippo signaling in preimplantation embryos. ...................... 53
1-2 Downstream cascades of WNT signaling pathway. ............................................ 54
2-1 Developmental changes in expression of selected genes involved in WNT signaling for embryos produced in vitro .............................................................. 86
2-2 Representative examples of localization of immunoreactive β-catenin at various stages of preimplantation development of embryos ............................... 87
2-3 Representative examples of localization of immunoreactive non-phospho (active) β-catenin during preimplantation development ...................................... 88
2-4 Lack of localization of active β-cateninin the nucleus of in vitro produced embryos after activation of canonical WNT signaling with the GSK3 inhibitor .... 89
2-5 Consequences of treatment of embryos with WNT agonists for immunolocalization of active β-catenin. .............................................................. 90
2-6 Localization of active β-cateninin mouse and bovine embryos by confocal microscopy. ........................................................................................................ 91
2-7 Immunoreactive phospho-JNK in blastocysts produced in vitro ......................... 92
2-8 Representative confocal microscopy images of immunoreactive active β-catenin in bovine embryonic fibroblast cells following activation of canonical WNT signaling. ................................................................................................... 93
3-1 Treatment of embryos at day 5 after insemination with DKK1 reduces amounts of immunoreactive β-catenin but does not prevent the WNT agonist (AMBMP) from increasing amounts of β-catenin. ............................................. 122
3-2 Immunolocalization of the transcription factors YAP1 and CDX2 in morulae and blastocysts. ................................................................................................ 123
3-3 Effect of treatment of embryos with DKK1 from day 5 to day 7 of development on accumulation of pJNK. ........................................................... 124
3-4 Effect of treatment of embryos with WNT7A from day 5 to day 7 of development on accumulation of β-catenin and pJNK ...................................... 125
4-1 Effect of addition of embryokines during day 5-7 after fertilization on birth weight ............................................................................................................... 138
14
5-1 Expression of the top 50 expressed genes in the oviduct at days 0, 3, 5 and 7 after ovulation ................................................................................................... 168
5-2 Expression of the top 50 expressed genes in the endometrium at days 0, 3, 5 and 7 after ovulation ......................................................................................... 169
5-3 Immunolocalization of CSF2, DKK1 and WNT5A in endometrium. ................. 170
5-4 Immunolocalization of WNT7A in endometrium ................................................ 171
5-5 Detection of CSF2 in uterine fluid by Western blotting ..................................... 172
15
LIST OF ABBREVIATIONS
AMBMP 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3methoxyphenyl)pyrimidine
BSA Bovine serum albumin
cDNA Complementary deoxyribonucleic acid
COC Cumulus oocyte complex
CT Cycle threshold
DAPI 4’,6-diamidino-2-phenylindole
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DPBS Dulbecco’s phosphate buffered saline
DPBS-PVP Dulbecco’s phosphate buffered saline- containing 1% (w/v) polyvinylpyrrolidone
EGA Embryonic genome activation
FBS Fetal bovine serum
FITC Fluorescein isothyocyanate
ICM Inner cell mass
IgG Immunoglobulin G
IVF In vitro fertilization
IVP In vitro produced
mRNA Messenger ribonucleic acid
PVP Polyvinylpyrrolidone
qPCR Quantitative real time polymerase chain reaction
RNA Ribonucleic acid
SOF-BE2 Synthetic oviductal fluid-bovine embryo 2
TE Trophectoderm
16
Note: Gene symbols are used without definition. Gene names can be retrieved from Pubmed https://www.ncbi.nlm.nih.gov/gene
17
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
WNT SIGNALING IN THE PREIMPLANTATION BOVINE EMBRYO
By
Paula Tríbulo
August 2017
Chair: Peter J. Hansen Major: Animal Molecular and Cellular Biology
Errors during preimplantation development result in embryonic mortality and/or
suboptimal phenotype after birth. Although WNT signaling regulates several
developmental processes, its specific role during preimplantation development remains
unclear. The aim of the research presented in this dissertation was to unravel the role of
WNT signaling during preimplantation development and evaluate the participation of
maternal cues on the regulation of embryonic WNT signaling pathways within the
embryo. First, it was found that the typical WNT signaling mediated by nuclear
localization of β-catenin is not fully functional during preimplantation development and
WNT signaling relies on non-nuclear β-catenin as well as β-catenin independent
pathways. Then, the consequences of endogenous and exogenous WNT signaling for
development in the preimplantation embryo were evaluated. Results indicate that
embryo-derived WNTs are dispensable for blastocyst formation, but participate in the
regulation of inner cell mass (ICM) proliferation, likely through a mechanism
independent of β-catenin. The reduction in blastocyst development upon stimulation of
WNT signaling with the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-
methoxyphenyl)pyrimidine (AMBMP) and the increase of embryos becoming blastocyst
18
when exposed to WNT7A lead to the hypothesis that maternally-derived WNTs can play
a positive or negative role in regulation of preimplantation development. Subsequently,
expression of WNT-related molecules was assessed in the reproductive tract during the
first seven days after ovulation, the time at which preimplantation development takes
place in the bovine. A large number of WNT and WNT-related proteins were detected
underpinning the hypothesis that the dam regulates WNT signaling in the embryo. An
additional objective of the studies presented here was to evaluate whether or not WNT
signaling during preimplantation programs the embryo to have different characteristics
after birth. In vitro produced embryos were treated with the WNT-regulatory molecule,
DKK1, and resulted in calves with reduced birth weights. This result illustrates the ability
of DKK1 to alter the pattern of development of the bovine embryo to affect postnatal
phenotype. Overall, results show that WNT signaling is maternally-regulated, and
although dispensable for blastocyst formation, it imposes changes in the
preimplantation embryo that modify the postnatal phenotype.
.
19
CHAPTER 1 LITERATURE REVIEW
Introduction
Successful development of a newly-formed embryo is a complex process. In the
cow, less than 50% of zygotes are viable 7 days after fertilization (Sartori et al., 2010)
and about 25% of embryos reaching the blastocyst stage are lost before calving
(Hansen, 2011). Maternal errors and inherent defects in the embryo cause these
embryonic losses. Knowledge on the regulatory mechanisms of embryonic development
and on the optimal maternal environment to maximize pregnancy outcomes is
incomplete. Although outcomes of procedures for vitro embryo production reveal that
maternal signals are not essential during the preimplantation period, the importance of
maternal cues as a modulator of development is revealed by the aberrant
characteristics of in vitro derived blastocysts for gene expression (Corcoran et al.,
2006; McHughes et al., 2009; Gad et al., 2012), metabolism (Khurana and Niemann,
2000), lipid content (Crosier et al., 2000; Sudano et al., 2012), ultrastructure (Boni et al.,
1999; Rizos et al., 2002), and DNA methylation (Niemann et al., 2010). Moreover, the
pregnancy rate of embryos produced in vitro is lower than that for embryos produced in
vivo (Lonergan et al., 2007; Pontes et al., 2009).
Another strong piece of evidence for the influence of maternal environment on
embryonic development is the need of synchrony between donor and recipient animals
in embryo transfer programs. (Newcomb and Rowson, 1975; Hasler et al., 1987). The
environment in which the embryo undergoes preimplantation development can affect
development even to the point of modifying phenotypic characteristics after birth. For
instance, the offspring of pregnant rodents fed a low protein diet exclusively during the
20
time when the embryo undergoes preimplantation development displayed
cardiovascular and metabolic diseases in adulthood (Kwong et al., 2000; Watkins et al.,
2008). Bovine embryos exposed to colony stimulating factor 2 (CSF2) for two days
during preimplantation development grow faster after birth than those not exposed to
CSF2 (Kannampuzha-Francis et al., 2015). Similarly, offspring from in vitro derived
embryos show altered postnatal phenotype compared to their in vivo counterparts such
as cardiometabolic dysfunction in humans (Ceelen et al., 2008), behavioral alterations
in anxiety and deficiencies in memory in mice (Fernandez-Gonzalez et al., 2004) and
large offspring syndrome in cattle (Behboodi et al., 1994; Numabe et al., 2000).
This dissertation focuses on the role of one potential maternal signaling system
for regulation of embryonic development – the WNT system. Originally described as
being involved in wing formation in Drospophila (Wingless) and mammary development
in mammals (Int), the WNT family of secretory ligands participates in diverse
developmental processes including cell proliferation (Logan and Nusse, 2004),
maintenance of pluripotency (Sato et al., 2004; Sokol, 2011), differentiation (Liu et al.,
2014) and migration (Morosan-Puopolo et al., 2014). WNT signaling has been proposed
as one system involved in embryo-maternal cross talk because WNT-related molecules
are produced in the reproductive tract and embryonic survival in mice can be reduced
through manipulation of WNT signaling pathways around the time of implantation
(Mohamed et al., 2004; Hayashi et al., 2007). WNTs are also involved in regulation of
pluripotency (reviewed by Nusse et al., 2008; Clevers et al., 2014) and may therefore be
important for some of the early differentiation events in the preimplantation embryo.
Despite the potential for WNTs to play an important role in development of the
21
preimplantation embryo, the specific role of these molecules during preimplantation
development is rudimentary.
The following literature review will begin with an overview of the key events that
take place during preimplantation development. The main focus will be on the bovine
embryo but information from other species is included when pertinent. Thereafter, an
overview of WNT signaling will be provided and then current knowledge of the role of
WNTs during preimplantation development in mammalian embryos will be summarized.
Preimplantation Embryonic Development in the Cow
Overview
Preimplantation development refers to early developmental processes in
mammalian embryos that take place before implantation and while the embryo transits
through the oviduct to reach the uterus. In the species that have been most studied, the
mouse and human, implantation takes place at the blastocyst stage of development so
the preimplantation period encompasses the time from fertilization to blastocyst
formation. In the cow, however, implantation does not formally occur (the trophoblast
does not penetrate the endometrial epithelium to invade the endometrial stroma) and
the first attachments of the trophoblast to the endometrium occur at about day 21 (King
et al., 1981). Thus, the preimplantation period is prolonged in the bovine and involves
many more differentiation events than simply formation of the blastocyst. For purposes
of this dissertation, however, and to make comparisons with the mouse and human
more appropriate, the preimplantation period in the cow will be considered as continuing
up to formation of the blastocyst.
22
Development of the bovine embryo to the blastocyst stage takes approximately 7
days, with embryos entering the uterus on day 5 after fertilization (Betteridge and
Fléchon, 1988). The fertilized egg, i.e. the zygote, undergoes subsequent cell divisions
to originate 2-, 4-, 8-cell developmental stages and so on. These cellular divisions are
often asynchronous allowing the existence of embryos with uneven number of
blastomeres (Betteridge & Fléchon 1988). One characteristic of cellular divisions during
the preimplantation period is that the size of individual cells is reduced as proliferation
advances, as a consequence of the constant size of the entire embryo. Therefore, these
divisions are referred to as cleavage divisions. Individual blastomeres are
distinguishable up to the 16-cell stage but thereafter, compaction occurs, individual cell
boundaries are obscured and the embryo acquires a blackberry (morus in Latin)
appearance and is termed a morula. Following the appearance of a blastocoelic cavity
and the first cell lineage commitment, the totipotent morula is transformed into a
multipotent blastocyst.
During preimplantation development there are four critical events including: 1)
the transition from maternal to zygotic control of development, also termed embryonic
genome activation; 2) compaction at the morula stage; 3) cell lineage commitment and
4) blastocyst formation.
Embryonic Genome Activation
Before the embryo is able to transcribe its own genome, cellular function and
proliferation is under the control of maternal mRNA accumulated and stored in the
oocyte during oocyte maturation (Schier, 2007; Fair, 2010). The transition from
maternally-derived mRNA to embryonic-synthesized transcripts has been termed
embryonic genome activation (EGA) or the maternal-zygote transition and is a key
23
event associated with successful early differentiation, implantation and fetal
development (Niemann and Wrenzycki, 2000). The timing for initiation of EGA is
species-specific, taking place at the 2 cell stage in the mouse (Wang and Dey, 2006; Li
et al., 2013), at the 4-8 cell stage in humans and swine (Braude et al., 1988; Sirard,
2012), and at the 8 cell stage in the bovine (Graf et al., 2014a).
Prior to EGA, developmental changes in protein synthesis depend on post-
transcriptional regulation of maternally-derived mRNA (Reyes and Ross, 2016).
Molecules involved in this process include cis-acting regulatory sequence elements,
proteins that bind to them or that modify RNA binding proteins and other proteins that
participate in translation. Cytoplasmic polyadenylation element (CPE) is one of the
regulatory elements present in mRNA molecules that have been widely studied. Via
binding to CPE binding protein (Cao and Richter, 2002), CPE labels maternal mRNA for
translation during oocyte maturation (Ma et al., 2013). Maternal mRNA lacking CPE
become deadenylated and translationally inactive (Fox and Wickens, 1990; Varnum and
Michael Wormington, 1990). Deadenylated mRNA are either subsequently degraded or
can be readenylated after fertilization (Paynton et al., 1988). Readenylation in mammals
has been proposed to be an evolutionary vestige of the mechanism used in lower
vertebrates for control of protein synthesis in the embryo; in these species,
readenylation of maternally derived RNA is a key event to allow development within
hours from fertilization (Bachvarova et al., 1985).
The processes implicated in EGA include degradation of oocyte-derived mRNA,
protein turnover, rearrangement of cellular organelles and cytoskeleton,
uncharacterized actions of specific maternal proteins composing the subcortical
24
maternal complex, epigenetic changes in the chromatin to allow transcription, and
acquisition of transcription machinery (reviewed by Stitzel and Seydoux, 2007). In the
mouse, these changes start with resumption of meiosis in the oocyte and redistribution
of mitochondria and endoplasmic reticulum (ER) (Voronina and Wessel, 2003).
Fertilization is also involved in EGA because it initiates Ca+ signaling inside the egg
(Malcuit et al., 2006) by activating phosphatidylinositol 3-kinase signaling (Miyazaki et
al., 1989). This mechanism induces the cell machinery to control subsequent events
including cell cycle resumption and internalization of ER (FitzHarris et al., 2003) and is
crucial for EGA (Zheng et al., 2010). Furthermore, fertilization triggers digestion of the
vast majority of maternal transcripts in the oocyte (Potireddy et al., 2006), another key
event for the success of EGA (Ma et al., 2013). Removal of maternal mRNA involves
loss of masking proteins that prevent transcripts from degradation in the oocyte, such as
mouse specific Y-box protein 2 (Medvedev et al., 2008) and activation of miRNA that
impair translation of maternal mRNA (reviewed by Lee et al., 2014). Chromatin
remodeling to allow transcription involves epigenetic changes whose regulation is
partially mediated by Brg1 protein. The key role of maternally-derived Brg1 in controlling
EGA is reflected by the massive reduction in transcription and developmental arrest at
the 2-cell stage in embryos derived from null mutant oocytes (Bultman et al., 2006).
These mutant embryos show reduced methylation of histone 3, indicating that the
mechanism mediating defective EGA involves epigenetic marks. Indeed, Brg1 changes
the conformation of DNA around histones.
The activation of embryonic transcription is a stepwise process involving two
major rounds of embryonic zygotic transcription. In the first, denominated as minor
25
EGA, there is a small set of de novo transcripts, followed by a major round of embryonic
transcription that reprograms the gene expression pattern (reviewed by Kanka, 2003).
In bovine embryos, the minor activation of transcription is observed at the 2-4 cell stage
(Memili and First, 2000; Kues et al., 2008), and is also thought to set the stage for the
major EGA (Graf et al., 2014a, b). This idea is supported by two findings. One is that the
approximately 400 genes initially transcribed (Graf et al., 2014a, b) are dispensable for
embryonic development since blocking transcription at this stage does not impair
blastocyst formation (Liu and Foote, 1997). The second is that these genes are involved
in functions including RNA processing, protein synthesis and protein transport (Graf et
al., 2014a) which are consistent with a role for preparation of the cell for the upcoming
massive transcription of the genome. Gene ontology analysis of the approximately 4200
genes activated in the major EGA indicates participation of the genes in RNA splicing;
mRNA transcription from RNA polymerase II promoter; regulation of transcription from
RNA polymerase II promoter; purine nucleotide biosynthesis; and 5S class rRNA
transcription from RNA polymerase III type 1 promoter.
Compaction
Compaction is the first morphological change in the preimplantation embryo and
is characterized by a reduction in the entire volume of the embryo as well as the loss of
clearly-identifiable boundaries between individual cells. The process is driven by
cytoskeletal changes in the embryo and is a prerequisite for subsequent differentiation
of embryos into differentiated cells of the blastocyst.
Most of the current understanding of compaction comes from the mouse model.
In this species, compaction starts at the 8-cell stage of development when polarization
is established in blastomeres by the emergence of an apical and a basolateral domain
26
of the cellular membrane (Fleming and Pickering, 1985; Johnson and McConnell, 2004).
At this moment, cellular contacts increase by close membrane apposition and
intercellular space is minimized. Consequently, blastomeres flatten against each other
becoming no longer individually distinguishable (Calarco and Brown 1969, Ducibella et
al., 1977). The cells of the morula eventually show variation in appearance with outer
cells remaining polar but with inner cells being not polarized.
The mechanisms involved in compaction include cellular adhesion, cortical
tension and filopodia. Early studies documented the key role of cell adhesion in
compaction because the process could be inhibited either by blocking Ca+-dependent
adhesion (Fleming et al., 2001) or targeting cell surface glycoproteins (Ducibella and
Anderson, 1975). E-cadherin is the essential glycoprotein mediating compaction since
the process is completely disrupted in its absence (Stephenson et al., 2010). Ligation of
extracellular domains of E-cadherins from adjacent cells result in formation of adherens
junctions while intracellular domains allow connection of the cellular membrane and the
cortex via interaction with the cytoskeleton (Hoffman and Yap, 2015).
During compaction other junctional complexes are established as well, including
desmosomes, tight and gap junctions (Bell et al., 2008). The role of these other
complexes in compaction is not clear. For instance, blockage of the main constitutive
protein of the gap junction, connexin-43 (Cx43), results in unraveling of compaction and
extrusion of blastomeres from the rest of the embryo (Becker and Davies, 1995).
Connexin-43 knockout mice, however, successfully progress through compaction (De
Sousa et al., 1997), suggesting the existence of compensatory mechanisms.
27
Even though cell adhesion is widely accepted to be critical for compaction
(Kemler et al., 1977; Vestweber et al., 1985), the role of E-cadherin in this process has
been questioned (Maître et al., 2012). The tension mediated by E-cadherin increases
contact surfaces between cells and is referred to as adhesive tension. Cortical tension,
on the other hand, is generated by a dynamic network of actin filaments connected to
the cell membrane that are contracted or expanded by myosin motor proteins of the
cytoskeleton (Pasternak et al., 1989) so as to reduce intercellular contacts, i.e.
conferring cells with a spherical shape. The differential interfacial tension hypothesis
postulates that cellular shape is dictated by the balance between cortical and adhesive
tensions (Brodland, 2002). Accordingly, cells allocate in such a pattern that maximizes
adhesive tension minimizing their cortical tension. Indeed, the role of cortical tension in
compaction has been recently reported (Maître et al., 2015).
Other players in the process of compaction are the filopodia which have been
recently proposed to participate in drawing adjacent cells closer to each other (Fierro-
González et al., 2013). Filopodia were first described as membrane protrusions
containing E-cadherin and proteins that link it to the actin cytoskeleton i.e. α- and β-
catenin observed in mouse embryos from 8-cell (at the onset of compaction) to 16-cell
stage (compacted) (Fierro-González et al., 2013). Interestingly, only a fraction of
blastomeres possess filopodia and they are extended to few neighboring cells
simultaneously. Further characterization of these structures is needed to understand
their role and mechanism of action during compaction.
Regulatory pathways involved in regulation and timing of compaction are
unknown. It has been proposed that the nucleus:cytoplasm ratio is responsible for
28
triggering compaction, as it is important in compaction in fish and amphibians (Newport
and Kirschner, 1982). Initial studies showed that artificially increasing the
nuclear:cytoplasm ratio by aspirating cytoplasmic material from 1-cell mouse embryos
results in early compaction at the 4-cell stage (Feng and Gordon, 1997). The
experimental design of this study does not allow determination as to whether aspiration
exerted its affect by altering the nuclear:cytoplasm ratio or by extraction of maternal
factors regulating embryonic morphogenesis. Moreover, restoring cytoplasmic material
has diverse outcomes depending upon the developmental stage of the embryo used as
the cytoplasm donor (Lee et al., 2001). Specifically, cytoplasm from advanced stage
donor embryos delayed embryonic morphogenesis, whereas cytoplasm from the 1-cell
donor hastened it. (Lee et al., 2001).
In the cow, compaction occurs at day 6 when the embryo has approximately 32
blastomeres (Betteridge & Flechón 1988). Comparison of the expression of CX43
during preimplantation development of in vivo- and in vitro-derived embryos suggests
that the mechanism of cell-cell communication differ between these embryos
(Wrenzycki et al., 1996). While in vivo derived embryos express CX43 throughout the
preimplantation development; morulae and blastocysts derived from culture do not
express CX43, but still develop to the blastocyst stage, suggesting redundant
mechanisms involved in cell-cell communication.
First Lineage Commitment: Trophectoderm Differentiation
Although segregation of the first two distinct cell populations of the embryo,
trophectoderm (TE) and inner cell mass (ICM), is not established until the blastocyst
stage, events underpinning cell fate take place earlier during preimplantation
development. Whether blastomere fate is established before, during or after compaction
29
is debated (Rossant and Tam, 2009; Zernicka-Goetz et al., 2009; Burton and Torres-
Padilla, 2014) as will be discussed further in this section.
Origin of ICM and TE
Using the mouse model, two main models have been proposed to explain TE
differentiation. The polarity model identifies polarization of blastomeres at the 8-cell
stage as a key regulator of cell lineage commitment (Johnson et al., 1981). The division
plane of the polar blastomeres dictates whether the two daughter cells are polarized TE
cells (as a consequence of cellular division along the apical-basal plane, i.e.
symmetrical division) or one TE and one ICM cell (as a consequence of cellular division
along the meridional plane, i.e. asymmetrical division). The positional model, on the
contrary, postulates that cell fate is dictated by the position of the cell within the embryo
at the 16-cell stage (Tarkowski and Wróblewska, 1967). Thus, inner cells of the 16-cell
stage embryo become ICM while outer cells become TE. In agreement with this model,
changing position of blastomeres at the 16 and 32-cell stages resulted in modification of
cell lineage (Hillman et al., 1972; Kelly et al., 1977; Suwinska et al., 2008).
It is important to bear in mind, however, that manipulation of cell position affects
both polarization and cell fate (Ziomek et al., 1982 a, b; Eckert et al., 2005).
Consequently, a more recent model has been proposed to refine the polarity model by
considering molecular aspects associated with polarity (Yamanaka et al., 2006). In this
case the presence or absence of an apical domain (characteristic of polar cells) is
proposed to drive cell fate decisions. The fact that a defined feature, i.e. apical domain,
dictates the fate of the cell is less questionable than the more subjective classification of
inner and outer cells, which is typically based on localization of nuclei and which can be
quite variable among blastomeres (Ajduk et al., 2014).
30
Recently, labeling cellular membrane and recording embryo development with
high resolution, followed by computationally segment of embryos allowed
characterization of ICM formation in live mouse by tracking individual cell shape and
position over time (Samarage et al., 2015). Behavior of most blastomeres (over 100
embryos were videotaped and studied) lead the authors to propose that ICM originates
from symmetrical cell divisions of polar cells at the 12-cell stage that are subsequently
internalized by apical constriction, a process driven by contraction of actomyosin
networks that shrink the apical surface.
Some scientists argue that cell fate commitment does not occur until the embryo
undergoes compaction. Evidence includes variable distribution of inner and outer cells
expressing caudal type homobox 2 (CDX2; TE marker) and Nanog homeobox (Nanog;
epiblast marker) before compaction (Ditrich 2007), plasticity in cell fate when
experimentally blastomeres are extracted or repositioned (Papaoiannou and Ebert
1986), and failure to identify uneven distribution of fate markers in mammalian embryos
at early stages of development (Kurotaki et al., 2005; Motosugi et al., 2005). Others
propose that molecular heterogeneities that predict cell fate can be observed as early
as the 2 to 8-cell stage (Gardner, 2001; Plachta et al., 2011; Shi et al., 2015). Examples
of predictors include sperm entrance position, which is proposed to dictate the plane of
cellular division establishing heterogeneity between blastomeres (Piotrowska and
Zernicka-Goetz, 2001), and timing of first cleavage (Piotrowska et al., 2001).
Transcription factors driving ICM-TE segregation
Whether because of position or polarity, cell fate is ultimately controlled by
expression of lineage-specific transcription factors. These transcription factors are
species-specific and have been substantially studied in mouse embryos. In bovine
31
embryos, however, little information is available and will be detailed towards the end of
this section.
In mouse embryos, markers for TE include Cdx2, Eomesodermin (Eomes), Gata
binding protein 3 (Gata3), Kruppel-like factor 5 (Klf5), and TEA domain family member 4
(Tead4) (Beck et al., 1995; Niwa et al., 2000; Strumpf et al., 2005); while POU domain,
class 5, transcription factor 1 (Pou5f1), Nanog, and SRY (sex determining region Y)-box
2 (Sox 2) are markers of cell pluripotency within the ICM (Cao, 2013). Expression of
Pou5f1and Cdx2 is ubiquitous at the 8-cell embryo but Cdx2 becomes restricted to
outer cells at the morula stage, while Pou5f1is confined to nuclei of the ICM at the
blastocyst stage (Niwa et al., 2000; Strumpf et al., 2005). Expression of Cdx2 and
Pou5f1 is mutually exclusive at the blastocyst stage, as indicated by Cdx2-/- mouse
embryos whose phenotype retains expression of Pou5f1 and Nanog in outer cells
(Strumpf et al., 2005).
The transcription factors driving TE differentiation regulate one another. Thus,
Gata3 and Cxd2 are regulated by Tead4 (Ralston et al., 2010; Hirate et al., 2012).
Moreover, Cdx2 is also regulated by Klf5 (Lin et al., 2010). Downstream of Cdx2 is
Eomes (Strumpf et al., 2005). Studies using knock out embryos for the genes encoding
these transcription factors show that in absence of either Klf5 or Tead4, embryos fail to
form blastocyst and neither Cdx2 nor Eomes are expressed (Yagi et al., 2007; Lin et al.,
2010). Subsequently, it was found that Tead4 directly activates Cdx2 enhancer (Rayon
et al., 2014). When Cdx2 or Eomes are deleted, blastocysts form but abnormalities in
TE result in impaired implantation (Strumpf et al., 2005). Interestingly, Klf5 and Tead4
are detected from the 2 cell stage through the blastocyst stage and they both localize to
32
the outer cells of the blastocyst (Yagi et al., 2007; Lin et al., 2010). Furthermore, Gata3
Cdx2 and Eomes are first expressed at the 4-cell, 8-cell and blastocyst stages of
development, respectively (Strumpf et al., 2005; Home et al., 2009).
Among the transcription factors characterizing ICM, Sox2 is the first to be
selectively expressed in inner cells, at the 16-cell stage (Guo et al., 2010), and is
confined to ICM progenitors before blastocyst formation (Wicklow et al., 2014).
Expression of Sox2, however, is observed as early as the unfertilized oocyte throughout
preimplantation development (Li et al., 2013). Uniform expression of Pou5f1is observed
from the 8-cell stage, but in contrast to Sox2, it remains ubiquitous up to the middle-to-
late blastocyst stage, when it becomes restricted to ICM (Dietrich and Hiiragi, 2007;
Palmieri et al., 1994). Similarly, Nanog has a wide distribution until late blastocyst stage
when it is found only in a sub population of the ICM (Strumpf et al., 2005; Dietrich and
Hiiragi, 2007; Wicklow et al., 2014).
In the cow, CDX2 and POU5F1 are co-expressed in all nuclei up to the morula
stage, similar to the situation in mouse embryos. In contrast to the mouse, they remain
co-expressed at the blastocyst stage due to lack of a mutation in the regulatory
sequence of POU5F1 present in the mouse that allows CDX2 binding and repression of
POU5F1 expression in TE cell (Berg et al., 2011). The key role of CDX2 on TE
differentiation was initially revealed by failure of TE development after CDX2 inactivation
(Berg et al., 2011). More recently, blastocyst development was observed in CDX2
depleted embryos, but embryos defects included epithelial integrity lost and
upregulation of NANOG (Goissis and Cibelli, 2014; Sakurai et al., 2016). Furthermore,
TE differentiation is characterized by increasing expression of CDX2 with decreasing
33
pluripotent capabilities that becomes irreversible by day 11 of development (Berg et al.,
2011). Another difference between mouse and bovine embryos is that TEAD4 is not a
master transcription factor in bovine embryos. Downregulation of TEAD4 neither impairs
blastocyst formation nor affects expression of CDX2, GATA3 or POU5F1 (Sakurai et al.,
2016). Master transcription factors responsible for maintenance of ICM remain unknown
in bovine. In late blastocysts, Nanog and Sox2 are confined to ICM as is the case for
mouse embryos (Kuijk et al., 2008).
Role of Hippo signaling in regulation of TE differentiation
As mentioned above, polarization, position and expression of master regulators
are involved in cell lineage segregation. Recently, the Hippo signaling pathway has
been implicated as a key connector of these regulators in the mouse embryo.
Hippo signaling participates in the regulation of expression of the master
transcription factor that mediates TE differentiation, Tead4. The core molecules of the
Hippo pathway are the protein kinases Mst1/2 and Lats1/2, their transcriptional co-
activators Yap1 and Taz, and the target transcription factors Tead1-4 (Figure 1-1). E-
cadherin-mediated cell-cell adhesion stimulates Hippo signaling (Kim et al., 2011),
which, when active, prevents nuclear translocation of Yap1, which is a transcriptional
co-activator of Tead4 (Zhao et al., 2007, 2010). Consequently, activation of the pathway
suppresses expression of target genes, among them, Tead4. Hippo signaling is spatially
regulated, and therefore differentially activated in polar and apolar cells. The Hippo
pathway components angiomotins (encoded by Amot) are proteins involved in
phosphorylation-mediated inactivation of Yap1. In preimplantation embryos Amot has a
highly distinctive distribution between polar and apolar cells (Hirate et al., 2013; Leung
and Zernicka-Goetz, 2013). In polar (outer) cells, angiomotin (Amot) is restricted to the
34
apical domain where its phosphorylation is impaired so that Hippo signaling is not
activated and Tead4 expression occurs (Hirate et al., 2013). In contrast, basolateral
localization of Amot in apolar cells results in activation of Hippo signaling and a block to
nuclear accumulation of Yap1 via phosphorylation of Lats 1/2 and Hippo-independent
binding of Yap (Chan et al., 2011; Paramasivam et al., 2011; Hirate et al., 2013).
Second Lineage Commitment: Hypoblast Differentiation
By the late blastocyst stage, a third cell type, the hypoblast, differentiates from
ICM (Kuijk et al., 2008). This cell lineage, called the primitive endoderm in mice, forms
an epithelium located on the surface of the ICM lying in contact with the blastocyst
cavity. When the hypoblast forms, the remaining cells of the ICM, which remain
pluripotent, are denominated as epiblast.
In mouse, segregation of hypoblast cells was thought to be driven by the position
of the cell within ICM (Enders et al., 1978). The “positional induction” model proposed
that cells on the surface of the ICM in contact with the blastocoel are sensitive to an
inductive signal that triggers cell differentiation, and that the hypothetical signal does not
rich deep in the ICM. This model was supported by the observation that cells isolated
from outer ICM differentiate into hypoblast (Dziadek, 1979). One assumption of this
model was that all cells within ICM have an equivalent capability to become either
epiblast or hypoblast (Zernicka-Goetz et al., 2009). Subsequently, it was discovered that
the ICM consists of a heterogeneous population of cells, characterized by a mix of cells
expressing different levels of the transcription factors Nanog and Gata6 as markers of
epiblast and hypoblast precursors. As a result, a “sorting model” has been proposed in
which hypoblast cells first differentiate and then move to the outer part of the ICM
(Rossant et al., 2003; Chazaud et al., 2006). Time-lapse dynamics of Gata6 localization
35
showed that hypoblast precursors move from deep to surface layers within ICM (Plusa
et al., 2008). This study also revealed that some cells expressing Gata6, change their
fate by switching to Nanog expression, or undergo apoptosis. The recent finding that
some ICM cells contribute to both cell types (Meilhac et al., 2009) has led to a new
model in which both cell movement and positional induction are proposed to participate
in lineage segregation (Meilhac et al., 2009).
It is now accepted that lineage allocation of cells in the ICM to hypoblast and
epiblast involves a temporal sequence of 1) initial co-expression of lineage-specific
transcription factors, 2) mutually-exclusive expression of transcription factors where
hypoblast and epiblast precursors are distributed in the ICM in a salt-and-pepper
manner, and 3) cell movement leading to the sorting and spatial segregation of the
epiblast and hypoblast cell lineages (Plusa et al., 2008; Meilhac et al., 2009). The
upstream mechanisms that lead to the mosaic pattern of distribution of hypoblast and
epiblast precursors, and the pathways required for a fated cell to reach its final position
remain unknown. One interpretation of the “salt-and-pepper” distribution of cells in the
ICM has been that formation of cells destined for hypoblast and epiblast is random. An
alternative view is the “social mobility” model in which it is proposed that a first wave of
cells becomes epiblast and the second becomes hypoblast. Those cells in the
inadequate location move, switch type, or undergo apoptosis (Zernicka-Goetz et al.,
2009). Nevertheless, the exact mechanisms that connect cell polarity, cell position, and
cell signaling to the cell fate in blastocysts remain to be determined.
One of the first lineage-specific transcription factors expressed in hypoblast is
Gata6, which has been observed around the 16 to 32-cell stage. Gata4, another
36
transcription factor of the Gata family, is expressed later, around the 64-cell stage, when
the salt-and-pepper distribution is observed, and cells are likely to have a defined fate.
Gata6 null mutant mice have defective visceral endoderm and die (Morrisey et al., 1998;
Koutsourakis et al., 1999). Once hypoblast precursors are positioned underlying
epiblast, Gata6 is co-expressed with Sox17, and this latter transcription factor is
required for maintainance of hypoblast commitment (Artus et al., 2011). Additionally,
other markers of hypoblast have been recognized including Creb312, Dab2, Fgfr2, Fn1,
Grb2, Pdgfrα, Runx1, Snai1, Tcf23 (Cai et al., 2008; Plusa et al., 2008; Guo et al.,
2010).
Fibroblast growth factor (FGF) has been implicated in regulation of hypoblast
formation since mutations in Fgf4 ligand (Feldman et al., 1995; Goldin and
Papaioannou, 2003), Fgfr2 receptor (Arman et al., 1998) or Grb2, a downstream
signaling molecule (Chazaud et al., 2006), result in lack of hypoblast differentiation.
During preimplantation embryonic development, Fgf4 and Fgfr2 are the most highly
expressed members of the FGF signaling system (Niswander and Martin, 1992; Arman
et al., 1998). Although ubiquitous initially, these molecules become asymmetrically
expressed by the 32-cell stage, with the ligand and receptor higher in epiblast or
hypoblast precursor cells, respectively (Guo et al., 2010). By the 64-cell stage,
expression of these genes becomes mutually exclusive, with Fgf4 only in epiblast and
Fgfr2 in hypoblast (Messerschmidt and Kemler, 2010; Frankenberg et al., 2011).
Examination of the genes regulated by Gata6 indicates that pathways
upregulated in cells undergoing hypoblast differentiation include those involved in the
Ras/Erk pathway, canonical WNT signaling, and a signaling axis linking G-protein
37
signaling to RhoA and the ERM protein moesin (Verheijen et al., 1999; Liu et al., 2002;
Krawetz and Kelly, 2008). It has been suggested that the coordinate signaling by these
independent pathways is required to induce cell differentiation by modulating expression
of target genes and triggering cytoarchitectural changes.
In cattle, the hypoblast begins to form as an epithelial layer by day 8 after
insemination (Kuijk et al., 2012). The only current marker for hypoblast in cattle is
GATA6. In bovine embryos GATA6 has been observed in almost all nuclei at Days 5
and 6 after insemination (Kuijk et al., 2012). Further, d 40% of ICM cells in blastocysts
at day 7 and 8 after fertilization expressing GATA6 and the epiblast marker NANOG
concomitantly on day 7, while only 7% of those cell retain the double expression on day
8 (Kuijk et al., 2012).
Similar to the mouse, hypoblast formation in bovine embryos is also associated
with the FGF/MAPK pathway. The proportion of ICM cells expressing epiblast-specific
transcription factor and hypoblast expressing GATA6 is altered by modulating
FGF/MAPK signaling. Thus, treatment of bovine embryos with FGF receptor or MEK
inhibitors reduces GATA6 positive cells (Kuijk et al., 2012). Different from the mouse,
however, GATA6 positive cells are still present when FGF receptors are inhibited,
suggesting that more upstream regulators are involved in driving hypoblast
differentiation (Kuijk et al., 2012). Since hypoblast cells are the only cell type of
blastocyst maintaining GATA6, it is reasonable to think that GATA6 regulates
expression of genes that play a central role on hypoblast function.
Blastulation
Concomitant with the first cell fate decision that segregates TE and ICM, the
embryo cavitates to form the blastocyst. The process, known as blastulation , involves
38
acquisition of intercellular junctional complexes to form a sealed, epithelial like layer of
cells around the embryo (Ducibella and Anderson, 1979), functional modification of
cytoskeletal elements (Ducibella et al., 1977); migration of lipid vesicles and
mitochondria to the cellular cortex (Wiley 1987); and concentration of Na+/K+-ATPase
within the plasma membrane lining the blastocoel (Borland et al., 1977). It is proposed
that the polarity of ion/solute transporters, generated as a consequence of blastomere
polarization, creates an osmotic gradient across TE cells promoting diffusion of water
and thereby leading to the formation of the blastocoel. Initially, the trans –trophectoderm
ionic gradient was thought to be energized by Na+/K+-ATPase since blastocoel
formation is abolished after blockage of Na+ (Betts et al., 1997). The rapid transport of
water that takes place during cavitation relative to the small osmotic gradient generated
by the Na+/K+-ATPase confined to the basolateral membrane domain promoted further
investigation that led to the discovery of the presence of aquaporins (AQP) in TE cells
(Barcroft et al., 2003). Aquaporins are transmembrane proteins functioning as molecular
water channels that allow water flow in direction of osmotic gradients. Additional
mechanisms exerted by other structures/molecules may exist since Aqp knockout
embryos develop to the blastocyst stage (Marikawa and Alarcon, 2012). Bovine
embryos express AQP at the blastocyst stage (Camargo et al., 2011
WNT Signaling
Overview
The gene encoding for WNT was discovered by two independent research
groups. Scientists studying breast cancer in a mouse model named it Int-1(Nusse and
Varmus, 1982); while the group observing a wingless phenotype in knock out
39
Drosophila named it wingless (Nüsslein-Volhard and Wieschaus, 1980). Subsequently,
the homology between Int-1 and wingless was found (Rijsewijk et al., 1987) and the
name WNT arose.
WNT signaling participates in multiple developmental events during
embryogenesis as well as adult tissue homeostasis. Functions of this complex signaling
pathway include regulation of cell proliferation (Logan and Nusse, 2004), maintenance
of pluripotency (Sato et al., 2004; Sokol, 2011), differentiation (Liu et al., 2014) and
migration (Morosan-Puopolo et al., 2014). Several Wnt loss of function mutations have
been generated in the mouse producing phenotypes revealing the key role of WNT on a
wide range of developmental processes (Table 1-1). The role of WNT signaling during
preimplantation development, however, has not been well elucidated and will be
discussed in detail later in this review.
The diverse functions of WNT signaling are the result of the complex nature of
the signaling pathway. The signaling system is regulated by 19 WNT ligands that
interact with a variety of receptors including 10 frizzled receptors (FZD), receptor
tyrosine kinase like orphan receptor 2 (ROR2), protein tyrosine kinase (PTK7), knypek
(KNY) and related to tyrosine kinases (RYK) and co-receptors such as LDL receptor
related protein (LRP 5/6), kremen and leucine rich repeat containing G protein-coupled
receptor 4 (LGR4) (reviewed by Cadigan and Nusse, 1997; Logan and Nusse, 2004).
Ligands may also interact with extracellular WNT regulators such as dickkopf proteins
(DKK 1-4), secreted frizzled-related proteins (SFRP 1-5), and WNT inhibitor factor
(WIF1). DKK family proteins influence the signaling of WNTs by binding to the FZD co-
receptors LRP5/6 (Mao et al., 2001) and kremen (Mao et al., 2002).
40
DKK1 has a strict inhibitory effect on FZD receptor (Kazanskaya et al., 2000) that
depends on the availability of the kremen 1 coreceptor. In addition to the inhibitory
effect, DKK1 can also activate alternative WNT downstream signaling mediated by
small GTPases (Caneparo et al., 2007). Similarly, DKK2 and DKK4 require kremen 2 to
inhibit WNT signaling, and cannot function with kremen 1 (Mao et al., 2002). The
biological roles of DKK3 are still unknown since it does not interact to kremen or LRP
co-receptors and does not inhibit WNT signaling (Mao et al., 2001). The SFRP prevent
WNT actions by either direct interaction with WNT ligands preventing WNT-FZD binding
or formation of a non-functional ligand-FZD complex (reviewed by Kawano and Kypta,
2003). Similarly, WIF1 binds to WNTs abrogating their ability to bind FZD; thus it inhibits
WNT signaling (Hsieh et al., 1999).
Currently, three different pathways are described to be activated upon WNT-
receptor interaction (Figure 1-2): WNT/β-catenin signaling pathway (MacDonald et al.,
2009) is the best described downstream pathways, and is often called the canonical
WNT signaling pathway, In addition, there are the planar cell polarity pathway (PCP)
(Veeman et al., 2003; Seifert and Mlodzik, 2007), and the calcium signaling pathway
(Kühl et al., 2000; Kohn and Moon, 2005). The specific phenotype induced by WNT
signaling depends upon the ligands and receptors in play, as well as other specific
characteristics of signaling molecules in the cell; thus, specific WNT-receptor interaction
can invoke different outcomes in different cell types (Amerongen et al., 2008).
Canonical Wnt signaling
Canonical WNT signaling is the best-described pathway downstream of WNT.
The central effector of this signaling pathway is β-catenin (CTNNB1), a dual-function
protein that not only modulates gene expression upon translocation to the nucleus after
41
WNT activation, but also serves as a constitutive protein for cellular structures involved
in cell-cell adhesion (review by Perez-Moreno et al., 2003).
The canonical signaling pathway is depicted in Figure 1-2 (reviews by Logan and
Nusse 2004; Cadigan and Nusse 2015 ). In the absence of WNT, β-catenin is degraded
in a complex reaction involving phosphorylation and targeting to the proteasome.
Casein kinase I (CKI) primes the serine-threonine rich substrate within the β-catenin
molecule and a subsequent dual phosphorylation reaction is performed by glycogen
synthase kinase 3 (GSK3). For these phosphorylations to occur, β-catenin binds first to
AXIN, a protein that presents a docking motif for GSK3 adjacent to the β-catenin binding
motif. Such modification is recognized by E3 ubiquitin ligase TrCP1 and, after
ubiquitination, β-catenin is degraded in proteasomes. Targeting cytosolic β-catenin for
degradation takes place in a multimeric protein complex, known as the β-catenin
destruction complex, and formed by oligomers of AXIN; those not only bring together
GSK3 and β-catenin, but also recruit adenomatous polyposis coli (APC), a protein with
11 binding sites for β-catenin.
The degradation of β-catenin is inhibited upon binding of a WNT molecule to a
FZD receptor and LRP5/6 co-receptor (Figure 1-2). Subsequently, dishevelled protein
(DSH) associated with the intracellular domain of FZD changes its conformation to
expose a high affinity binding motif for AXIN. Consequently, AXIN oligomers are
removed from the β-catenin destruction complex to halt β-catenin targeting for
degradation. Cytosolic amounts of β-catenin increase, and eventually the protein
translocates to the nucleus where it first displaces GROUCHO to then interact with
transcription factors lymphoid enhancer-binding factor 1 (LEF1) and T-cell factor (TCF)
42
to regulate transcription of genes involved in cell proliferation and pluripotency such as
cyclin D1 (Tetsu and McCormick, 1999) and c-Myc (He et al., 1998),
WNT/planar cell polarity pathway
Cells and tissues experience two types of polarization: polarity along the apical-
basolateral axis, known as epithelial cell polarity; and polarity across the plane of the
epithelium named, termed tissue polarity or planar cell polarity (PCP). The latter is
regulated by the homonymous signaling pathway in which WNTs play a major role.
Interestingly, PCP is not restricted to epithelial tissues; it regulates cell migration and
cell intercalation in mesenchymal cells (Shih and Keller, 1992). Historically, knowledge
of the PCP pathway comes from arthropods, and later from sophisticated genetic
experiments in Drosophila (reviewed by Adler, 2002; Klein and Mlodzik, 2005). More
recently, PCP has been identified as a key regulatory mechanism for developmental
processes in vertebrates. In zebrafish and Xenopus it controls convergent extension
during gastrulation and neurulation (reviewed by Wallingford, 2005). In mammals, PCP
regulates a number of essential developmental processes including neural tube closure
and left-right patterning (Gray et al., 2011). The pathway has also been implicated in
malignancy since alterations of the PCP pathway can confer ability of cancer cells to
migrate (reviewed by Camilli and Weeraratna, 2010).
Several core components of PCP pathway are also involved in canonical WNT
signaling pathway such as FZD receptors and dishevelled (Krasnow and Adler, 1994;
Axelrod et al., 1998) but the downstream effects of PCP activation are independent of β-
catenin (Figure 1-2). In addition to FDZ receptors, PCP can be activated through Kny
(Caneparo et al., 2007), PTK7, RYK and ROR (reviewed by Cadigan and Nusse, 1997;
Logan and Nusse, 2004). Receptor activation by WNTs recruits dishevelled and thereby
43
activates small GTPases Rac and RhoA (reviewed by Veeman et al., 2003) which
culminate in cytoskeleton modifications and/or regulation of gene expression via the
MAP3K-JNK pathway (Miyagi et al., 2004).
Calcium signaling
The least characterized downstream signaling pathway involved in WNT are
those using Ca2+ as a second messenger (Figure 1-2) (review by Kühl et al., 2000).
Currently, evidence of increased intracellular Ca2+ release as a consequence of WNT
stimulation comes mainly from studies in developmental biology models, with few
studies in cultured human cells. Overexpression of WNT5A (Slusarski et al., 1997) and
WNT11 (Westfall et al., 2003) augment frequency of calcium fluxes in zebrafish
blastulae. Similarly, in Xenopus embryos, Wn5a and Wnt11 activate Ca2+/calmodulin-
dependent protein kinase II (CamKII ; Kühl et al., 2000) and protein kinase C (PKC;
Sheldahl et al., 1999). Furthermore, WNT11 acts via CamKII to regulate cell movement
in Xenopus embryos (Garriock and Krieg, 2007). Recombinant Wnt5a activates Ca2+
transients in mammary epithelial cells (Dejmek et al., 2006) and thyroid carcinoma cells
(Kremenevskaja et al., 2005). In addition, activation of FZD receptors in absence of
ectopic WNT ligands activate CamKII and PKC, as it has been observed for FZD2
(Slusarski et al., 1997), FZD3 (Sheldahl et al., 1999) FZD4 (Robitaille et al., 2002) and
FZD6 (Sheldahl et al., 1999).
WNT Signaling in Embryonic Stem Cells
Stem cells (SC) are undifferentiated cells that can give rise to one or more
differentiated cells of specific phenotypes. Cells of the early embryo are totipotent since
they can give rise to all cell lineages in the body. Once the blastocyst has formed, cells
of the ICM become pluripotent – while they can form all cell lineages in the fetus, the
44
ability to differentiate into placental tissue is lost. Stem cells in fetal and adult tissues
become more restricted. For example, spermatagonial stem cells can give rise to cells
of the spermatogenic lineage. One characteristic of stem cells is the capacity for self-
renewal so that cell division can be accompanied either by differentiation or
maintenance of the stem cell phenotype.
Embryonic stem cells are cells derived from the epiblast of blastocysts (ESC) and
those from epiblast of the post-implantation embryo (EpiSC) (reviewed by Nichols and
Smith, 2009). As compared to ESC, EpiSC are less pluripotent with the former being
referred to as in a naïve pluripotent state and the latter having a primed pluripotent
state. Alternatively ESC are referred to as pluripotent while EpiSC are multipotent. Cells
derived from ESC can form cells of each of the three primary germ layers (i.e.
endoderm, mesoderm, and ectoderm). Moreover, ESCs can be incorporated into the
fetus when they are introduced into preimplantation embryos to make blastocyst
chimeras. In contrast, EpiSCs cannot contribute to chimeras (reviewed by Rossant,
2008) nor can they give rise to ESCs. Both ESC and EpiSC express core pluripotency
transcription factors Pou5f1, Nanog and Sox2 but ESCs have an unique open genome
with minimal repression factors (Niwa, 2007) while EpiSCs have undergone some
epigenetic changes to support differentiation towards different cell types (Tesar et al.,
2007).
WNT signaling is often implicated in control of self-renewal and proliferation of
stem cells. In adult tissues, lineage tracing on the basis of WNT target genes revealed
the critical role of WNT signaling in SC located in a wide range of organs including
intestine, stomach, skin, hair follicle and mammary gland (reviewed by Clevers et al.,
45
2014). The role of WNT signaling in maintenance of pluripotency was initially proposed
based on observations that activation of WNT signaling through inhibition of GSK-3β
was sufficient to maintain pluripotency in mouse ESC (mESC) and human ESC (Sato et
al., 2004; Ogawa et al., 2006). In contrast, spontaneous expression of FGF4, and
consequent activation of the mitogen-activated protein kinase/extra cellular signal-
related kinase (MAPK/ERK) signaling pathway triggers differentiation of mESC in
culture (Kunath et al., 2007). Therefore, the standard procedure for mESC culture is the
2i strategy that consists of dual inhibition of GSK-3β and MAPK/ERK (reviewed by Ying
et al., 2008).
The gold standard method to assess pluripotency of ESC is by evaluation of their
ability to form teratomas after being injected in mice. Teratomas are benign tumors that,
because they derive from pluripotent ESC, are composed of differentiated tissues of
endo-, meso-, and ectodermal origin. A number of mutations in APC in mESC result in
activated β-catenin mediated WNT signaling pathway (Kielman et al., 2002). These
mutant mESC had less ability to differentiate than their wild type counterparts
supporting the notion that WNT/β-catenin signaling pathway maintains pluripotency.
Despite the evidence mentioned above, the role of WNT/β-catenin pathway on
maintenance of pluripotency in SC is controversial. One argument that call the role of
WNT/β-catenin in ESC into question is that neither WNT ligand nor receptors have been
shown to actively prevent differentiation (reviewed by Nusse et al., 2008). Another piece
of evidence is that GSK3 participates in multiple pathways unrelated to WNT by
phosphorylating a number of cellular substrates that can potentially control pluripotency
in a WNT independent manner (review by Sokol et al., 2011). Further, because the
46
cellular context determined by the array of receptors, co-receptors and WNT regulatory
molecules dictate the outcome of WNT signaling, it is likely that WNT signaling control
pluripotency in some, but not all, SC.
Nevertheless, the mechanism whereby WNT signaling maintains SC pluripotency
may involve inhibition of differentiation through suppression of genes required for
differentiation. One of the target genes of canonical WNT signaling, TCF3, regulates
gene expression of ESC transcription factors. Activation of WNT signaling results in the
release of TCF3-mediated repression, and consequent expression of the pluripotent
markers POU5F1, NANOG and SOX2 (Cole et al., 2008). Furthermore, autocrine and
paracrine actions of WNTs prevent the transition from ESC to EpiSC (Berge et al.,
2014).
WNT Signaling during Preimplantation Development
The preimplantation mouse embryo expresses WNT related genes throughout
the preimplantation development (Lloyd et al., 2003; Kemp et al., 2005). Among the
genes expressed are Wnt1, Wnt3a, Wnt4, Wnt6, Wnt7a, Wnt7b, Wnt9a, Wnt10b, Sfrp1,
Sfrp2, and Dkk1. The role of WNTs and WNT signaling in development of the
preimplantation mouse embryo, however, is not clear. Kemler et al. (2004) generated
mutant embryos whose β-catenin was resistant to ubiquitination and thus it accumulates
in large amounts in the cell. Blastocyst formation was not affected by stabilization of β-
catenin although gastrulation was defective. Results from that study indicate that
nuclear β-catenin is dispensable for preimplantation embryonic development. In another
study, Xie et al. (2008) evaluated the effect of blocking WNT/β-catenin by culturing
embryos in the presence of recombinant DKK1 (which blocks WNT-FZD-LRP
47
inteactions) from the 2-cell to the blastocyst stage. There was no effect on blastocyst
formation.
To further evaluate whether or not WNT signaling during preimplantation
reverberates later on development, 1-day pregnant mice were intravenously injected
with an adenovirus vector carrying Dkk1 cDNA, thereby creating conditional WNT
inactivation (Xie et al., 2008). Blastocysts collected from these mutant mice were
transferred to wild type mice at day 4, and the reciprocal transfer was also performed to
distinguish between blastocyst activation and uterine receptivity defects. Results
showed no differences in pregnancy when wild-type embryos were transfer to either
mutant or wild-type recipients. In contrast, implantation was impaired in the group of
embryos that underwent preimplantation development in a mother subjected to WNT
inactivation and that were subsequently transferred to wild type females. Evaluation of
these embryos developed in a mutant mother revealed that at the blastocyst stage
nuclear localization of β-catenin was blocked and the target gene of β-catenin, c-Myc,
was downregulated. In addition, wild type blastocysts, which successfully implanted
showed an overall downregulation of β-catenin independent signaling. In particular,
there was low expression of the small GTPase RhoA. The authors concluded that
activation of WNT/ β-catenin concomitant with inactivation of β-catenin independent
WNT signaling in TE cells is required for proper function of the TE during implantation.
More recently, the role of endogenous WNT signaling in embryonic and
extraembryonic requirement for WNT ligand secretion was studied (Biechele et al.,
2013). Two types of mutant mice were generated. One type consisted of Porcn allele
deletion in zygotes and extra embryonic tissue precluding endogenous WNT secretion,
48
i.e. loss of function for endogenous WNTs. The other mutant type expressed a stable
form of β-catenin, i.e. over activation of WNT/β-catenin signaling. Porcn mutants
successfully developed to the blastocyst stage and had normal cell numbers and cell
fate distribution compared with wild type embryos. Stabilization of β-catenin increased
the number of ICM cells, but blastocysts had all three cell types at the blastocyst stage.
Those authors concluded that Porcn-dependent WNT signaling is dispensable for
blastocyst formation and does not participate in cell fate allocation. In addition, the
observation that expression of WNT target genes was not affected in neither of these
two phenotypes led to the conclusion that WNT/ β-catenin is not fully functional during
preimplantation development.
In pig and human embryos WNT signaling during preimplantation development is
involved in TE differentiation, yet the mechanism underlying this function may differ
between species. Pig embryos cultured in the presence of DKK1 developed into
blastocysts with increased number of CDX2+ blastomeres and increased frequency of
hatching (Lim et al., 2013). In the same study, stabilization of β-catenin with a GSK3
inhibitor (LiCl) reduced CDX2 expression and number of CDX2+ blastomeres. For
human embryos, in contrast, stabilization of β-catenin, by either inhibition of its
degradation (GSK3 inhibitor I-azakenpaullone) or WNT3 stimulation, causes
upregulation of the TE marker EOMES, although there is no effect on other TE markers
(CDX2 and KRT18) or on the number of CDX2+ cells (Krivega et al., 2015). In addition,
loss of function of β-catenin caused by chemical induction of degradation using
Cardamonin caused downregulation of CDX2 expression and reduction in the number
of CDX2+ cells (Krivega et al., 2015). Neither gain nor loss of function of WNT signaling
49
during preimplantation development affected expression of pluripotency markers
(Krivega et al., 2015).
In bovine preimplantation embryos, results of the consequence of overactivation
of WNT signaling have been inconsistent. Use of the agonist 2-amino-4-(3,4-
methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) blocks
development of the embryo to the blastocyst stage in a concentration dependent
manner (Denicol et al., 2013). The endogenous inhibitor of WNT/ β-catenin signaling,
DKK1, negated the negative effect of AMBMP suggesting that the molecule acts
through canonical WNT signaling (Denicol et al., 2013). Similarly, inhibition of β-catenin
degradation using one GSK3 inhibitor (LiCl) inhibited development of embryos to the
blastocyst stage (Aparicio et al., 2000). In contrast, another GSK3 inhibitor
(CHIR99021) improved the ability of embryos to develop to the blastocyst stage
(Aparicio et al., 2000). The observed inconsistency in results probably reflects
experimental use of inhibitors or activators of canonical WNT signaling that could have
effects on multiple signaling pathways, as well as generate different outcomes
depending on the cellular features such as abundance of regulatory molecules and
receptors. WNT signaling may also be involved in cell lineage commitment in the bovine
embryo. Culture of bovine embryos in the presence of DKK1 increased the proportion of
CDX2+ (TE marker) and GATA6+ (hypoblast marker) cells without affecting blastocyst
development (Denicol et al., 2013, 2014). Furthermore, embryos exposed to DKK1
during the morula to blastocyst transition had better ability to establish pregnancy after
transfer to recipients than control embryos (Denicol et al., 2014).
50
There are some indications that canonical WNT signaling is aberrant in the
preimplantation embryo. In the study of mouse embryos by Kemler et al. (2004)
described above, nuclear localization of β-catenin was not observed at any
developmental stage in either wild type or mutant embryos. In the human, as well, there
was lack of nuclear localization of β-catenin in the embryo even after stabilization of β-
catenin with a mutation, and unchanged expression of a classical β-catenin target gene
(TCF1) (Krivega et al., 2015). In contrast, Xie et al. (2008), also working in the mouse,
found nuclear localization of β-catenin from the 1-cell to the blastocyst stage, however,
by the blastocyst stage, only TE cells displayed nuclear β-catenin.
Evidence for Association of Endometrial Expression of DKK1 and Fertility
Two independent studies indicate that endometrial expression of the WNT
inhibitor, DKK1, is reduced in sub-fertile bovine females. In one study, endometrial
expression of DKK1 at day 17 of the estrous cycle was lower for lactating versus non-
lactating cows (Cerri et al., 2012). Lactation is often considered as inducing subfertility
in dairy cows (reviewed by Sartori et al., 2010; Hansen, 2011). A second line of
evidence comes from a study where inherent fertility of heifers was determined on the
basis of outcomes of four consecutive artificial inseminations (Minten et al., 2013).
Pregnancy was terminated between inseminations in cases where females became
pregnant. Subsequently, heifers were categorized as high fertile (pregnant 4 times) sub-
fertile (pregnant 1-2 times), and infertile (no pregnancies). To investigate whether
embryos or uteri from these heifers explain the differences in their ability to become
pregnant subsequent studies were performed. No differences where observed in
quantity or quality of embryos, or pregnancy rates after follicular superestimulation of
the classified heifers and embryo transfer. In contrast, when these categorized heifers
51
were recipients of embryos, pregnancy rates were greatest for those in the high fertility
group. Subsequently, reproductive tracts from cows of the three categories were
obtained on day 14 of the estrous cycle to compare the transcriptome. Results show
that expression of DKK1 was highest in fertile heifers, intermediate in infertile heifers
and lowest in sub-fertile heifers.
Results of these studies implicate WNT signaling in general and DKK1 in
particular as a possible important determinant of fertility.
52
Table 1-1. Phenotypes generated by mutation of Wnts in mouse
Gene Phenotype Reference Wnt1 Deletion portion midbrain, cerebelum (McMahon and Bradley,
1990) Wnt2 Placental defects (Monkley et al., 1996) Wnt3 Early gastrulation
No formation of primitive streak (Barrow et al., 2003) (Liu et al., 1999)
Wnt3a Underdevelopment of hippocampus Defect in vertebral patterning
(Lee et al., 2000) (Ikeya and Takada, 2001)
Wnt4 Absence of kidneys Failure in mullerian duct formation
(Stark et al., 1994) (Vainio et al., 1999)
Wnt5a Delayed osteoblast differentiation Impaired lung morphogenesis
(Yang, 2003) (Li et al., 2002)
Wnt5b Delayed osteoblast differentiation (Yang, 2003) Wnt6 Defective decidualization (Wang et al., 2013) Wnt7a malformed female reproductive tracts
Lack of mullerian regression in male (Miller and Sassoon, 1998) (Parr and McMahon, 1998)
Wnt7b Chorio-allantoic fusion defects (Parr et al., 2001) Wnt8a viable (Vendrell et al., 2013) Wnt8b viable * Wnt9a Skeletal abnormalities (Später et al., 2006) Wnt9b Defective urogenital development (Carroll et al., 2005) Ant10b Defective myocyte differentiation (Vertino et al., 2005) Wnt11 Defective ureters – kidney hypoplasia (Majumdar et al., 2003) Wnt 16 viable *
*reviewed by van Amerongen and Berns (2006)
53
Figure 1-1. Position-dependent Hippo signaling in preimplantation embryos. Hippo
signaling is activated in inner apolar cells, and is turned off in outer polar cells. The result is inhibition of Yap phosphorylation when signaling is off, so that YAP can interact with Tead4 and activate
54
Figure 1-2. Downstream cascades of WNT signaling pathway. WNT/β-catenin inactive:
in absence of WNT ligand , cytosolic β-catenin is targeted by a destruction complex formed by AXIN, GSK3 and APC for proteosomal degradation. Note
that WNT/-catenin can also be inactivated by DKK1 interacting with LRP5/6 and kremen1 co-receptors to impair WNT-FZD-LRP complex formation. WNT/β-catenin active: As a result of WNT-FZD-LRP binding, the β-catenin destruction complex is disassembled and β-catenin accumulates and becomes translocated to the nucleus where it regulates transcription by interacting with LEF and TCF transcription factors. WNT/PCP signaling is activated by diverse ligand-receptor interactions that recruit dishevelled (Dsh) and activates small GTPases RAC1 and RHOA. Outcomes of this signaling include cytoskeleton changes and regulation of transcription. WNT/calcium signaling can be activated by WNT-FZD interaction as well as FZD activation. It results in intracellular release of calcium via inositol 3 phosphate, calmodulin and protein kinase C. Abreviations:IP3: Inositol 3 phosphate – DAG: Diacylglycerol - CamKII: calmodulin-dependent protein kinase II – PKC: protein kinase C – KNY: knypek - ROR2: receptor tyrosine kinase like orphan receptor 2 - DSH: - JNK: c-Jun N-terminal kinase– LEF: lymphoid enhancer binding factor 1 – TCF: transcription factor 7 – GSK3: glycogen synthase kinase 3 - LRP5/6: low density lipoprotein receptor-related protein 5/6 - APC: adenomatosis polyposis coli - DKK1: dickkopf-related protein 1 CK1:casein kinase 1 -
55
Objectives of the Present Investigations
The overall objective of the research presented in this dissertation is to
understand the role of WNT signaling during preimplantation development of the bovine
embryo and the maternal contribution to the regulation of this signaling pathway. In
cattle, exposure of embryos to the endogenous regulator of WNT signaling, DKK1,
induces cell lineage commitment and confer embryos with better competence to
establish and maintain pregnancy after transfer to recipient animals (Denicol et al.,
2014). In contrast, overactivation of WNT signaling sometimes inhibited development
(Denicol et al., 2013, Aparicio et al., 2010). Such results suggest overactivation of WNT
signaling may be detrimental to embryonic development in the cow. Further evidence
for this idea comes from the findings that fertility in cattle is related to endometrial
expression of DKK1, which encodes for a secreted inhibitor of canonical WNT signaling
(Cerri et al., 2012; Minten et al., 2013).
Four series of experiments were designed to understand the role and regulation
of WNT signaling during preimplantation development and consequences of that
signaling for development through the prenatal period. In Chapter 2, WNT signaling was
characterized during preimplantation development. It was hypothesized that 1)
canonical WNT signaling (i.e. WNT signaling mediated by nuclear localization of β-
catenin) is attenuated in the preimplantation embryo and 2) WNT can activate other
signaling pathways in the embryo. Actions of embryo-derived and exogenous WNTs on
the preimplantation embryo were evaluated in Chapter 3. The role of embryo-derived
WNTs was determined by evaluating consequences of inhibition of WNT secretion by
addition of a PORCN inhibitor (necessary for WNT secretion) (Takada et al., 2006) as
well as the WNT inhibitor DKK1. Actions of exogenous WNT (such as might be secreted
56
by the endometrium) were determined by testing effects of AMBMP and WNT7A, on
embryonic development.
Recently, it was shown that one embryokine, CSF2, can affect embryonic
development from day 5-7 of development in a way that alters characteristics of the
resultant calf (Kannampuzha-Francis et al., 2015). In particular, calves born following
embryo transfer of an embryo treated with CSF2 grew faster after three mo of age than
calves born following transfer of a control embryo. In Chapter 4, it was tested whether
actions of DKK1 during the transition from morula to the blastocyst stage would alter
phenotype after birth.
Because results of earlier chapters suggested a role for endometrial-derived
WNT in embryonic development, an experiment described in Chapter 5 was conducted
to survey oviductal and endometrial expression of genes for a number of WNT and
other growth factors during the first 7 days after ovulation.
57
CHAPTER 2 WNT REGULATION OF EMBRYONIC DEVELOPMENT LIKELY INVOLVES
PATHWAYS INDEPENDENT OF NUCLEAR β-CATENIN
Introduction
WNT signaling is a complex signaling system regulated by 19 WNT ligands that
interact with a variety of receptors including FZD, ROR, PTK7 and RYK (Cadigan and
Nusse, 1997; Logan & Nusse, 2004). Among the downstream signaling cascades are
the canonical pathway involving binding of WNT to FZD and recruitment of the co-
receptor LRP5 or LRP6 (MacDonald et al. 2009), the planar cell polarity pathway
(Veeman et al., 2003; Seifert and Mlodzik, 2007), and calcium signaling pathway (Kühl
et al. 2000; Kohn and Moon, 2005). The specific phenotype induced by WNT signaling
depends upon the ligands and receptors in play, as well as other specific characteristics
of signaling molecules in the cell; thus, a specific WNT-receptor interaction can invoke
different outcomes in different cell types (Amerongen et al. 2008).
Canonical WNT signaling is the most well described pathway for WNT signaling
and is crucial for a number of developmental processes through regulation of cell
proliferation (Logan and Nusse, 2004), maintenance of pluripotency (Sato et al. 2004;
Sokol, 2011), differentiation (Liu et al. 2014) and migration (Morosan-Puopolo et al.
2014). The central effector of this signaling pathway is β-catenin, a protein that not only
modulates gene expression upon translocation to the nucleus after WNT activation, but
also serves as a constitutive protein for adherens junctions involved in cell-cell adhesion
(Fleming et al. 2001). Nuclear accumulation of β-catenin is triggered by inhibition of its
degradation by the proteasome induced by a complex consisting of CKI, GSK3 and
APC. Once in the nucleus, β-catenin displaces GROUCHO to interact with the
transcription factors LEF1 and TCF7 to regulate transcription of genes involved in cell
58
proliferation and pluripotency such as CCNDBP1 (Tetsu and McCormick, 1999) and
MYC (He et al. 1998), respectively.
WNTs are important regulators of mammalian development but their role during
the preimplantation period has not been resolved. In the mouse, inhibition of canonical
WNT signaling does not impair blastocyst development (Huelsken et al., 2000; Kemler
et al., 2004; Xie et al., 2008; Lyashenko et al., 2011) or affect identity, expansion, or
self-renewal of embryonic stem cells (ESC) (Lyashenko et al., 2011; Wray et al.,
2011b). In other species, the role of canonical WNT signaling in the preimplantation
embryo is less clear because of the experimental use of inhibitors or activators of
canonical WNT signaling that could have effects on multiple signaling pathways. In the
cow, for example, one inhibitor of GSK3B (which causes activation of the canonical
pathway) increased competence of embryos to develop to the blastocyst stage whereas
another inhibitor reduced development (Aparicio et al. 2000). A physiological antagonist
of canonical WNT signaling, DKK1, enhanced the ability of porcine embryos to undergo
hatching (Lim et al. 2013), increased trophectoderm (TE) differentiation in pig and cattle
embryos (Lim et al. 2013; Denicol et al. 2014), and increased competence of bovine
embryos to establish pregnancy after transfer to recipient females (Denicol et al. 2014).
While such results suggest that activation of canonical WNT signaling may inhibit TE
differentiation, DKK1 can also regulate other signaling pathways independent of
canonical WNT signaling (Caneparo et al. 2007; Tahinci et al. 2007).
In the human embryo, accumulation of β-catenin in the nucleus in response to
inhibition of GSK3B depends upon stage of development, with accumulation being
attenuated after day 3 of development and absent in blastocysts (Krivega et al. 2015).
59
Such a result is consistent with findings in the mouse that canonical WNT signaling is
not required for development, at least after day 3, and that developmental changes in
the embryo cause a dampening of canonical WNT signaling.
For the current study, the bovine embryo was used as a model to test the overall
hypotheses that 1) canonical WNT signaling (i.e. WNT signaling mediated by nuclear
localization of β-catenin) is attenuated in the preimplantation embryo and 2) WNT can
activate other signaling pathways in the embryo, as evaluated for activation of JNK.
These hypotheses were evaluated in several experiments to characterize
developmental changes in expression of genes involved in WNT signaling, localization
of β-catenin in blastomere nuclei, and accumulation of phospho-JNK in the nucleus after
WNT activation.
Materials and Methods
Embryo Production
Bovine embryos were produced in vitro from oocytes obtained from Bos
(admixture of B. taurus and B. indicus) ovaries collected at a local abattoir. Procedures
for oocyte recovery and maturation were as described elsewhere (Dobbs et al. 2013).
Following oocyte maturation, oocytes were fertilized for 8-10 h in groups of up to 300
oocytes with sperm pooled from three randomly selected B. taurus and B.indicus bulls
using procedures described elsewhere (Denicol et al. 2014). Groups of 25-30
presumptive zygotes were placed in 50 µL microdrops of SOF-BE2 (Kannampuzha-
Francis et al. 2017) covered with mineral oil (Sigma-Aldrich, St. Louis, MO, USA) and
cultured at 38.5oC in a humidified atmosphere of 5% O2 and 5% CO2 with the balance
N2. Treatments were applied to cultured embryos by removing 5 µL of culture medium
and adding the treatment in a volume of 5 µL.
60
For immunofluorescence experiments, embryos were produced in vitro following
procedures described above with a few modifications. Oocytes were harvested using
BoviPROTM oocyte washing medium (MOFA Global, Verona, WI, USA) and fertilization
of matured oocytes was performed using IVF-TL (Parrish et al. 1986) (Caisson
Laboratories, Logan, UT, USA) containing PHE [80 µL of 0.5 mM penicillamine, 0.25
mM hypotaurine, and 25 µM epinephrine in 0.9% (w/v) NaCl as described by (Ortega et
al. 2016).
Developmental Changes in Expression of Selected Genes Involved in WNT Signaling for Embryos Produced in Vitro (Experiment 1)
To prepare pools of matured oocytes, cumulus oocyte complexes (COCs) were
harvested at the end of oocyte maturation (20-22 h). Cumulus cells were removed by
vortexing for 5 min in HEPES-SOF medium (Denicol et al. 2014) containing 1,000 U/ ml
of hyaluronidase. Denuded oocytes were washed three times in Dulbeccos’s
phosphate-buffered saline (DPBS) containing 1% (w/v) polyvinylpyrrolidone (DPBS-
PVP), incubated in 0.1% (w/v) proteinase solution (protease from Streptomyces griseus;
Sigma-Aldrich, St Louis, MO, USA) in DPBS to remove the zona pellucida, washed
three times in DPBS-PVP, and suspended in groups of 30 in 100 µL extraction buffer
from the PicoPure RNA isolation kit (Applied Biosystems, Foster City, CA, USA).
Samples were stored at -80oC.
Embryos were produced by in vitro fertilization in 19 replicates. Embryos were
harvested from culture drops at the following stages: 2-cell [28 – 32 h post insemination
(hpi)]; 3-4 cell (44 – 48 hpi); 5-8 cell (50-54 hpi); 9-16 cell (72 hpi); morula (120 hpi); and
blastocyst (168 hpi). Embryos were collected, processed as for denuded oocytes to
remove the zona pellucida, suspended in groups of 30 in 100 µL extraction buffer from
61
the PicoPure RNA isolation kit (Applied Biosystems), and stored at -80°C. A separate
pool of bulls was used for each replicate, resulting in a total of 19 different bulls.
Transcript abundance was examined for seven genes related to WNT signaling
by quantitative real time PCR (qPCR). Genes included two transcription factors (LEF1
and TCF7), two transcription factor inhibitors [AES and LOC505120 (GROUCHO-like)],
two canonical WNT co-receptors (LRP5 and LRP6), and a soluble inhibitor of canonical
WNT signaling (DKK1) as well as three reference genes (GAPDH, SDHA and YWHAZ).
The reference genes were chosen because expression is stable over preimplantation
development (Goossens et al. 2005), and it was verified that developmental stage did
not affect expression of any of these three genes. Primers are listed in Table 2-1.
Primers for DKK1, GAPDH, LRP6, SDHA, and YWHAZ were previously published
(Goossens et al. 2005; Denicol et al. 2013) while those for AES, LEF1, LOC505120,
LRP5, and TCF7 were designed using software from Integrated DNA Technologies
(Coralville, Iowa, USA). Primers were synthesized by Integrated DNA Technologies. All
newly-designed primer pairs were validated using cDNA from pools of day 7 bovine
blastocysts by generation of a standard curve (efficiency varied from 92.4 - 108.5%),
evaluation of melt curves and sequencing of PCR amplicons. Sequences were mapped
to the B. taurus genome using the Basic Local Alignment Search Tool of the National
Center for Biotechnology Information. All sequences aligned to the corresponding gene.
RNA of pools of oocytes and embryos was extracted using the PicoPure RNA Isolation
kit (Applied Biosystems) following the manufacturer’s protocol. DNase treatment was
performed using the QIAGEN DNase kit (Valencia, CA, USA) and mRNA was reverse
transcribed using the High Capacity cDNA Reverse Transcription Kit of Applied
62
Biosystems. The qPCR utilized SsoFast EvaGreen Supermix reagent (Bio-Rad,
Hercules, CA, USA) and was performed with a Bio-Rad CFX96-Real-Time system using
conditions described previously (Dobbs et al. 2013). Two technical replicates were
performed for each sample and the mean cycle threshold (CT) calculated. Mean CT
values greater than 35 were considered non-detectable and assigned a value of 35 for
statistical analysis.
A total of five biological replicates containing 30 oocytes or embryos each were
subjected to qPCR. Data analyzed were ΔCT values, which were calculated by
subtracting the geometric mean of the three reference genes from the mean CT value of
the sample. For graphical purposes, the relative transcript abundance was calculated as
the 2 ΔCT. Therefore, abundance of each mRNA type is expressed relative to expression
of reference genes.
Data were analyzed by least-squares analysis of variance using the GLM
procedure of SAS for Windows, version 9.4 (SAS Institute Inc., Cary, NC, USA).
Assumptions of analysis of variance were tested using the Univariate procedure of SAS.
Results are reported as least-squares means ± standard error of the mean. The level of
significance was P < 0.05.
Characteristics of the WNT Signaling System in the Morula and ICM and TE of in-Vitro Produced Embryos as Revealed by RNA-Seq (Experiment 2)
Data sets of the transcriptome of three pools of in-vitro produced morulae
collected at day 6 after insemination, and three pools of ICM and TE purified from in-
vitro produced blastocysts at day 8 after insemination, were examined for stage and cell
type effects on expression of 80 genes involved in WNT signaling. Reads were mapped
to Btau_4 (http://genome.ucsc.edu/). Procedures and data for ICM and TE have been
63
published previously (Ozawa et al. 2012) and raw data was deposited in the DDBJ
Sequence Read Archive at http://www.ddbj.nig.ac.jp/index-e.html (Submission
DRA000504). The samples of morulae (100 per pool) were produced using the same
procedures and in the same replicates of in-vitro fertilization as for ICM and TE. Data
were processed following the same bioinformatic methods as reported earlier (Ozawa et
al. 2012). Details of the magnetic activated cell sorting procedure used to separate ICM
from TE were previously published (Ozawa & Hansen, 2011).
RNA-seq data was obtained using a SOLiDTM v4 sequencer (Applied
Biosystems). Data on a subset of genes involved in WNT signaling were evaluated for
treatment effects by least-squares analysis of variance using the GLM procedure of
SAS for Windows, version 9.4. The dependent variable was number of reads of the
transcript and the independent effect was cell type (morula, ICM and TE). The total
transcript reads per sample was used as a covariate. Orthogonal contrasts were used to
determine whether transcript abundance differed between morulae and blastocysts
(morula vs. ICM+TE) or between ICM and TE. Results are reported as least-squares
means ± standard error of the mean. The level of significance was P < 0.05.
Localization of Total and Active β-catenin in Bovine Preimplantation Embryos as Determined by Immunofluorescence (Experiments 3 and 4)
Embryos produced in vitro were harvested at different stages of development
using the same schedule as described earlier. Embryos were washed three times in
cold DPBS- PVP, fixed in 4% (v/v) paraformaldehyde in DBPS/PVP for 15 min, and
washed in DPBS/PVP three times. Immediately thereafter, embryos were incubated in
permeabilization solution [DPBS containing 0.5% (v/v) Triton X-100] for 30 min at room
temperature, followed by incubation for 1 h in blocking buffer [DPBS containing 5%
64
(w/v) bovine serum albumin (BSA)]. Embryos were then incubated overnight at 4°C with
1 µg/ ml of primary antibody in antibody buffer [DPBS containing 0.1% (v/v) Tween 20
and 1% BSA (w/v)]. For detection of total β-catenin (Experiment 3), rabbit polyclonal
anti-human β-catenin antibody (Abcam, Cambridge, MA, USA) was used. For detection
of active β-catenin (Experiment 4), rabbit polyclonal anti-human non-phospho (active) β-
catenin antibody (Ser33/37/Thr41; Cell Signaling Technology, Beverly, MA, USA) was
used, and for negative control, primary antibody was replaced with the same
concentration of rabbit IgG. After three washes in washing buffer [DPBS containing
0.1% (v/v) Tween 20 and 0.1% BSA (w/v], oocytes and embryos were incubated with 1
µg/ ml goat anti-rabbit IgG conjugated with Alexa Fluor 555 (Life Technologies,
Carlsbad, CA, USA) for 1 h at room temperature in the dark. Primary and secondary
antibodies were diluted in antibody buffer. Nuclear labeling was achieved by incubation
with 1 μg/ ml Hoechst 33342 (Sigma-Aldrich) for 15 min at room temperature. Embryos
were finally rinsed in DPBS/PVP and placed on a slide containing SlowFade Gold
antifade reagent (Life Technologies), covered with a coverslip, and observed with a 40x
objective using a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Göttingen,
Germany) and Zeiss filter sets 02 [4=,6=-diamidino-2-phenylindole (DAPI)], and 04
(rhodamine).
Digital images were acquired using AxioVision software (Zeiss) and a high-
resolution black and white Zeiss AxioCam MRm digital camera. For Experiment 3,
evaluation of total β-catenin was replicated 7 times with a total of 450 embryos. For
Experiment 4, evaluation of active β-catenin was replicated 5 times with a total of 417
embryos.
65
Changes in Immunoreactive Active β-catenin in Embryos Following Activation of Canonical WNT Signaling (Experiments 5-8)
For Experiment 5, in-vitro produced embryos were cultured in drops as described
above. Culture drops were randomly assigned to stage and treatments. Developmental
stages included 3-4 cell (44-48 hpi), 5-8 cell (50-54 hpi), 9-16 cell (72 hpi), and compact
morula (120 hpi). At the corresponding time for each developmental stage, 5 µL of SOF-
BE2 in the culture drop were replaced by either 5 µL GSK3 inhibitor [CHIR-99021 HCL;
Tocris Bioscience, Bristol, UK], 5 µL of the nuclear export inhibitor leptomycin (Sigma),
5 µL leptomycin + GSK3 inhibitor, or 5 µL vehicle (SOF-BE2 containing 0.04 % (v/v)
ethanol). Final concentrations were 10 µM for the GSK3 inhibitor and 22 ng/ ml for
leptomycin. After 30 min of treatment while embryos were maintained in culture
conditions (30 min was chosen because leptomycin B increases nuclear NFkB p65 in
HeLa cells at this time; (Wolff et al. 1997), embryos were fixed and labeling for
immunofluorescence was carried out as described earlier using antibody against non-
phospho (active) β-catenin or the corresponding rabbit IgG. A total of 191 embryos were
evaluated.
For Experiment 6, canonical WNT signaling in 5-8 cell embryos and morulae was
stimulated by addition of final concentrations of either 100 ng/ ml human recombinant
WNT1 (Sigma-Aldrich; 99% identical amino acid sequence to bovine WNT1), 0.7 µM of
the WNT agonist 2-amino-4-(3,4-methylenedioxy)benzylamino)-6-(3-
methoxyphenyl)pyrimidine (AMBMP, Sigma-Aldrich) or SOF-BE2 containing 0.1% (v/v)
DMSO (vehicle). Embryos were harvested 1, 6 and 24 h after treatment and analyzed
by immunofluorescence for nuclear β-catenin (n=36 embryos). For Experiment 7,
compact morulae were treated with 0.7 µM AMBMP or vehicle at day 5 after
66
insemination. Blastocysts were harvested 48 h later for assessment of nuclear active β-
catenin (n=78 embryos). For experiment 8, embryos were treated with 10 µM GSK3
inhibitor (CHIR-99021) on day 6 after insemination or were used as a control group that
received an equivalent amount of vehicle (SOF-BE2 containing 0.04 % (v/v) ethanol).
Blastocysts were harvested 24 h after treatment for assessment of nuclear active β-
catenin (n=15 embryos).
Localization of Active β-catenin in Mouse and Bovine Embryos as Evaluated by Confocal Microscopy (Experiment 9 and 10)
For Experiment 9, cryopreserved 5-8-cell mouse embryos (B6C3F1 x B6D2F1)
were obtained from Embryotech Laboratories (Haverhill, MA, USA) and thawed
following the supplier’s instructions. Embryos were washed with HEPES-TALP and
incubated in 50 µL oil-covered microdrops of EmbryoMax KSOM medium with amino
acids (EMD Millipore, Billerica, MA, USA) for 3 h at 38.5°C in a humidified atmosphere
of 5% CO2 and 5% O2. The 5-8 cell embryos were fixed in 10% (v/v) formalin for 30 min,
permeabilized in 2.5% (v/v) Tween 20 in DPBS for 20 min, blocked with DPBS
containing 5% (w/v) BSA for 1 h, and incubated overnight with 1 µg/ ml purified mouse
monoclonal IgG against active β-catenin [anti-active-β-catenin (anti ABC) clone 8E7;
Millipore] at 4°C. After sequential washes with DPBS containing 0.1% (w/v) BSA and
0.1 % (v/v) Tween 20, embryos were incubated in affinity-purified goat anti-mouse IgG
coupled to fluorescein isothiocyanate (FITC; Abcam) for 1 h at room temperature,
followed by 5 min incubation in 1 µg/ ml Hoechst 33342. Embryos were suspended in
10 µL drops of ProLong® Gold anti-fade mounting medium (ThermoFisher Scientific) in
chamber slides. Embryos (n=5) were observed using a spinning disk confocal scanner
mounted to an Olympus DSU-IX81 inverted fluorescent microscope. Digital images
67
were captured with a 60X objective and DAPI and FITC filter sets, using an attached
Hamamatsu C4742-12AG monochrome CCD camera.
For Experiment 10, bovine embryos were produced in vitro and harvested at the
morula stage on day 5 and blastocyst stage on day 7 after insemination. Labeling of
active β-catenin was performed following same procedure and antibody as for
Experiment 4 except that embryos were suspended in 10 µL drops of ProLong® Gold
anti-fade mounting medium (ThermoFisher Scientific) in chamber slides. Embryos (n=4)
were observed and images captured using the spinning disk confocal scanner and
camera mentioned above, with 40 or 60X objectives and DAPI and Texas Red filter
sets.
Nuclear Localization of β-catenin in Bovine Embryonic Fibroblast Cells Following Activation of Canonical WNT Signaling (Experiment 11)
Cells of the bovine embryonic fibroblast (BEF) cell line (Ozawa et. al 2012) were
studied to verify nuclear labeling of non-phospho (active) β-catenin in non-embryonic
cells. Cells were cultured with Dulbeccco’s modified Eagle’s Medium (Gibco,
ThermoFisher Scientific) containing 10% (v/v) fetal bovine serum and 1% (v/v)
antibiotic-antimycotic (10,000 units/ ml penicillin, 10 mg/ ml streptomycin and 25 µg/ ml
amphotericin B; Sigma-Aldrich). Cells were initially cultured in cell culture flasks
(Corning, Corning, NY, USA) at 38.5°C in a humidified atmosphere of 5% CO2. At 90%
confluency, cells were trypsinized [0.25% (w/v) trypsin, Life Technologies], mixed with
an equal volume of culture medium, centrifuged for 10 min at 1750 x g, resuspended in
culture medium, and counted (Automated Cell Counter, Bio-Rad, Richmond, CA, USA).
Cells were seeded in 8-chamber slides (Sigma-Aldrich) at a density of 10,000 cells/well
and allowed to adhere overnight in culture medium. Culture medium containing
68
treatments were used to replace medium. Treatments included 10 µM GSK3 inhibitor
(CHIR-99021), 0.7 µM AMBMP or vehicle [0.5% (v/v) DMSO]. Concentrations were
chosen based on effect of these molecules in other studies (Denicol et al. 2013;
Lappas, 2014). Fixation and immunolabeling of cells was performed 24 h after treatment
using 4% paraformaldehyde for 20 min, followed by permeabilization with DPBS
containing 0.5% (v/v) Triton X-100 during 30 min, and blocking for 1 h with DPBS
containing 5% (w/v) BSA. Incubation with primary antibody [rabbit anti-human polyclonal
non-phospho (active) β-catenin (Ser33/37/Thr41); Cell Signaling Technology] or rabbit
IgG proceeded overnight at 4°C. Incubation with goat anti-rabbit IgG conjugated with
Alexa Fluor 555 proceeded for 1 h, followed by DNA labeling using Hoechst 33342.
Cells were observed and images captured following methods described for mouse
embryos; DAPI and Texas red fluorescence filters were used. Image analyses consisted
of quantification of proportion of cells depicting nuclear β-catenin.
A total of 41 images were analyzed. The proportion of cells showing nuclear
localization of active β-catenin was analyzed by logistic regression using the LOGISTIC
procedure of SAS for Windows, version 9.4 (SAS Institute Inc.) including treatment as a
fixed effect. The PDIFF means separation test was used to determine which treatments
differed from control cells. The level of significance was P<0.05.
Non-Canonical WNT Signaling Mediated by Phosphorylation of JNK (i.e., MAPK8) by WNT11 in Bovine Blastocysts (Experiment 12- 14)
Experiment 12 was performed to determine whether WNTs could activate one
component of the planar cell polarity pathway in bovine embryos. Embryos were
produced in vitro as described. At day 7 after insemination, culture drops were randomly
assigned to treatments. In each culture drop, 5 µL of SOF-BE2 were replaced by either
69
5 µL human recombinant WNT11 (R&D systems, Minneapolis, MN, USA; 98% amino
acid sequence identity with bovine WNT11) or 5 µL vehicle [SOF-BE2 containing 0.01
% (w/v) BSA (in addition to BSA included in SOF-BE2 formulation)]. Final
concentrations of WNT11 were 0.5, 1 and 2.5 µg/ml. Blastocysts were harvested 6 h
after treatment and fixed in 4% (v/v) paraformaldehyde. Immunolabeling was performed
as described for β-catenin except that the primary antibody was 1 µg/ ml anti-phospho-
JNK [(Thr183/Tyr185,Thr221/Tyr223), Millipore] and 1 µg/ ml rabbit IgG served as a
negative control. Note that the phosphorylation site is conserved among human, mouse,
and bovine and that the amino acid sequence identity between these species is 97-99
%. Embryos were then incubated with 1 µg/ ml goat anti-rabbit IgG conjugated with
Alexa Fluor 555 (Life Technologies), and nuclear labeling was performed using Hoechst
33342. Embryos were evaluated using a Zeiss Axioplan 2 epifluorescence microscope
as described earlier. Quantification of intensity of labeling with antibody was performed
using ImageJ software (U.S. National Institutes of Health, Berthesda, MD, USA -
version 1.70_02). Total immunoreactive phospho-JNK was measured as follows: after
selection of the area encompassing the entire embryo, the mean intensity was obtained
using the Measure Analysis feature and background intensity was obtained from the
area surrounding the embryo. The latter was subtracted from the embryo intensity
before statistical analysis. A total of 49 embryos were analyzed. Data were analyzed by
least-squares analysis of variance using the PROC GLM procedure of SAS for
Windows, version 9.4 (SAS Institute Inc.) including WNT11 concentration as a fixed
effect. Differences between individual concentrations of WNT11 and control embryos
were assessed using the PDIFF mean separation test of SAS.
70
Experiment 13 was performed to test whether WNT11 affected competence of
embryos to develop to the blastocyst stage and allocation of blastocyst blastomeres to
TE and ICM. Treatments (vehicle and 2.5 µg/ ml WNT11) were applied at day 5 after
fertilization as described above. Blastocyst development was assessed on day 7 after
fertilization, and blastocysts with clearly-delineated blastocoels were harvested, fixed,
permeabilized, and blocked as described above. Trophectoderm cells were identified by
localization of a transcription factor crucial for differentiation of TE [caudal type
homeobox 2 (CDX2); (Berg et al, 2011)]. Labeling was achieved by sequential
incubation with mouse anti-human polyclonal CDX2 antibody ready to use (Biogenex,
Fremont, CA, USA) and 1µg/ ml goat anti-mouse IgG conjugated with FITC
(Abcam).Total number of cells was determined by counting DNA-labeled nuclei by
labeling with 1µg/ml Hoechst 33342 after immunolabeling for CDX2. Number of ICM
was calculated as the difference between total cells and TE. The experiment was
performed in 5 replicates with a total of 484 COCs and semen from 13 different bulls.
Blastocyst cell number was evaluated for 74 embryos Data were analyzed for effect of
treatment using Proc Glimmix of SAS for Windows, version 9.4 (SAS Institute Inc.).
Each embryo was considered an observation and development (0=not developed to
blastocyst; 1=developed to blastocyst) was considered a binary variable. Results are
presented as least-squares means ± standard error of the mean.
Results
Developmental Changes in Expression of Selected Genes Related to WNT Signaling for Embryos Produced In Vitro (Experiment 1)
Results on expression of seven genes related to WNT signaling in embryos
produced in vitro are shown in Figure 2-1. Expression of each gene was affected by
71
stage of development (P= 0.004 for LRP5 and P<0.0001 for the other genes). There
were several distinct developmental patterns in gene expression. The first was a
continuous decline in transcript abundance from the oocyte to the morula stage. Only
AES showed this pattern and, for this gene, there was a slight increase in transcript
abundance by the blastocyst stage. A second pattern, shown for DKK1, was a
continuous increase from the oocyte stage to the 9-16 cell stage followed by a decline in
transcript abundance. For the other five genes, there was a slight increase in transcript
abundance from the oocyte to the 2-cell or 4-cell stage followed by a decline in
transcript abundance after the 2-cell, 4-cell or 5-8 cell stage. Transcripts for two genes,
DKK1 and LEF1, were not detectable at the blastocyst stage.
Characteristics of the WNT Signaling System in the Morula and ICM and TE of in Vitro Produced Embryos as Revealed by RNA-Seq (Experiment 2)
A RNA-Seq database of the transcriptomes of in vitro produced day 6 morulae
and isolated TE and ICM of day 8 blastocysts (Ozawa et al. 2012) was assessed for
expression of 80 genes associated with WNT signaling. Genes were considered
expressed if the average number of reads was > 5. Results are summarized in Table 2-
2.
Only 7 of 19 WNT genes were expressed with the remaining 12 WNT genes
having < 5 reads or being not detected. Of the 7 WNT that were expressed, only one,
WNT6, varied in expression between groups. Expression was higher for TE than ICM or
morula. A key gene involved in post-translational modification of WNTs, PORCN, was
highly expressed and transcript abundance was higher for TE than morula or ICM.
A total of 6 of 10 FZD receptor genes were expressed in the morula or
blastocyst. Expression of FZD1 and FZD7 was lower for ICM than for TE. In contrast,
72
expression of FZD6 was higher for both ICM and TE of the blastocyst than for the
morula. The FZD co-receptor gene LRP6, was abundantly expressed but number of
reads for LRP5 was < 5.
Among the genes involved in canonical WNT signaling that were expressed were
β-catenin and genes involved in the β-catenin destruction complex (APC, AXIN1, and
GSK3B). DVL1, which acts to bind FZD proteins and transmit information about WNT
binding, was not considered expressed (< 5 reads). Two other disheveled genes (DVL2
and DVL3) were expressed, however. The only gene involved in β-catenin metabolism
that varied with stage of development was APC, which was expressed more in TE than
ICM.
Expression of the two WNT regulated transcription factor genes, TCF7 and LEF1
was low In addition, expression of TCF7L1 was relatively low in both morula and
blastocysts. In contrast, TCF7L2 was highly expressed although expression declined
from the morula to the blastocyst stage for both ICM and TE. In contrast, the
transcription factor inhibitor, AES, increased in transcript abundance from the morula to
blastocyst stage for both ICM and TE.
Expression was also examined for several genes that can promote or antagonize
canonical WNT signaling. Among such genes that were affected by developmental
stage, were the WNT stimulatory molecule, RSPO3 and one of its receptors, LGR4.
Expression of both genes was significantly lower for ICM and TE of the blastocyst than
for morula. Two antagonists of canonical WNT signaling also declined from the morula
to blastocyst stage, DKK1 and WIF1. KREMEN1, which can function as a DKK1
73
receptor, was also reduced in expression for ICM and TE of the blastocyst as compared
to the morula.
Note that the pattern of gene expression was generally consistent with the earlier
experiment (Figure 2-1). In the first experiment, expression of six genes declined from
the day 5 morula to the day 7 blastocyst. A similar decline occurred from the day 6
morula to day 8 blastocyst for DKK1 and LEF1 . LRP6 did not change in expression,
LOC505120 and TCF7 were not detected by RNA-seq and LRP5 was barely detectable.
There was one gene whose expression increased very slightly from the morula to
blastocyst stage in the first experiment, AES, and a larger increase was observed when
comparing the day 6 morula to day 8 blastocyst.
Localization of Total and Active β-catenin in Bovine Preimplantation Embryos as Determined by Immunofluorescence (Experiments 3 and 4)
Expression of genes coding for molecules involved in WNT signaling was
indicative that canonical WNT signaling is partially silenced at the morula and blastocyst
stages. To further explore this idea, two experiments were conducted to evaluate
whether a key feature of canonical WNT signaling, nuclear localization of β-catenin,
occurs during preimplantation embryonic development.
For Experiment 3, an antibody recognizing both active (non-phosphorylated) and
inactive (phosphorylated) β-catenin was used to determine localization of total β-
catenin. Representative images are shown in Figure 2-2. Regardless of stage of
development, most immunoreactive β-catenin was localized to cell membranes;
immunoreactive protein in the nucleus was absent in all but the occasional cell.
Since nuclear β-catenin was not observed at any developmental stage,
Experiment 4 was performed using an antibody specific for active β-catenin (i.e., non-
74
phosphorylated β-catenin) (Figure 2-3). As for total β-catenin, most active β-catenin was
localized to plasma membranes and, except for the scattered nucleus, nuclear
localization was absent at all developmental stages.
Failure of Canonical WNT Activators to Induce Localization of Nuclear Active β-catenin (Experiments 5 to 8)
One possible reason for the lack of nuclear β-catenin in embryos is absence of
stimulation by canonical WNTs. To test this hypothesis, three experiments were
conducted to evaluate localization of active β-catenin after stimulation of WNT signaling.
In Experiment 5, embryos were treated with a GSK3 inhibitor at the 3-4 cell, 5-8 cell, 9-
16 cell and morula stages of development. Inhibition of GSK3 leads to accumulation of
β-catenin and import into the nucleus (Yuan et al. 2005). Some control and GSK3
inhibitor-treated embryos were also treated with leptomycin to block nuclear exportins.
This treatment was added to enhance nuclear localization of β-catenin in case nuclear
β-catenin induced by GSK3 inhibition is rapidly exported from the nucleus.
Representative images are shown in Figure 2-4. Active β-catenin remained localized to
non-nuclear regions of the cells and to the plasma membrane in particular. With the
exception of the occasional cell, there was no accumulation of β-catenin in the nucleus
at any stage of development.
For Experiment 6, embryos at the 5-8 cell or morulae stages of development
were treated with either the WNT agonist AMBMP or human WNT1 and localization of
active β-catenin determined after 1, 6, 24, and 48 h of incubation. Although both
treatments increased intensity of labeling for active β-catenin in the plasma membrane,
there was no accumulation of detectable β-catenin in the nucleus (Figure 2-5A). For
Experiments 7 (results not shown) and 8 (Figure 2-5B), treatment of morulae with
75
AMBMP at day 5 or GSK3 inhibitor on day 6 after insemination also failed to increase
nuclear labeling with β-catenin in resultant blastocysts at day 7. Both treatments did
increase immunoreactive active β-catenin localized in the plasma membrane (Figure 2-
5B).
Localization of Active β-catenin in Mouse and Bovine Embryos Evaluated by Confocal Microscopy (Experiment 9 and 10)
As found for bovine embryos using epifluorescence microscopy, there was no
observable active β-catenin in the nucleus of mouse 5-8 cell embryos (Figure 2-6A).
Similarly, no active β-catenin was observed in nuclei of bovine embryos when embryos
were examined by confocal microscopy (Figure 2-6B).
Nuclear Localization of β-catenin in Bovine Embryonic Fibroblast Cells Following Activation of Canonical WNT Signaling (Experiment 11)
To test whether absence of nuclear localization of active β-catenin was a unique
feature of preimplantation embryos, localization of the protein was also examined in
BEF cells derived from bovine embryonic fibroblasts. In these cells, punctuate labeling
of active β-catenin was observed in the nucleus of a fraction of cells (Figure 2-8). The
proportion of cells depicting nuclear localization of active β-catenin was 310/763
(40.6%) for control cells vs. 67/148 (45.3%) for cells treated with AMBMP (P=0.29 for
difference from control) and 750/839 (89.4%) for cells treated with GSK inhibitor
(P<.0001 for difference from control).
Actions of WNT11 on Phosphorylation of the Non-canonical Signaling
Protein JNK and Development to the Blastocyst Stage (Experiment 12-13)
Immunoreactive phospho-JNK was localized in nuclei (Figure 2-7A). Moreover,
the pattern of nuclear labeling of phospho-JNK was punctuated. The degree of labeling
varied between cells although labeling was not consistently elevated in TE or ICM.
76
Treatment of blastocysts with human recombinant WNT11 caused a significant increase
in intensity of labeling of phospho-JNK at 2.5 µg/ ml (P<.0001) but not at 0.5 or 1 µg/ ml
(Figure 2-7B).
In Experiment 13, effect of WNT11 on development and blastocyst cell number
was determined (Table 2-3). Treatment with WNT11 increased the proportion of oocytes
that developed to the blastocyst stage (P=0.042) but had no effect on number of ICM,
TE or total cells in the resulting blastocysts.
Discussion
The mouse embryo does not require canonical WNT signaling for either
development to the blastocyst stage or ESC identity, expansion, or self-renewal
(Huelsken et al. 2000; Kemler et al. 2004; Xie et al. 2008; Lyashenko et al. 2011) The
situation for other species is less clear. Here it is shown that one of the characteristics
of preimplantation development in the cow is a temporal decrease in expression of key
genes involved in WNT signaling along with a paucity of nuclear β-catenin, even after
stimulation of the embryo with molecules that activate canonical WNT signaling. These
observations are consistent with the idea that, like the mouse, canonical WNT signaling
is dispensable for blastocyst development in the cow. In contrast, non-canonical WNT
signaling improved embryonic development because WNT11 increased the proportion
of embryos becoming blastocysts while also increasing phosphorylation of JNK, a
central player in the WNT/planar cell polarity (PCP) pathway (Zeke et al. (2016).
Observed changes in gene expression also mean that, similar to the human (Krivega et
al. 2015), characteristics of WNT signaling are likely to change during development. By
the blastocyst stage, WNT signaling may play different roles in the ICM and TE because
of differences in expression of several important genes in the WNT signaling system.
77
A key observation of the current series of experiments was, with rare exceptions,
the absence of observable immunoreactive β-catenin in the nucleus of embryos at every
stage examined. Failure to observe nuclear β-catenin was not because of failure of the
antibodies used to recognize the molecule because immunoreactive total and active β-
catenin could be localized to the plasma membrane. Lack of nuclear β-catenin was
observed even after embryos were treated with molecules expected to activate
canonical WNT signaling including a GSK3 inhibitor, the WNT agonist AMBMP (Liu et
al. 2005) or the canonical WNT1 (Shimizu et al., 1997; Yuan et al. 2005). The lack of
nuclear β-catenin was not due to rapid export from the nucleus because inhibition of
nuclear exportins with leptomycin did not lead to accumulation of β-catenin in the
nucleus. Failure of the molecules to induce nuclear localization was not because the
molecules were inactive because all three WNT activators increased active β-catenin
associated with the plasma membrane and because GSK3 inhibition increased the
percent of cells with nuclear β-catenin in cells of the BEF cell line. Immunolabeling of
nuclear active β-catenin in BEF cells was characterized by a punctuate pattern
resembling that previously described in newly differentiated chondrocytes (Guo et al.
2004) and intrahepatic cholangiocarcinoma cells (Wang et al. 2015).
An absence of β-catenin in the nucleus of the preimplantation embryo may be a
widespread phenomenon in the mammal, at least for certain stages of development. In
the human embryo, accumulation of β-catenin in the nucleus in response to inhibition of
GSK3B depends upon stage of development, with accumulation being attenuated after
day 3 of development and absent in blastocysts (Krivega et al. 2015). In the pig,
immunoreactive nuclear β-catenin was faint in expanded blastocysts and absent in
78
hatching blastocysts (Lim et al. 2013). Moreover, accumulation in the nucleus was not
induced by LiCl inhibition of GSK3 (Lim et al. 2013).
Results with respect to the mouse are contradictory. No nuclear β-catenin was
detected in mouse blastocysts in one study (Kemler et al. 2004) whereas active β-
catenin was observed in the nucleus of embryos at the 1-cell, 2-cell, 4-cell, 8-cell,
morula and blastocyst stage of development in another (Xie et al. 2008). Present results
fail to replicate findings of nuclear β-catenin in mouse 5-8 cell embryos even though the
antibody used in the present experiment was the same as used earlier (Xie et al. 2008).
The findings that β-catenin does not translocate to the nucleus in the bovine
embryo after treatment with canonical WNT activators does not mean that WNTs are
not involved in regulation of embryonic development. In addition to canonical signaling,
there is a variety of other signaling cascades activated by WNTs termed non-canonical
pathways (Filmus et al. 2008; Chien et al. 2009; van Amerongen and Nusse, 2009;
Gao, 2012). Some of these pathways use FZD as a receptor (PCP and Ca++ mediated
signaling) whereas others use other receptor molecules such as ROR and RYK.
Individual WNTs preferentially stimulate canonical or non-canonical signaling depending
upon ability to bind FZD and recruit LRP5/6 and other coreceptor molecules. Thus,
some documented actions of WNTs on the preimplantation embryo, for example,
promotion of TE development in human embryos by WNT3 (Krivega et al. 2015), could
involve signaling through one or more pathways independent of accumulation of β-
catenin in the nucleus. Here it was shown that WNT11, which is considered to
preferentially activate non-canonical pathways (Flaherty & Dawn, 2008; Uysal-Onganer
& Kypta, 2012), can activate a key component of the PCP pathway in bovine
79
blastocysts by phosphorylating the signaling kinase JNK in the nucleus. Activation of
JNK has been implicated in actions of WNT11 in other cells (Pandur et al. 2002; Cha et
al. 2008; Chen et al. 2014; Geetha-Loganathan et al. 2014) although, under certain
circumstances, WNT11 can inhibit JNK signaling (Railo et al. 2008). The observation
that activated JNK was localized to the nucleus suggests that the protein translocate to
the nucleus after activation, as has been described for other cells (Schreck et al. 2011;
Coffey, 2014). Furthermore, WNT11 participates in regulation of preimplantation
developmental processes since addition of exogenous WNT11 to the culture medium
resulted in higher proportion of inseminated oocytes that developed to the blastocyst
stage Further investigation is needed to unravel the downstream effect of this WNT in
the preimplantation bovine embryo, but the nuclear localization of phospho-JNK in
response to WNT11 suggests the presence of a JNK-nuclear substrate usually
associated with an effect on gene expression [reviewed in Zeke et al. (2016)].
In addition, although WNT activation did not cause accumulation of β-catenin in
the nucleus in bovine embryos, it did increase β-catenin in the embryo, with the protein
being localized primarily to the plasma membrane. Similar effects have been observed
in human embryos (Krivega et al. 2015). Thus, certain actions of WNT on the embryo
could conceivably involve signal transduction pathways utilizing membrane-bound β-
catenin. In mouse embryonic stem cells, β-catenin bound to E-cadherin is required for
expression of Klf4 and Nanog via STAT3 phosphorylation (Hawkins et al., 2012). It may
also be possible that activation of WNT signaling does lead to some accumulation of β-
catenin in the nucleus but at amounts too low to be detected by immunofluorescence.
Further investigation is needed to understand the alternative WNT signaling pathways
80
regulating developmental processes, as well as the role of each of these pathways in
preimplantation embryo development.
Analysis of gene expression during development is consistent with a reduction in
WNT signaling as the embryo develops. In Experiment 1, transcript abundance for all
genes examined declined to a nadir at the morula or blastocyst stage of development.
This was true for the WNT coreceptors, LRP5 and LRP6, the canonical WNT antagonist
DKK1, two WNT-dependent transcription factors, LEF1 and TCF7, as well as two
repressors of WNT-dependent transcription factors, LOC505120 (encodes for
GROUCHO-like protein) and AES. The decline in gene expression is not an artifact of in
vitro fertilization or culture because similar developmental patterns of gene expression
were seen for 6 of the 7 genes for embryos that developed in vivo (Supplemental Table
S2-3 in Jiang et al. 2014). The only exception was for AES, which rose in transcript
abundance at the blastocyst stage for in vivo embryos (Jiang et al., 2014a) but
remained low for in vitro produced embryos. It is possible that the developmental
decline in abundance of most transcripts examined is part of the large-scale destruction
of maternally-derived mRNA in the oocyte after fertilization (Tadros & Lipshitz, 2009;
Graf et al., 2014)
More research is required but analysis of differences in gene expression between
the ICM and TE of the blastocyst are consistent with the idea that WNT signaling
functions differently in the two cell types. Genes upregulated in the TE included three
receptors or co-receptors (FZD1, FZD7 and LRP6) and two genes involved in inhibition
of canonical WNT signaling (APC and SFRP1). Expression of WNT6 was also
upregulated in the TE. This WNT, which can promote differentiation of primitive
81
endoderm (Krawetz & Kelly, 2008), functions as a canonical WNT when binding FZD
1/2/7 and as a non-canonical WNT when binding FZD 5/8 (Schmidt et al. 2007;
Lhomond et al. 2012; Li et al. 2014). Perhaps WNT6 is secreted by TE cells to
participate in differentiation of cells of the ICM to primitive endoderm.
In conclusion, the accumulation of β-catenin in the nucleus in response to
canonical WNT activators is blocked in the preimplantation bovine embryo. Moreover,
there is a decline in expression of several genes important for canonical WNT signaling
as the embryo advances in development. In contrast, at least one non-canonical
signaling pathway involving JNK and the PCP pathway can be activated in the bovine
preimplantation embryo. Moreover, WNT11, which causes JNK activation, improves
competence of the embryo to develop to the blastocyst stage. Thus, some actions of
WNTs on the preimplantation embryo are likely to involve signaling through
mechanisms independent of nuclear β-catenin. Differences in gene expression between
the TE and ICM mean that, by the blastocyst stage, WNT signaling may play different
roles in the ICM and TE.
82
Table 2-1. Primer sequences used for real-time PCR. Gene
Gene ID
Reference sequence
Primers
Size (bp)
AES 505375 NM_001128497.1 F:5'-GACAAACACTGAGAGAGGAAGG-3' 95 R:5’-CCAGTAGGCAGCTACCATAAAT-3’ DKK1 50445 NM_001205544.1 F: 5’-GACTGGTGGAGGCGCTCGGA-3’ 137 R:5’-GCTGTGCCCAGAGCCGTCAT-3’ GAPDH 281181 XM_618013 F:5’-ACCCAGAAGACTGTGGATGG-3’ 175 R:5’-CAACAGACACGTTGGGAGTG -3’ GROUCHO 505120 XM_002694911.2 F:5'-AGTCGGCCAACTTTCCAGGACTTA-3' 180 R: 5’-AAGATGCAGCATTCGGTTTCAGCC-3’ LEF1 535399 NM_001192856.1 F:5'-CTGACGCATCCTTCCAATTCT-3' 182 R: 5’-CATCCCGACCACTGTGTAATC-3’ LRP5 534450 XM_002699405.2 F:5'-AGTATACTGC CAGCTCCGCG -3' 120 R:5’-TTCAGTCCGC CGTGGCGCTG-3’ LRP6 53628 XM_002687783.2 F:5’-AGTGCCCTGGAACATGTGGTAGAA-3’ 102 R:5’-ATTGGTTCCTGTGTCTGCCCAGTA-3’ SDHA 281480 NM_174178 F:5’GCAGAACCTGATGCTTTGTG-3’ 185 R:5’-CGTAGGAGAGCGTGTGCTT-3’ TCF7 782690 NM_001099186.2 F:5'-GCATGGTCACAACAACCAAGCTCA-3' 121 R:5’-TGTGGGTAGAAGCTTCCCTTGGTT-3’ YWHAZ 287022 XM_005215615.1 F:5'-GCATCCCACAGACTATTTCC-3' 120 R:5'- GCAAAGACAATGACAGACCA-3'
83
Table 2-2. Effect of stage of development and cell lineage [inner cell mass (ICM) vs trophectoderm (TE)] on expression of genes involved in WNT signaling for day 6 morulae and day 8 blastocysts produced in vitroa.
Number of reads
P value
Gene
Morula
ICM
TE
Morula vs (ICM+TE) ICM vs TE
WNTs b WNT2 18.49 15.19 17.66 0.778 0.784 WNT2B 22.00 21.00 50.67 0.079 0.952 WNT6 0.20 2.96 14.51 <.0001 <.0001 WNT8A 5.82 0.27 1.12 0.079 0.664 WNT10A 395.0 468.0 652.7 0.068 0.548 WNT11 101.0 91.33 119.7 0.376 0.745 WNT16 4.09 23.65 12.26 0.236 0.409
WNT processing
PORCN 196.97 213.19 450.17 0.016 0.004 Frizzled receptors and LRP co-receptors c
FZD1 145.00 82.33 140.00 0.159 0.016 FZD3 9.62 3.43 5.29 0.098 0.585 FZD6 62.29 108.59 132.79 0.032 0.367 FZD7 31.21 18.56 55.90 0.231 0.001 FZD8 91.00 50.49 58.17 0.385 0.877 FZD10 73.33 59.33 75.33 0.539 0.415 LRP6 302.90 264.38 412.72 0.390 0.024 Proteins involved in canonical signaling d APC 14.43 12.47 19.77 0.247 0.006 AXIN1 30.56 18.00 31.44 0.450 0.186 CNBP 11.79 9.83 9.38 0.469 0.901 β-catenin 584.6 5897.4 6838.01 0.673 0.540 DVL2 9.89 4.84 6.60 0.315 0.717 DVL3 49.99 37.32 66.36 0.913 0.203 GSK3B 1144.6 549.73 720.33 0.060 0.539 Transcription factors e LEF1 61.55 1.64 3.14 0.096 0.968 TCF7L1 12.16 16.53 10.31 0.859 0.486 TCF7L2 556.98 211.86 116.16 0.003 0.339
84
Table 2-2. Continued
Genes
Number of reads
P value
Morula
ICM
TE
Morula vs (ICM+TE)
ICM vs TE
Transcription factors inhibitors f AES 48.68 136.79 181.86 0.04 0.402 TLE3 17.67 89.67 122.00 0.154 0.187 R-spondin signaling g LGR4 166.23 60.65 102.46 0.032 0.289 LOC100337123 h 11.74 3.25 5.67 0.002 0.174 RSPO1 202.67 187.00 211.00 0.703 0.749 RSPO2 11.91 0.62 4.04 0.118 0.514 RSPO3 1925.6 643.08 301.03 0.019 0.540 Other soluble WNT regulatory proteins i DKK1 15.71 3.22 1.06 0.056 0.761 NDP 1498.1 1510.6 1779.3 0.670 0.530 SFRP1 124.75 48.81 143.11 0.145 0.006 SFRP2 61.00 16.00 25.67 0.700 0.236 SFRP3 63.47 23.03 16.83 0.236 0.882 SFRP4 31.67 25.67 38.67 0.127 0.393 WIF1 96.78 30.92 33.30 0.040 0.937 Other WNT signaling proteins j DACT2 49.56 41.19 43.25 0.367 0.830 KREMEN1 53.14 9.67 15.87 0.011 0.643 RYK 97.14 46.14 79.40 0.172 0.265 a Data are least-squares means of number of reads. b The following genes had < 5 reads: WNT1, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT7A, LOC100337066 (WNT7B paralog), WNT8B, WNT9A, WNT9B,and WNT10B. c The following genes had < 5 reads: FZD2, FZD4, FZD5, FZD9, and LRP5. d The following genes had < 5 reads: AXIN2, DVL1. e The following genes had < 5 reads: TCF7 f The following genes had < 5 reads: LOC505120(GROUCHO ortholog), TLE1, TLE2, TLE4, and TLE6. g The following genes had < 5 reads: LGR5, LGR6 and RSPO5. h RSPO3 like. i The following genes had < 5 reads: DKK4 and SFRP5 j The following genes had < 5 reads: ANKRD6, DACT1, DACT3, FRAT1, KREMEN2, NKD2, VANGL1, and VANGL2
85
Table 2-3. Effect of 2.5 µg/ ml WNT11 from day 5 to day 7 after insemination on development of embryos to the blastocyst stage at day 7 after inseminationa
Blastocyst cell number
Treatment Percent
blastocyst
Total TE ICM
Vehicle 17.8 ± 2.5 127 ± 6 89 ± 5 38 ± 3
WNT11 25.5 ± 2.7 138 ± 6 93 ± 4 45 ± 3
P-value 0.042 0.210 0.529 0.137
a Data are the least-squares means ± SEM of results. Data on percent blastocyst represent the percent of inseminated oocytes that developed to the blastocyst stage. The total number of oocytes was 484 . Blastocyst cell number was evaluated for 74 embryos.
86
Figure 2-1. Developmental changes in expression of selected genes involved in WNT signaling for embryos produced in vitro (Experiment 1). Expression was assessed by qPCR. Expression of each gene was affected by stage of development (P= 0.004 for LRP5 and P<0.0001 for the other genes). Data are presented as least-squares means + SEM of results from 5 replicates. Blast, blastocyst (168 hpi).
87
Figure 2-2. Representative examples of localization of immunoreactive β-cateninat
various stages of preimplantation development of embryos produced in vitro (Experiment 3). Embryos were labeled with antibody to β-catenin (red) and DNA (blue). The total number of embryos evaluated was 450.
88
Figure 2-3. Representative examples of localization of immunoreactive non-phospho
(active) β-catenin during preimplantation development of embryos produced in vitro (Experiment 4). Embryos were labeled with antibody to non-phospho (active) β-catenin (red) and DNA (blue). A total of 417 embryos was evaluated.
89
Figure 2-4. Lack of localization of active β-cateninin the nucleus of in vitro produced
embryos after activation of canonical WNT signaling with the GSK3 inhibitor (Experiment 5). Leptomycin was added to reduce nuclear export of β-catenin. Shown are representative images of individual embryos labeled with antibody to non-phospho (active) β-catenin (red) and DNA (blue) treated at the 9-16-cell stage and harvested 24 h after stimulation. Similar results were seen for embryos treated at other stages of development (total number of embryos examined = 191).
90
Figure 2-5. Consequences of treatment of embryos with WNT agonists for
immunolocalization of active β-catenin. Shown in panel A) (Experiment 6; n=36 embryos) are representative images of individual embryos produced in vitro that were treated at the 5-8 cell stage with vehicle, the WNT agonist AMBMP or recombinant WNT1, harvested 24 h later and labeled with non-phospho (active) β-catenin (red) and DNA (blue). Shown in panel B) (Experiment 8; n=15 embryos) are representative images of individual embryos produced in vitro that were treated at day 6 after insemination with vehicle or GSK3 inhibitor, harvested 24 h later and labeled with non-phospho (active) β-catenin (red) and DNA (blue).
91
Figure 2-6. Localization of active β-cateninin mouse and bovine embryos by confocal microscopy. Shown in panel A) (Experiment 10) are images of 2 individual 5-cell mouse embryos labeled with non-phospho (active) β-catenin (green) and Hoechst (blue). A total of 5 embryos were examined. Shown in panel B) (Experiment 11) are representative confocal images of bovine embryos at the morula stage captured with 40X magnification (top), at the blastocyst stage captured with 40X magnification (middle), and at the blastocyst stage captured with 60X magnification (bottom). A total of 4 embryos were examined.
92
Figure 2-7. Immunoreactive phospho-JNK in blastocysts produced in vitro (Experiment
12). Shown in panel A) are representative images of embryos labeled with phospho-JNK (red) and Hoechst (blue) that were treated at the blastocyst stage with 0, 0.5, 1 or 2.5 µg / ml of recombinant WNT11 and harvested 6 h later. Shown in panel B) are average values of intensity of fluorescence of phospho-JNK in the whole area of the embryo (n=49 embryos). ***, P<0.0001 from 0 μg/ ml.
93
Figure 2-8. Representative confocal microscopy images of immunoreactive active β-catenin in bovine embryonic fibroblast cells following activation of canonical WNT signaling. Cells were labeled with antibody to non-phospho (active) β-catenin (red) and Hoechst (blue) after 24 h stimulation with either vehicle, the WNT agonist AMBMP or a GSK3 inhibitor. Arrows indicate nuclei depicting nuclear β-catenin, while arrow heads indicate nuclei with no accumulation of β-catenin.
94
CHAPTER 3 CONSEQUENCES OF ENDOGENOUS AND EXOGENOUS WNT SIGNALING FOR
DEVELOPMENT IN THE PREIMPLANTATION BOVINE EMBRYO
Introduction
The WNT are a family of 19 extracellular growth factors whose secretion
depends upon palmitoylation mediated by acytransferases (Willert et al., 2003; Ho and
Keller, 2015). WNT signaling participates in a number of developmental processes
including cellular proliferation (Logan and Nusse, 2004), maintenance of pluripotency
(Sato et al., 2004; Sokol, 2011), cellular differentiation (Liu et al., 2014), asymmetrical
cell division (Sawa, 2012), the epithelial-mesenchymal transition (Kleber and Sommer,
2004), and axis elongation (Silhankova and Korswagen, 2007; Zinovyeva et al., 2008).
The outcome of WNT signaling depends upon the cellular context established by the
availability of receptors, co-receptors and molecules involved in different signaling
pathways. WNTs may interact with any of ten frizzled (FZD) receptors as well as
alternative receptors including ROR, PTK7 and RYK to activate different downstream
signaling cascades (Cadigan and Nusse, 1997; Logan and Nusse, 2004). The most
well-described signaling pathway, the so-called canonical pathway, is mediated by
nuclear β-catenin. This protein has a dual function because it provides an indirect link
between cadherin and the contractile cortical actin cytoskeleton (Abe et al., 2013) and
can also localize into the nucleus where it interacts with transcription factors to regulate
gene expression (He et al., 1998; Tetsu and McCormick, 1999). Other downstream
signaling pathways activated by WNTs include the planar cell polarity pathway (PCP)
(Veeman et al., 2003; Seifert and Mlodzik, 2007), and Ca+ mediated cascade (Kühl et
al., 2000; Kohn and Moon, 2005). More recently, non-nuclear β-catenin has also been
associated with downstream signaling (Hawkins et al., 2012).
95
The role of endogenous and maternally-derived WNTs in preimplantation
development is unclear. Blastocyst-stage embryos express only a subset of WNT
genes. Of the 19 WNT ligands, only 10, 8 and 6 are expressed in mouse, human and
bovine blastocysts respectively (Lloyd et al., 2003; Kemp et al., 2005; Ozawa et al.,
2012; Denicol et al., 2013a; Yan et al., 2013). There also is limited or no detectable
nuclear accumulation of β-catenin in these three species even in the presence of WNT
agonists (Kemler et al., 2004; Krivega et al., 2015). In mouse embryos, inhibition of
secretion of endogenous WNT does not affect blastocyst formation (Biechele et al.,
2013) and neither does depletion of β-catenin (Huelsken et al., 2000). Furthermore,
inhibition of β-catenin mediated WNT signaling with DKK1 does not impair blastocyst
formation either in mouse, cow, or pig embryos (Li et al., 2008; Xie et al., 2008b;
Denicol et al., 2013a; Lim et al., 2013). Taken together, evidence suggests that
endogenous WNT signaling mediated through nuclear accumulation of β-catenin is
dispensable for blastocyst formation. A role for embryo-derived WNT acting through
other pathways is possible however.
Moreover, maternally-derived WNTs could also contribute to regulation of
embryonic development although the consequences of maternal WNT for the embryo
are unclear. There may also be a physiological role for the WNT antagonist DKK1 in
preimplantation development. A product of the uterine endometrium (Kao et al., 2002;
Tulac et al., 2003; Peng et al., 2008; Cerri et al., 2012), DKK1 can both block
intracellular accumulation of β-catenin by interfering with the formation of a WNT-FZD-
LRP5/6 complex (Logan and Nusse, 2004; MacDonald et al., 2009) and can itself signal
through activation of JNK and the PCP pathway (Caneparo et al., 2007; Killick et al.,
96
2014) . Exposure of pig embryos to DKK1 increased the number of TE cells in
blastocyst, without changing the number of ICM, thereby increasing the proportion of
cells in the blastocyst that were TE (Lim et al., 2013). Similarly, exposure of bovine
embryos to DKK1 increased the proportion of blastocyst blastomeres classified as TE
and improved competence of embryos to establish pregnancy after transfer (Denicol et
al., 2014). In the pig, DKK1 increased the proportion of blastocysts that hatched in vitro
(Lim et al., 2013).
The purpose of the series of experiments documented in this chapter was to
determine consequences of activation and inhibition of β-catenin dependent and -
independent WNT signaling on development of bovine preimplantation embryos and
allocation of cells in the blastocyst into ICM and TE lineages. Results indicate a limited
role of embryo-derived WNTs in blastocyst development and suggest that, depending
on the type of ligand, maternally-derived WNT can potentially either promote or inhibit
competence of the embryo to become a blastocyst.
Materials and Methods
Embryo Production Using Non-Sex Sorted Sperm
Formulation of media used for production of bovine embryos in vitro are
described elsewhere (Ortega et al., 2016). Cumulus-oocyte complexes (COC) were
obtained from cattle ovaries (including Bos taurus and cattle that are an admixture of B.
taurus and B. indicus) collected at a local abattoir by bisecting follicles 3 to 8 mm in
diameter with a scalpel. Procedures for oocyte recovery and maturation, fertilization and
embryo culture were performed following procedures described elsewhere (Dobbs et
al., 2013) with a few modifications. Oocytes were harvested using BoviPROTM oocyte
washing medium (MOFA Global, Verona, WI, USA) and matured for 20-22 h in groups
97
of 10 in oocyte maturation medium (OMM). Groups of up to 300 matured oocytes were
then fertilized for 8-10 h in 300 µl IVF-TL (Caisson Laboratories, Logan, UT, USA) to
which sperm (final concentration, 1 x 106 cells/ml) and 80 µl of a solution of PHE (0.5
mM penicillamine, 0.25 mM hypotaurine, and 25 µM epinephrine) were added. Sperm
used for each fertilization procedure consisted of a pool from three B. taurus or Brangus
bulls that were randomly selected from available bulls. A different assortment of bulls
was used for each procedure. Sperm from frozen-thawed straws were purified before
fertilization using an Isolate gradient [(Irvine Scientific, Santa Ana, CA; 50% (v/v) and
90% (v/v) isolate] and diluted in IVF-TALP (Caisson Laboratories). After removal of
cumulus cells, groups of 25-30 presumptive zygotes were placed in 50 µl microdrops of
SOF-BE2 covered with mineral oil (Sigma-Aldrich, St. Louis, MO, USA) and cultured at
38.5oC in a humidified atmosphere of 5% O2 and 5% CO2 with the balance N2. Unless
stated otherwise, treatments were administered on day 5 of development [120 hours
post insemination (hpi)]. The procedure consisted of removing 5 µl of culture medium
and replacing it with 5 µl of culture medium containing ten times the desired
concentration of the treatment in the drop.
Embryo Production Using Sex-Sorted Sperm
Procedures were as described above except for semen preparation and
fertilization. Commercially-available X and Y-sorted sperm from Angus sires were
obtained from ABS Global (De Forest, WI, USA) and Genex Cooperative, Inc.
(Shawano, WI, USA). Separated pools of X and Y-sorted sperm from the same two
bulls, randomly-selected from those available, were used in each fertilization procedure.
The total number of bulls used was six. Sperm were purified before fertilization using
Puresperm® 40/80 gradient column (Nidacon International AB, Mölndal, Sweden).
98
Sperm was first centrifuged (2,600 x g for 5 min) in 2.0 ml microcentifuge tubes
containing 250 µl sperm over two layers of 200 µl of Puresperm® (top layer of
Puresperm®40 and bottom layer of Puresperm®80). The pellet representing the bottom
100 µl was transferred to a new microcentrifuge tube, washed in 1000 µl of IVF-TL that
had been pre-equilibrated at 38.5°C under 5% CO2, and centrifuged at 600 x g for 3
min. Fertilization of groups of 30 matured cumulus-oocyte complexes was performed in
60 μL oil-covered microdrops of IVF-TL medium containing 3.5 µl of PHE. Final
concentration of sperm in the fertilization drop was 2 x 106 cells/ml. Fertilization was
carried out for 18–20 h at 38.5°C and a humidified atmosphere of 5% (v/v) CO2.
Treatments were administered as described for embryos produced with non-sex sorted
semen.
Immunolabeling of Protein in Bovine Embryos
Procedures for labeling embryos against β-catenin and pJNK were as follows.
Embryos were fixed in 4% (v/v) paraformaldehyde, and permeabilized in Dulbeccos’s
phosphate-buffered saline (DPBS) containing 0.5% (v/v) Triton X-100. Blocking was
performed using DPBS containing 5% (w/v) bovine serum albumin (BSA), and
incubation with primary antibody diluted in antibody buffer [DPBS containing 0.1% (v/v)
Tween 20 and 1% BSA (w/v)] was performed overnight at 4 °C. Total immunoreactive β-
catenin was detected using 1µg/ml rabbit polyclonal anti-human β-catenin (Abcam).
Immunoreactive pJNK was detected using 1 µg/ml rabbit polyclonal anti-phospho-JNK
[(Thr183/Tyr185,Thr221/Tyr223) (Millipore, Billerica, MA, USA)]. Note that the
phosphorylation site is conserved among human and bovine JNK and that the amino
acid sequence identity between these species is 99%. Incubation with labeled second
antibody (goat anti-rabbit IgG conjugated with Alexa Fluor 555 (Life Technologies;
99
1µg/ml diluted in antibody buffer) proceeded for 1 h at room temperature. Nuclear
labeling was performed with 1µg/ml Hoechst 33342 (Sigma-Aldrich) in antibody buffer.
Slides were mounted using SlowFade Gold antifade reagent (Life Technologies,
Carlsbad, CA, USA), and observed with a 40x objective using a Zeiss Axioplan 2
epifluorescence microscope (Zeiss, Göttingen, Germany) and Zeiss filter sets 02
[4=,6=-diamidino-2-phenylindole (DAPI)], 03 (FITC filter), and 04 (rhodamine). Digital
images of individual blastocysts were acquired using AxioVision software (Zeiss) and a
high-resolution black and white Zeiss AxioCam MRm digital camera. For negative
control, IgG of the same species was used to replace primary antibody using the same
concentration.
For dual immunolocalization of YAP1 and CDX2, embryos were processed as
described above with few modifications. After overnight incubation with rabbit
monoclonal anti-human YAP1 (Cell Signaling Technology, Beverly, MA, USA) at a
concentration of 0.01µg/ml. embryos were washed 3 times with washing buffer [DPBS
containing 0.1% (v/v) Tween 20 and 0.1% BSA (w/v] and incubated with secondary
antibody [goat anti-rabbit IgG conjugated with Alexa Fluor 555 (Life Technologies;
1µg/ml] for 1 h at room temperature. Embryos were washed again 3 times and
incubated for 1 h with primary antibody against CDX2 (mouse anti-human polyclonal
CDX2 antibody, ready to use; Biogenex, Fremont, CA, USA) and 1 h with 1µg/ml goat
anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC; Abcam, Cambridge,
MA, USA). Nuclear labeling, slide mounting, and image acquisition were performed as
describe above.
100
Quantification of intensity of labeling in either the entire embryo or in the nuclei
was performed using ImageJ software (U.S. National Institutes of Health, Berthesda,
MD, USA).
For labeling of the entire embryo, the area encompassing the entire embryo was
selected and the mean intensity obtained using the Measure Analysis feature of
ImageJ. Background intensity was obtained from the area surrounding the embryo
using the same technique and the value subtracted from embryo intensity. Labeling in
the nucleus was determined using a similar technique except that the software was
used to isolate nuclear regions within each embryo based on labeling with Hoechst
33342.
Number of ICM and TE cells was determined for embryos labeled with anti-CDX2
as described above. Those cells with nuclei labeled with CDX2 were considered TE and
the number of ICM cells was determined by subtracting number of TE cells from the
total number of cells determined by counting number of nuclei labeled with Hoechst
33342.
Experiment 1: Effect of Activation of Canonical WNT Signaling by Inhibition of GSK3 on Development
Embryos were cultured in 50 µl microdrops of SOF-BE2. Treatments were either
10 µM CHIR99021 (Tocris Bioscience, Avonmouth, Bristol, UK; final concentrations in
the drop) or vehicle (SOF-BE2). The concentration of inhibitor was chosen because it
was effective at blocking lipopolysaccharide-induced inflammation in adipose tissue
(Lappas, 2014). Blastocyst development was evaluated on day 7 of development (168
hpi). The experiment was performed in 5 replicates using a total of 803 COC and 10
different bulls.
101
Experiment 2: Effect of Activation of Canonical WNT Signaling by the Agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) in the Presence or Absence of DKK1 on Development and β-catenin Labeling
The source of AMBMP was Calbiochem (San Diego, CA, USA). Human
recombinant DKK1 was purchased from R&D Systems (Minneapolis, MN, USA). Both
reagents were reconstituted as previously described (Denicol et al., 2013a). Embryos
were cultured in 50 µl microdrops of SOF-BE2. Treatments were either vehicle [SOF-
BE2 containing 0.1% (v/v) DMSO], 0.7 µM AMBMP, 100 ng/ml DKK1, or 0.7 µM
AMBMP plus 100 ng/ml DKK1 (final concentrations in the drop). Blastocyst
development was evaluated on day 7 of culture (168 hpi), and a fraction of blastocysts
with a clearly delineable blastocoel were randomly selected for immunolabeling of β-
catenin. The experiment was performed in 7 replicates using a total of 1,006 COC and
10 different bulls. A total of 165 blastocysts were analyzed for immunolabeling of β-
catenin.
Experiment 3: Effects of Inhibition of Endogenous WNT Signaling with Wnt-C59 or DKK1 on Ability of Embryos to Develop to the Blastocyst Stage and Blastocyst Cell Number
Wnt-C59 [2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridine-3-
yl)phenyl)acetamide] was purchased as a 10 mM stock in dimethyl sulfoximine (DMSO)
from Cellagen Technology (San Diego, CA, USA). The Wnt-C59 was serially diluted to
100 nM in SOF-BE2 containing 0.001% (v/v) DMSO so that the final concentration of
Wnt-C59 in the culture drop was 10 nM. The concentration of inhibitor was chosen
because it blocked WNT activation in HeLa cells (Proffitt et al, 2013). Treatments were
vehicle [SOF-BE2 containing 0.01% (w/v) BSA (in addition to the BSA included in SOF-
BE2 formulation) and 0.0001% (v/v) DMSO]; 10 nM Wnt-C59; or 100 ng/ml DKK1 (final
102
concentrations in the drop). Blastocyst development was assessed on day 7 of
development (168 hpi). A fraction of blastocysts with a clearly delineable blastocoel was
randomly selected and subjected to immunolabeling to determine the numbers of TE
and ICM cells. The experiment was performed in 5 replicates with a total of 905 COC
and 13 different bulls. Treatment effect was evaluated for 91 blastocysts.
Experiments 4-5: Effects of DKK1 on Development and Blastocyst Cell Number
In each experiment, treatments were 100 ng/ml DKK1 and vehicle [SOF-BE2
containing 0.01% (w/v) BSA (in addition to the BSA included in SOF-BE2 formulation)].
The concentration of DKK1 was chosen because it was effective at blocking actions of
WNT signaling agonist on development of bovine embryos to the blastocyst stage
(Denicol et al., 2013a).
Both experiments 4 and 5 were designed to test whether DKK1 alters
competence of embryos to become blastocysts and the number of TE and ICM cells in
the blastocyst. An additional goal of experiment 5 was to determine whether effects of
DKK1 varied with embryo sex. Experiment 4 was performed in 10 replicates with a total
of 1,545 COC and conventional semen from 7 bulls. Experiment 5 was replicated 5
times with a total of 1,348 COC and X- and Y-sorted semen from 6 bulls. Blastocyst
development was evaluated on day 7 of development (168 hpi) and a fraction of
blastocysts with a clearly delineated blastocoel (n=89 for Experiment 4 and 204 for
Experiment 5) were randomly selected for labeling with anti-CDX2 and Hoechst 33342
to determine number of TE and ICM.
103
Experiment 6: Effects of DKK1 on Developmental Changes in YAP1 and CDX2 Localization in Morulae and Blastocysts
The transcription factor YAP1 plays an important role in TE formation in the
mouse by interacting with TEAD4 to induce transcription of CDX2 (Nishioka et al., 2007;
Yagi et al., 2007). For Experiment 6, it was determined whether DKK1 modifies the
developmental pattern of immunoreactive YAP1 and CDX2 from day 5 to day 7 of
development (when the embryo transitions from the morula to the blastocyst
stage). Embryos were treated with either 100 ng/ml DKK1 or vehicle [SOF-BE2
containing 0.01% (w/v) BSA (in addition to the BSA included in SOF-BE2 formulation)].
Morulae on day 5 (6 hours after treatment), morulae on day 6 (24 hours after treatment)
and blastocysts with a clearly delineated blastocoel on day 7 (48 hours after treatment)
were harvested and fixed for immunolocalization of YAP1 and CDX2. Number of cells
was counted based on DNA staining; number of YAP1+ and CDX2+ were also
quantified. Intensity of the nuclear YAP1 and nuclear CDX2 was obtained. Data are
presented as absolute and relative number of CDX2+ and YAP1+ nuclei. This
experiment was performed in 5 replicates with a total of 1,510 COC and semen from 7
bulls. A total of 232 individual embryos were assessed for immunofluorescence.
Experiment 7: Effects of DKK1 on Activation of JNK
Experiment 7 tested the hypothesis that DKK1 could activate JNK signaling in
bovine embryos. Activation was assessed as the accumulation of pJNK. Treatments,
which were added at day 5 of development, included vehicle [SOF-BE2 containing
0.01% (w/v) BSA (in addition to the BSA included in SOF-BE2 formulation)], and 100
ng/ml DKK1 (final concentration in the drop). Morulae were harvested 6 hours after
adding the treatment and immunolabeled for pJNK. The experiment was performed in 4
104
replicates with a total of 587 COC and semen from 11 different bulls. Treatment effect
on JNK accumulation was evaluated in 91 embryos.
Experiments 8 - 11: Embryo Responses to WNT7A
For these experiments, treatments were 66 ng/ml human recombinant WNT7A
(eBioscience Inc., San Diego, CA, USA) or vehicle [SOF-BE2 containing 0.01 mM
NaPO4, 0.5 mM NaCl and 0.0005 % (w/v) CHAPS in water]. The concentration of
WNT7A was chosen because it is the upper limit of the range suggested for biological
activity of the product by the manufacturer, as determined by inhibition of Wnt-3a-
induced alkaline phosphatase production in MC3T3-E1 cells, and 50 ng/ml was effective
activating Akt/mTOR anabolic growth pathway in skeletal muscle (von Maltzahn et al.,
2012). Blastocyst rate was assessed on day 7 of development (180 hpi) and a fraction
of blastocysts with clearly delineated blastocoel was randomly selected for further
analyses.
For experiment 8, embryos were produced in vitro as described above with few
modifications. Oocytes were matured in BO-IVM® (IVF Bioscience, Falmouth, Cornwall,
UK) for 24 h, and fertilization proceeded for 12-14 h. Culture drops were randomly
assigned to one of four treatments in a 2 x 2 arrangement with two treatments
(recombinant WNT7A or vehicle) and treatment on either day 1 (15 hpi) or day 5 of
development (115 hpi). Blastocysts were harvested for gene expression analysis. Pools
of 10 blastocysts were washed three times in DPBS-PVP, incubated in 0.1% (w/v)
proteinase solution (protease from Streptomyces griseus; Sigma-Aldrich) in DPBS to
remove the zona pellucida, washed three times in DPBS-PVP, and snap frozen in 5 µl
DPBS-PVP. Samples were stored at -80oC until processed for gene expression analysis
105
by Fludigm qPCR. This experiment was performed in 6 replicates with a total of 1,566
COC and semen from 8 bulls.
For experiments 9 and 10, embryos were produced in vitro. Treatments were
added on day 5 of development (120 hpi). Blastocysts were harvested to determine
number of ICM and TE cells (experiment 9) and intensity of total β-catenin (experiments
10) by immunolabeling as described above. Experiment 9 was performed in 7 replicates
with a total of 1,413 COC and semen from 7 bulls. A total of 54 individual blastocysts
were assessed by immunofluorescence. Experiment 10 was performed in 10 replicates
with a total of 1,471 COC and semen from 11 bulls. A total of 239 individual blastocysts
were assessed by immunofluorescence to quantify total β-catenin.
For experiment 11, embryos were produced following procedures for experiment
8. Treatments were added on day 5 of development (120 hpi), and blastocysts were
harvested on day 7 of culture for immunolabeling of pJNK as described above. A total of
42 individual blastocysts were assessed by immunofluorescence to quantify total pJNK.
Statistical Analysis
Effects of treatment on the percent of oocytes or cleaved embryos developing to
the blastocyst stage were evaluated using Proc Glimmix of SAS for Windows, version
9.4 (SAS Institute Inc, Cary, NC, USA). Each embryo was considered an observation
(0=not developed to blastocyst; 1=developed to blastocyst). The analysis was
performed with the dependent variable considered as a binomial distribution, and
treatments as fixed effects. Results are presented as least-squares means ± standard
error of the mean.
Data on intensity of immunolabeling were analyzed by least-squares analysis of
variance using the PROC MIXED procedure of SAS. Treatments were fixed effects and
106
replicate was considered a random effect. Results are presented as least-squares
means ± standard error of the mean.
Results
Effect of Activation of Canonical WNT Signaling on Development (Experiments 1 and 2)
Two experiments were conducted to determine whether activation of canonical
WNT signaling would affect development of embryos to the blastocyst stage. In
Experiment 1, addition of the GSK3 inhibitor CHIR 99021 to cultured embryos from day
5 to day 7 of development decreased the proportion of oocytes and cleaved embryos
becoming blastocysts (P=0.03 and P=0.05, respectively; Table 3-1). In Experiment 2,
canonical WNT signaling was activated by addition of the WNT agonist AMBMP. To test
whether AMBMP acts by increasing β-catenin accumulation, embryos were treated with
DKK1 which blocks coactivation of WNT receptors. Immunoreactive β-catenin was
localized primarily to the plasma membrane and was never found in the nucleus even
after addition of AMBMP (Figure 3-1A). Intensity of immunoreactive β-catenin was
increased by AMBMP (P<0.0001) and decreased by DKK1 (P=0.0001). There was no
interaction between AMBMP and DKK1 because AMBMP increased immunoreactive β-
catenin even in the presence of DKK1. Note, however, that the amount of β-catenin in
embryos treated with AMBMP combined with DKK1 was similar to the amount of β-
catenin in embryos treated with vehicle alone. Thus, while DKK1 did not prevent actions
of AMBMP to increase β-catenin, the total amount of β-catenin was not elevated as
compared to controls (Figure 3-1B). AMBMP reduced development to the blastocyst
stage and, while DKK1 alone did not alter development, the effect of AMBMP was
107
blocked by DKK1 (DKK1 by AMBMP interaction: P=0.04 for blastocysts/oocyte and
P=0.03 for blastocysts/cleaved; Table 3-2).
Effects of Inhibition of Endogenous WNT Signaling with Wnt-C59 or DKK1 on Ability of Embryos to Develop to the Blastocyst Stage and Blastocyst Cell Number (Experiment 3)
Neither Wnt-C59, which blocks acylation and secretion of WNTs, nor DKK1,
which interferes with activation of the WNT-FZD-LRP5/6 receptor complex, altered the
proportion of oocytes or cleaved embryos developing to the blastocyst stage (Table 3-
3). However, blastocysts formed in the presence of Wnt-C59 had increased number of
ICM cells (P=0.02) and tended to have reduced TE:ICM ratio (P=0.06) relative to
embryos treated with vehicle. There was no effect of DKK1 on numbers of ICM or TE
cells in the blastocyst (Table 3-3).
Effects of DKK1 on Development and Blastocyst Cell Number (Experiments 4-5)
There was no effect of addition of DKK1 from day 5 to day 7 of development on
the proportion of oocytes or cleaved embryos that became blastocysts or on the
numbers of ICM or TE cells in the resulting blastocysts. This was true whether embryos
were produced using conventional semen (Table 3-4) and for male and female embryos
tested separately after production using sexed semen (Table 3-5). The only exception
was a tendency for female embryos to have smaller TE:ICM ratio (P=0.10)
Effects of DKK1 on Developmental Changes in Immunoreactive YAP1 and CDX2 in Morulae and Blastocysts (Experiment 6)
The transcription factor YAP1 plays an important role in TE formation in the
mouse by interacting with TEAD4 to induce transcription of CDX2. For Experiment 6, it
was tested whether DKK1 would modify developmental changes in the number of cells
positive for YAP1 and CDX2 from day 5 to 7 of development. Representative examples
108
of labeling are shown in Figure 3-2A. Both YAP1 and CDX2 were localized exclusively
to nuclei. For day 5 morulae, nuclei positive for YAP1 and CDX2 were not frequent and
labeling was faint. By day 6, however, there were abundant numbers of YAP1+ and
CDX2+ cells. By day 7, both type of cells were confined to the TE. Quantification of
nuclei that were positive for YAP1 and CDX2 indicated that the number of cells positive
for both markers increased during development (Figure 3-2B, 3.2F). However, the
proportion of total cells that were YAP1+ declined after day 5 (Figure 3-2C) while the
proportion of total cells that were CDX2+ increased from day 5 to 7 (Figure 3-2G).
Similarly, the percent of CDX2+ cells that were also YAP1+ declined over time (Figure
3-2H) while the percent of YAP1+ cells that were also CDX2+ increased (Figure 3-2G).
Thus, as the embryo developed, an increasing number of CDX2+ cells lost expression
of YAP1. Intensity of labeling for YAP1 and CDX2 in nuclei positive for the marker
increased during development (Figure 3-2E and 3-2I).
There was no effect of DKK1 on total number of blastomeres or on the number or
proportion of blastomeres that were YAP1+ or CDX2+. Similarly, DKK1 did not affect
intensity of CDX2+ cells.In contrast, the intensity of YAP1 labeling in YAP1+ cells was
reduced by DKK1 (P=0.04; Figure 3-2E).
DKK1 Does not Activate pJNK (Experiment 7)
Addition of DKK1 to embryos at day 5 of development did not increase
accumulation of immunoreactive pJNK in blastocysts at day 7 of development (Figure 3-
3).
Regulation of WNT Signaling by WNT7A (Experiments 8 and 11)
Results of Experiment 8 are summarized in Table 3-6. Treatment of embryos
with WNT7A beginning at either day 1 (20 hpi) or day 5 (115 hpi) increased the
109
proportion of oocytes (P=0.0005) and cleaved embryos (P=0.02) that became a
blastocyst at day 7 of development. There was no effect of day of treatment or
interaction of day with treatment.
Subsequent experiments with WNT7A involved treatment on day 5 of
development. In Experiment 9, WNT7A again increased the proportion of oocytes
(P=0.02) and cleaved embryos (P=0.04) that became a blastocyst at day 7 of
development. There was, however, no effect of WNT7A on blastocyst cell number
(Table 3-7).
Effects of WNT7A on accumulation of β-catenin and pJNK were evaluated in
Experiments 10 and 11. There was no effect of WNT7A on total immunoreactive β-
catenin on day 7 (Figure 3-4B), Moreover, there was no nuclear localization of β-catenin
regardless of treatment (Figure 3-4A-B). Treatment with WNT7A actually reduced JNK
signaling as indicated by a reduction in immunoreactive pJNK in either the total area of
the embryo (P<.0001) or in nuclei (P<.0001; Figure 3-4C-E).
Discussion
Collectively, data suggest that embryo-derived WNTs are dispensable for
blastocyst formation in bovine embryos, but participate in regulation of ICM proliferation,
likely through a mechanism independent of β-catenin. In contrast, exogenous WNTs,
can regulate competence of the embryo to develop to the blastocyst stage, with WNT
agonists that increase intracellular β-catenin inhibiting development and WNTs like
WNT7A that do not regulate intracellular β-catenin improving competence of the embryo
to develop to the blastocyst stage.
There are two lines of evidence that endogenous WNT are not required for
development to the blastocyst stage. Competence of embryos to develop to the
110
blastocyst stage was not reduced by inhibition of secretion of endogenous WNTs
through a Wnt-C59-mediated block of PORCN. Moreover, inhibition of β-catenin
mediated WNT signaling with DKK1 did not alter the proportion of oocytes or cleaved
embryos becoming a blastocyst. In earlier studies as well, there was no effect of DKK1
on development (Xie et al., 2008b; Denicol et al., 2013a).
The lack of role for endogenous WNTs in development to the blastocyst is also
true for mouse embryos. Blastocyst formation is not impaired in either β-catenin
deficient mice (Huelsken et al., 2000) or embryos in which Wnt signaling is blocked (Xie
et al., 2008b) or in which Porcn-dependent Wnt signaling is inhibited (Biechele et al.,
2013). There was also no effect of Dkk1 on development of the mouse embryo (Xie et
al., 2008b).
Although the cow parallels the mouse with respect to the dispensability of WNT
signaling for development to the blastocyst stage, there may be divergence between
species in role of endogenous WNT in formation of the ICM. As shown here, inhibition
of PORCN increased the number of cells in the ICM of the bovine embryo, indicating
that endogenous WNTs limit the number of these cells. In contrast, numbers of ICM and
TE cells were unperturbed in Porcn-mutant mouse blastocysts (Biechele et al., 2013).
The number of cells in the ICM could be determined by the proportion of blastomeres in
the morula that remain pluripotent after differentiation of the TE or by the degree of
proliferation and apoptosis of cells in the ICM. Specific WNT can promote differentiation
(Krivega et al., 2015), and apoptosis (Famili et al., 2015) and decrease proliferation (Qin
et al., 2016).
111
Treatment of embryos with DKK1, which was shown to reduce β-catenin in the
embryo, did not affect the number of ICM or TE cells. Thus, it is likely that the
endogenous WNT regulating number of ICM cells acts through a mechanism
independent of β-catenin. There was also no effect of DKK1 on TE cell number in the
blastocyst. Additional evidence against a role for DKK1 in differentiation of the bovine
blastocyst was the finding that DKK1 reduced accumulation of YAP1, a transcription
factor important for TE formation in mouse (Nishioka et al., 2009) and did not affect the
amount of the transcription factor CDX2 that is responsible for TE differentiation
(Sakurai et al., 2016). These findings stand in contrast to earlier studies in cattle
(Denicol et al., 2014) and pigs (Lim et al., 2013) that DKK1 increases the number of TE
cells in the blastocyst. In addition, DKK1 increased expression of AMOT in bovine
morulae (Denicol et al., 2015); this gene participates in TE formation in mice (Hirate et
al., 2012). The reason for the discrepancy between current findings and earlier ones
with respect to actions of DKK1 on TE numbers is not known. Although sex can affect
response of the bovine embryo to embryokines (Siqueira and Hansen, 2016), there was
no effect of DKK1 on TE numbers in either male or female embryos.
Although embryo-derived WNT have little effect on the competence of an embryo
to become a blastocyst, activation of WNT signaling, either by treatment with AMBMP or
WNT7A, can modify the proportion of embryos developing to the blastocyst stage. The
uterine endometrium expresses a wide number of WNT ligands including WNT1,
WNT5A, WNT6, WNT7A, WNT8A, WNT9A and WNT9B (Mamo et al., 2012; Tribulo et
al., 2015) and it is likely that maternally-derived WNT participate in embryonic
development. Consequences of maternal WNT signaling are likely to depend on a
112
complex array of factors including the abundance of specific WNT ligand, receptor and
co-receptor availability, and presence of WNT regulatory molecules such as DKK1 and
soluble frizzled receptors, which are also expressed in the endometrium (Tribulo et al.,
2015).
Results of the present study indicate that WNTs that increase β-catenin decrease
developmental competence because two treatments that increase cellular β-catenin,
GSK3 inhibitor and the WNT mimetic AMBMP, decreased the proportion of embryos
that developed to the blastocyst stage. In an earlier study, as well, AMBMP decreased
development of bovine embryos (Denicol et al., 2013a). Effects of AMBMP were
decreased by DKK1, which also decreased the amount of β-catenin in the embryo. This
result is an indication that actions of AMBMP involve accumulation of β-catenin and that
maternally-derived molecules like DKK1 can modify responses of the embryo to WNTs
that increase cellular β-catenin. As reported earlier (Tribulo et al., 2017), β-catenin was
not localized in the nucleus of the embryo after treatment with GSK inhibitor or AMBMP
and thus it is likely that β-catenin acts independent of a nuclear site of action.
Not all maternally-derived WNT are likely to inhibit development. Present results
indicate that WNT7A, which does not affect amounts of β-catenin in the embryo but
does decreases phosphorylation of JNK, increased the proportion of embryos that
developed to the blastocyst stage. WNT7A is not expressed in the bovine
preimplantation embryo (Denicol et al., 2013a) but is highly expressed in bovine
endometrium (Tribulo et al., 2015). Although it has been described that WNT signaling
mediated by JNK is required for cavity formation in mouse embryos (Lu et al., 2008; Xie
113
et al., 2008b), the role of WNT signaling mediated by pJNK remains unknown in the
bovine.
One of the objectives of the series of experiments documented here was to
identify downstream pathways affected by DKK1. This molecule, which can block
canonical WNT signaling by interfering with recruitment of the LRP5/6 co-receptor to the
WNT-FZD ligand receptor (Bafico et al., 2001; Nusse, 2001) maybe an important
determinant of fertility in the cow. Bovine embryos treated with DKK1 were more likely
to establish and maintain pregnancy after transfer to recipient cows than embryos not
treated with DKK1(Denicol et al., 2014). Also, expression of DKK1 in endometrium was
lower for heifers diagnosed as infertile compared to heifers considered fertile (Minten et
al., 2013) and was lower for endometrium of lactating cows than non-lactating cows
(Cerri et al., 2012).
Actions of DKK1 are complex because, in addition to interfering with WNT-FZD
signaling, DKK1 can act as a WNT agonist to activate non-canonical signaling pathways
such as WNT/PCP pathway (Caneparo et al., 2007). In the present work the ability of
DKK1 to reduce accumulation of β-catenin was documented, showing that it can reduce
β-catenin and block actions of molecules like AMBMP. Treatment with DKK1 did not
prevent AMBMP from increasing β-catenin. Thus, AMBMP acts to increase β-catenin in
the cell by acting downstream from formation of the WNT-receptor-co-receptor complex.
However, DKK1 did block actions of AMBMP on development, probably because the
total β-catenin accumulated in embryos treated with AMBMP and DKK1 in combination
was the same as for embryos treated with vehicle alone. In contrast to regulation of β-
catenin in the embryo, DKK1 had no effect on JNK phosphorylation even though DKK1
114
can activate JNK signaling in other cellular systems (Killick et al., 2014; Krause et al.,
2014). The lack of effect of DKK1 on activation of JNK in the bovine embryo may reflect
insufficient amounts of one or more molecules involved in the pathway by which DKK1
activates JNK signaling.
To our knowledge, data presented here are first description of localization of
YAP1 in the bovine embryo. This transcription factor plays an important role in TE
formation in the mouse by interacting with TEAD4 to induce transcription of CDX2. The
pattern of expression of YAP1 from the morula stage at day 5 to the blastocyst stage at
day 7 indicates that YAP1 accumulation in the nucleus precedes that of CDX2, as
revealed by higher number of YAP1+ than CDX2+ nuclei at day 5 morulae, and
presence of nuclear YAP1 in every nucleus that was CDX2+ at this developmental
stage. As embryos developed, however, the proportion of blastomeres that were YAP1+
declined and fewer nuclei were YAP1+ than were CDX2+. By the blastocyst stage, both
YAP1 and CDX2 were localized in the TE. This observation, as well as the observation
that over 90% of YAP1+ nuclei in the blastocyst were also CDX2+, is consistent with a
role of YAP1 in CDX2 expression and TE differentiation. Functional studies are needed
to determine whether or not YAP1 is required for CDX2 expression in bovine embryos.
Taken together, data suggest that embryo-derived WNTs are dispensable for
blastocyst formation in bovine embryos but do participate in formation of the ICM. In
contrast, exogenous WNTs can affect embryonic development in a positive or negative
manner depending upon nature of the WNT ligand and the downstream outcome. This
latter result implies that maternally-derived WNT could play important roles in
development of the preimplantation embryo.
115
Table 3-1. Effect of exposure of embryos to GSK3 inhibitor from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stagea
Treatment P-value Vehicle GSK3 inhibitor
Blastocysts/oocyte (%) 28.5 ± 2.3 21.9 ± 2.0 0.03 Blastocysts/cleaved embryo (%) 34.5 ± 2.7 27.4 ± 2.4 0.05 a Data are the least-squares means ± SEM of results from 5 replicates.
116
Table 3-2. Effect of treatment of embryos with the WNT agonist AMBMP and the endogenous regulator of WNT signaling, DKK1 from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stagea
Treatments Blastocysts/oocyte (%)
Blastocysts/cleaved embryo (%)
AMBMP DKK1
- - 24.3 ± 4.3 31.9 ± 5.1 + - 18.7 ± 3.6 23.0 ± 4.2 - + 18.4 ± 3.5 25.3 ± 4.5 + + 21.7 ± 4.0 28.3 ± 4.8
Statistical significance (P)
AMBMP 0.62 0.29 DKK1 0.72 0.86 Interaction 0.04 0.03 a Data are the least-squares means ± SEM of results from 7 replicates.
117
Table 3-3. Effects of inhibition of endogenous WNT signaling from day 5 to day 7 of development with either Wnt-C59 or DKK1, on ability of embryos to develop to the blastocyst stage, and cell number of blastocysts at day 7 of development.a
Treatment
Development Blastocyst cell number
TE:ICM ratio
Blastocysts/ oocyte (%)
Blastocysts/ cleaved embryo (%) Total TE ICM
Vehicle 16.6 ± 2.3 23.9 ± 3.9 133.6 ± 9.1 88.2 ± 6.1 43.3 ± 4.4- 2.2 ± 0.1 --
Wnt-C59 19.2 ± 2.2 26.4 ± 3.7 141.7 ± 8.1 89.1 ± 5.2 52.7 ± 4.1b 1.7 ± 0.1c-
DKK1 18.1 ± 2.1 26.1 ± 3.3 127.8 ± 9.0 81.2 ± 6.0 45.2 ± 4.4- 1.8 ± 0.1
a Data are the least-squares means ± SEM of results from 5 replicates. b Differs from control (P=0.02). c Differs from control (P=0.06).
118
Table 3-4. Effect of exposure of embryos to DKK1 from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stage and cell number of day 7 blastocystsa.
Treatment
Development Blastocyst cell number
TE:ICM ratio
Blastocysts/ oocyte (%)
Blastocysts/ cleaved embryo (%) Total TE ICM
Vehicle 25.1 ± 1.6 34.2 ± 2.0 129.3 ± 11.2 83.1 ± 7.6 46.6 ± 4.0 2.0 ± 0.1
DKK1 23.2 ± 1.5 31.8 ± 1.9 134.6 ± 11.6 85.9 ± 7.8 48.7 ± 4.2 1.8 ± 0.1
a Data are the least-squares means ± SEM of results from 10 replicates. There were no effects of treatment (P>0.10).
119
Table 3-5. Effect of treatment of embryos with DKK1 from day 5 to day of development on the ability of male and female embryos to develop to the blastocyst stage and cell number of day 7 blastocystsa.
Treatment Sex
Development Blastocyst cell number
TE:ICM
ratio
Blastocysts/ oocyte (%)
Blastocysts/ cleaved embryo (%) Total TE ICM
Vehicle
Female 20.6 ± 2.1 23.6 ± 2.5 148.2 ±7.1 88.9 ±4.7 59.5 ±4.1 1.7b ±0.1
Male 14.3 ± 1.9 17.1 ± 2.2 144.0 ±7.4 83.7 ±4.9 60.3 ±4.2 1.5c± 0.1
DKK1
Female 17.1 ± 2.0 20.1 ± 2.4 135.6 ±6.8 93.1 ±5.0 51.6 ±4.0 1.9b ±0.1
Male 15.9 ± 2.0 19.3 ± 2.4 153.1 ±7.6 83.6 ±4.4 60.1 ±4.3 1.6c ±0.1
a Data are the least-squares means ± SEM of results from 5 replicates. Treatment consisted of 100 ng/ml DKK1 (dickkopf-related protein 1). bc indicate tendency of sex effect on TE:ICM ratio P=0.10
120
Table 3-6. Effect of treatment of embryos with recombinant WNT7A from day 1 to day 7 of development or from day 5 to 7 of development (i.e., during the morula to blastocyst transition) on the ability of embryos to develop to the blastocyst stagea.
Timing of treatment Treatment
Development
Blastocysts/oocyte (%)
Blastocysts/cleaved embryo (%)
day 1-7 Vehicle 24.4 ± 2.2 37.5 ± 3.6 WNT7A 31.0 ± 2.4 44.2 ± 3.6
day 5 to 7 Vehicle 20.1 ± 2.0 31.7 ± 3.6 WNT7A 29.8 ± 2.3 42.8 ± 3.6
Statistical significance (P) WNT7A 0.0005 0.02 day 0.26 0.33 Interaction 0.51 0.54 a Data are the least-squares means ± SEM of results from 6 replicates.
121
Table 3-7. Effect of treatment of embryos with recombinant WNT7A from day 5 to day 7 of development on the ability of embryos to develop to the blastocyst stage and cell number of day 7 blastocystsa.
Treatment
Development Blastocyst cell number
TE:ICM ratio Blastocysts/
oocyte (%)
Blastocysts/ cleaved embryo
(%)
Total TE ICM
Vehicle 24.5 ± 1.7 32.1 ± 2.2 125.8 ±11.2 76.9 ±8.9 48.4 ±5.9 1.7 ± 0.2 WNT7A 30.7 ± 1.7 39.2 ± 2.2 129.4 ±11.6 74.1 ±8.1 56.9 ±4.6 1.5 ± 0.2 P-value 0.02 0.04 0.75 0.70 0.24 0.36 a Data are the least-squares means ± SEM of results from 4 replicates.
122
Figure 3-1. Treatment of embryos at day 5 after insemination with 100 ng/ml DKK1
reduces amounts of immunoreactive β-catenin but does not prevent the WNT agonist (AMBMP) from increasing amounts of β-catenin. A) Representative images of embryos immunolabeled for β-catenin (red) and DNA (blue). B) Quantification of intensity of β-catenin. Immunoreactive β-catenin was affected by AMBMP (***P<0.0001) and DKK1 (****P=0.0001) but not by the AMBMP by DKK1 interaction (P=0.9). Data are least-squares means ± SEM of results from 7 replicates, with a total of 165 labeled blastocyst
123
Figure 3-2. Immunolocalization of the transcription factors YAP1 and CDX2 in morulae
and blastocysts. A) Shown are representative images of individual embryos immunolabeled for YAP1 (red), CDX2 (green), and DNA (blue) at Days 5 (morulae), 6 (morulae) and 7 (blastocyst) of development. B to I) Effect of treatment of embryos with DKK1 at day 5 of development on immunoreactive YAP1 and CDX2 at Days 5 (morulae), 6 (morulae) and 7 (blastocysts) of development. Data represent the absolute B) and relative C) (percent of total cells) number of cells positive for YAP1, absolute F) and relative G) number of cells positive for nuclear CDX2. Panels D and H show quantification of dual labeling for YAP1 and CDX2 expressed relative to YAP1+ D) and CDX2+ nuclei H). Panels E and I show intensity of labeling of nuclei for YAP1 E) and CDX2 I). Data are least-squares means ± SEM of results from 5 replicates. Embryos treated with vehicle are represented by closed circles and solid lines while embryos treated with DKK1 are represented by broken lines and open circles. H). Data are least-squares means ± SEM of results from 5 replicates, with a total of 232 labeled embryos. Asterisks indicate effect of DKK1 on intensity of YAP1 immunofluorescence (P=0.04).
124
Figure 3-3. Effect of treatment of embryos with DKK1 from day 5 to day 7 of
development on accumulation of pJNK. A) Representative images of individual blastocysts immunolabeled for pJNK (red) and DNA (blue). B) Quantification of intensity of pJNK in whole embryonic area.(left pannel) Quantification of intensity of pJNK in nuclear area (right pannel). Data are least-squares means ± SEM of results from four replicates with a total of 91 labeled blastocysts.
125
Figure 3-4. Effect of treatment of embryos with WNT7A from day 5 to day 7 of
development on accumulation of β-catenin and pJNK. A) Representative images of individual blastocysts immunolabeled for β-catenin (red) and DNA (blue). B) Quantification of intensity of β-catenin in whole embryonic area. Data are least-squares means ± SEM of results from ten replicates with 239 labeled embryos. C) Representative images of individual blastocysts immunolabeled for pJNK (red) and DNA (blue). D) Quantification of intensity of pJNK in whole embryonic area. E) Quantification of intensity of pJNK in nuclear area. Data are least-squares means ± SEM
126
CHAPTER 4 CONSEQUENCES OF EXPOSURE OF EMBRYOS TO DICKKOPF-RELATED
PROTEIN 1 AND COLONY STIMULATING FACTOR 2 ON BLASTOCYST YIELD, PREGNANCY RATE, AND BIRTH WEIGHT OF THE CALF
Introduction
Embryonic development is under regulation of maternally-derived molecules
called embryokines. These molecules can affect competence of the embryo to develop
to the blastocyst stage, establish pregnancy after transfer to females and even change
postnatal phenotype. In the cow, both insulin-like growth factor-1 (IGF1) (Block et al.,
2008, 2011) and the cytokine colony stimulating factor 2 (CSF2) can increase the
percent of embryos becoming blastocyst in culture and increase the proportion of
embryos that establish pregnancy after transfer to females (de Moraes and Hansen.,
1997; Loureiro et al., 2011a, b; Denicol et al., 2014) s. In addition, while not affecting
competence to form a blastocyst, the WNT regulatory protein DKK1 can increase the
percent of embryos establishing pregnancy after transfer (Denicol et al., 2014).
Moreover, exposure of embryos to CSF2 from day 5 to 7 of culture improves postnatal
growth of the resultant calves (Kannampuzha-Francis et al., 2015).
The existence of embryokines leads to the possibility that addition of appropriate
embryokines into culture systems for in vitro produced embryos may increase the
outcomes of this increasingly-important procedure. Embryos produced in vitro have
several abnormal features compared to embryos produced in vivo including altered
gene expression (Corcoran et al., 2006; McHughes et al., 2009; Gad et al., 2012),
metabolism (Khurana and Niemann, 2000), lipid content (Crosier et al., 2000; Sudano et
al., 2012), ultrastructure (Boni et al., 1999; Rizos et al., 2002), and DNA methylation
(Niemann et al., 2010). Moreover, the pregnancy rate of embryos produced in vitro is
127
lower than for embryos produced in vivo (Lonergan et al., 2007; Pontes et al., 2009).
However, most embryos produced in vitro under commercial conditions are cultured in
medium containing fetal bovine serum (FBS) and it is possible that the existence of an
array of growth factors and other regulatory molecules in serum preclude additional
actions of exogenously added embryokines. Indeed, the beneficial effects of CSF2 on
blastocyst yield were abrogated when embryos were cultured in serum containing
medium (de Moraes and Hansen, 1997).
Here the effect of CSF2 and DKK1 alone and in combination on in vitro embryo
production in beef cattle was tested. The working hypothesis was that co-exposure of
embryos to CSF2 and DKK1 will improve development of preimplantation embryos to
the blastocyst stage and will confer greater ability to successfully establish and maintain
pregnancy. It was also hypothesized that exposure of preimplantation embryos to CSF2
in combination with DKK1 will improve birthweight of the offspring. The experiment was
performed using culture medium containing FBS to evaluate the value of adding these
embryokines under conditions commonly used in commercial in vitro fertilization (IVF)
laboratories.
Materials and Methods
Animals and Experimental Design
The experiment was conducted using 70 cows selected for oocyte collection
while housed on pasture on four commercial beef farms located near Córdoba,
Argentina (31.4201° S, 64.1888° W). Donor cows were included in the study from
August 2015 to August 2016. Donors were both heifers and multiparous cows of various
breeds including Angus (n=7), Red Angus (n=3), Bonsmara (n=14), Brahman (n=2),
128
Brangus (n=19), Red Brangus (n=8), and Braford (n=17). Body condition score at the
time of oocyte retrieval ranged between 3 and 4 (1-5 scale; Ferguson et al., 1994).
Each donor female was subjected to 1 to 4 rounds of oocyte retrieval using
transvaginal, ultrasound-guided ovum pickup procedures, referred to as ovum pick-up
(OPU). Oocytes were recovered by OPU at each farm, transported in cryotubes
(Corning, NY, USA) with maturation medium (Bioklone, Jaboticabal, SP, Brazil) and
conditioned atmosphere (5% (v/v) CO2 and 7% (v/v) O2) in an oocyte transport
incubator (Cryologic Pty Ltd., Blackburn, Victoria, Australia) and subjected to in vitro
maturation and fertilization at the IVF laboratory of the Instituto de Reproducción Animal
Córdoba (IRAC), Córdoba, Argentina. For each IVF session, spermatozoa from a single
bull were used to fertilize all viable cumulus-oocyte complexes from a single donor. Sire
(n=27 total) was selected by the cow owners. Different sires were used for individual
donors. Moreover, the same sire was not always used for an individual oocyte donor for
each OPU/IVF session. Blastocysts were then transferred to recipient cows located at
one of the four commercial farms.
The study was conducted as a randomized block design. Within each farm,
donors were randomly allocated to one of the four treatment groups (vehicle, CSF2,
DKK1, CSF2 + DKK1). Embryos from a given donor were always allocated to the same
treatment for each round of OPU.
Oocyte Retrieval
The complete procedure for OPU was performed while donor cows were under
epidural anesthesia (5-7 ml 2% (w/v) lidocaine, (Lidocaina VT, Laboratorio Vetue,
Venado Tuerto, Argentina). All visible follicles were aspirated using a transvaginal
ultrasound-guided 5 MHz microconvex probe (Mindray DP-30, Mindray Medical
129
International Ltd, Shenzhen, China) mounted to a transvaginal handle (WTA, Cravinhos,
Brazil) equipped with a disposable 18 ga x 3.81 cm vacutainer needle attached to a 50
ml Falcon® tube (Fisher Scientific, Altham, MA, USA) and a vacuum pump (SV-003,
WTA) via a clean silicon tubing. The handle-mounted-probe was positioned dorsally to
the vaginal fornix and one ovary held against the probe through rectal manipulation.
Ovarian follicles were targeted with the needle across the vaginal wall, and aspiration
was performed with a vacuum of 75-80 mm Hg, and 10-15 ml/min flow. Cumulus oocyte
complexes (COC) were collected in a phosphate-buffered saline based collection
medium (PICTOR-GEN®; Biogen Argentina SA, Córdoba, Argentina) supplemented with
heparin (10 IU/ml, Sobrius, Fada Pharma, Ciudad Autónoma de Buenos Aires,
Argentina). Immediately after OPU of both ovaries, the aspiration fluids were filtered (50
µm, Millipore, Alphaville, SP, Brazil). The COC were washed with collection medium
and COC retrieved by searching in a petri dish (Falcon) under a stereomicroscope.
Oocyte Classification, Transport and Maturation
Upon recovery, COC were classified based on appearance of the oocyte
cytoplasm and number of cumulus cell layers as described previously (Hasler et al.,
1995). Grade 1, 2 and 3 COC were considered viable, while grade 4 COC (which
ordinarily include denuded and expanded COC, as well as those with pycnotic
cytoplasm) were subdivided into two categories based on cytoplasmic appearance.
Thus, COC with pycnotic cytoplasm were discarded while COC that were either
denuded or expanded with homogenous cytoplasm were manipulated with their viable
counterparts. Viable and non-pycnotic grade 4 COC were transported in cryotubes
(Corning), containing 400 µl maturation medium (Bioklone) covered with 120 µl mineral
130
oil (Sigma, USA) with an atmosphere of 5% (v/v) CO2 and 7% (v/v) O2. Cryotubes were
prepared at the laboratory and kept at 4˚C. Immediately before OPU, cryotubes were
equilibrated in a 37˚C water bath. After transferring oocytes into the cryotubes, the
atmosphere was re-conditioned to 5% CO2 and 7% O2 and tubes were placed into an
oocyte transport incubator (Cryologic Pty Ltd.) set at 38˚C.
Upon arrival to the IVF laboratory, groups of up to 27 COC from the same donor
were transferred to equilibrated 100 µl drops of maturation medium. When the number
of COC from a donor was larger than 27, COC were split into groups of the same size.
Maturation was conducted for a total of 22 to 26 h (including transportation time) in an
incubator at 38.5˚C with an atmosphere of 5% CO2 in humidified air.
Embryo Production
Matured COC were washed in TL sperm medium (Bioklone) and placed in 50 µl
drops of IVF medium (Bioklone) covered by mineral oil, to which sperm (final
concentration, 2 x 106 cells/ml) were added. The bull used to provide sperm for each
fertilization procedure was chosen by the client. Sperm from frozen-thawed straws were
purified using Percoll before insemination. Co-culture of gametes proceeded for 24 h at
38.5˚C in a humidified atmosphere of 5% (v/v) CO2. After removal of cumulus cells by
pipetting, presumptive zygotes were placed in 100 µl oil-covered drops of culture
medium [synthetic oviduct fluid (SOF) containing FBS, Bioklone]. At 3 and 5 days after
insemination, culture medium (50 µl/drop) was replaced by the same volume of fresh
SOF medium. At day 5, the fresh medium contained either vehicle [Dulbecco’s
phosphate-buffered saline with 0.1% (w/v) bovine serum albumin (DPBS-BSA), final
concentration in the drop =0.002% (v/v)], CSF2, DKK1 or CSF2 and DKK1 at twice the
final concentration to achieve final concentrations of 10 ng/ml CSF2 and 100 ng/ml
131
DKK1 in a culture medium consisting of 0.001% (v/v) DPBS-BSA in culture medium
(Bioklone).
The source of lyophilized human recombinant DKK1 was R&D Systems
(Minneapolis, MN, USA). Protein was reconstituted using DPBS-BSA and stored at 10
µg/ml in aliquots at -20˚C until use. Lyophilized recombinant bovine CSF2 was from
Kingfisher Biotech, Inc. (Saint Paul, MN) and was described by the supplier as GM-
CSF. It was reconstituted in DPBS-BSA to 1 µg/ml and stored at -20˚C in aliquots until
use.
The proportion of cleaved zygotes was assessed 3 days after insemination.
Embryonic development was assessed 7 days after insemination using developmental
stage classifications described by the International Embryo Transfer Society
(Stringfellow and Givens, 2010). Transferable embryos had a quality score of grade 1 or
2 and one of the following stages: early blastocyst (Stage 5), blastocyst (stage 6),
expanded blastocyst (stage 7) and hatching blastocyst (stage 8). Embryos were washed
in Hepes-SOF medium (Bioklone), and individually packed in straws using Hepes-SOF
medium. Embryos were transported in an embryo transport incubator (WTA) at 38.5˚C
to the farm, which was located 2 to 15 hours from the laboratory.
Embryo Transfer and Pregnancy Diagnosis
Recipient animals were synchronized to be receptive to a day 7 blastocyst using
different hormonal treatments according to the characteristics of the herd. A total of 452
embryos were transferred including Bos taurus [Angus (n=31), Red Angus (n=34), and
Bonsmara (n=103)], Bos indicus [Brahman (n=11)], and Brahman-influenced [Brangus
(n=88), Red Brangus (n=46), Braford (n=139)] embryos. Pregnancy diagnosis was
performed by transrectal ultrasonography using a 5 MHz linear-array transducer
132
(Chison D 600 Vet; Chison Medical Imaging co., China) between day 30 and 40 of
gestation.
Birthweights of the Offspring
Birthweight data were available on a subset of 31 male and female calves from
five breeds (Angus, Red Angus, Brangus Negro, Red Brangus and Bonsmara). Weights
were measured within 72 h after birth.
Statistical Analyses
Binary responses were analyzed by multivariable logistic regression using the
GLIMMIX procedure of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) with
binomial distribution. Three response variables were investigated including proportion of
either putative or cleaved zygotes that developed to the blastocyst stage at day 7 after
insemination, and proportion of transferred blastocysts that established pregnancy. The
final statistical model for each variable was built using a backward selection method in
which variables were continuously removed when P > 0.10. Class variables tested for
effect on embryonic development and pregnancy included farm and breed of the donor.
Covariates tested for effect on embryonic development included number of retrieved
COC, number of viable COC, average quality score of COC, percent of viable COC that
cleaved. Covariates tested for effect on pregnancy also included number of blastocysts
at day 7 after insemination, percent of viable COC becoming a blastocyst on day 7, and
the percent of cleaved zygotes becoming a blastocyst on day 7. Data are presented as
least-squares means ± SEM.
Treatment effects on birthweight were determined by ANOVA using the MIXED
procedure of SAS including the fixed effects of DKK1, CSF2, interaction of DKK1 by
CSF2, sex and breed.
133
Results
In Vitro Production of Embryos
Overall, the average number of recovered COC per donor per OPU procedure
was 23.3, of those, 80.4 % were classified as viable (including grade 4 with
homogenous oocyte nucleus) and subjected to in vitro maturation. The overall
proportion of putative zygotes (i.e., those matured COC that were exposed to sperm)
that cleaved by day 3 after insemination was 59.4%. Moreover, 32% of putative zygotes
developed to the blastocyst stage by day 7 after insemination. Neither treatment nor any
covariate affected (P>0.05) any characteristic of in vitro production (Table 4-1).
Pregnancy Rate
A total of 452 embryos were transferred to recipients. Overall, the proportion of
transferred embryos that established pregnancy was 42.2%. There was no effect of
treatment on pregnancy rate (Table 4-1) and no covariate was found to affect the effect
of treatment related to pregnancy rate.
Postnatal Characteristics
In a dataset of 31 calves (n=15 males and 16 females), birthweight was affected
by the main effect of DKK1 (P=0.04), but not by CSF2 or the interaction between DKK1
and CSF2 (Figure 4-1). In particular, birthweight was lower for calves derived from
embryos treated with DKK1 from day 5 to 7 after insemination regardless of whether
CSF2 was also in the culture medium.
Discussion
Both CSF2 and DKK1 have been shown to modify embryonic development in
cattle when embryos are treated from day 5 to 7 of development (i.e., during the period
when the embryo transitions from the morula to the blastocyst stage). Treatment with
134
CSF2 increased the percent of embryos developing to the blastocyst stage, particularly
when the level of development in control embryos was low (de Moraes and Hansen,
1997; Loureiro et al., 2009). Moreover, embryos treated with CSF2 were more likely to
establish pregnancy rate after transfer to females (Loureiro et al., 2009; Denicol et al.,
2014). Treatment of embryos with DKK1 has been reported to increase subsequent
pregnancy rates following embryo transfer (Denicol et al., 2014). Despite the effects of
these two embryokines, there was no beneficial effect of either molecule on embryonic
development or post-transfer survival in the present experiment. Such a result is
interpreted to indicate that the characteristics of the embryo culture system can modify
effectiveness of specific embryo regulatory molecules in a commercial embryo transfer
production facility. Nonetheless, there were effects of one of the molecules, DKK1, on
subsequent development. In particular, calves born from embryos cultured with DKK1
were lighter at birth than embryos cultured in absence of exogenous DKK1. Thus,
actions of specific molecules on development of the preimplantation embryo can cause
long-term changes affecting postnatal phenotype.
The most likely explanation for failure to repeat effects of CSF2 and DKK1 on
embryonic development and competence for establishment of pregnancy is the
presence of FBS in the medium used to produce embryos. Addition of serum to embryo
culture medium can increase the percent of embryos advancing to a transferrable stage
of development (Van Langendonckt et al., 1997; Rizos, 2002) and is commonly added
to commercial embryo culture systems for that purpose. It is likely that CSF2 and DKK1
were ineffective at stimulating development to the blastocyst stage and increasing
pregnancy rate because other growth factors in serum were exerting similar actions on
135
the embryo. Indeed, for CSF2 beneficial effects on blastocyst yield occurred in the
absence of serum but not in the presence of 5% (v/v) bovine steer serum (de Moraes
and Hansen, 1997). The use of FBS can be associated with detrimental effects on
embryonic quality as reflected by accumulation of lipid droplets (Abe et al., 2002),
alterations in the embryonic transcriptome (Cagnone and Sirard, 2014) and
exacerbation of fetal growth leading to the large offspring syndrome reviewed by Young
et al. (1998). Perhaps discovery of additional molecules that can serve as embryokines
can lead to a replacement of serum in embryo culture medium with specific bioactive
molecules.
There are other some differences between earlier studies and the present
experiment that could be responsible for differences in results. The studies in which
effects of CSF2 and DKK1 on pregnancy rate after transfer were observed involved
dairy animals (vs. beef cattle in the present experiment), use of sexed semen to
produce predominately-female embryos (vs. conventional semen), and a source of
CSF2 different than the one for the present experiment. For CSF2, embryonic
responses vary between female and male embryos (Siqueira and Hansen, 2016) but
effects of DKK1 on morula gene expression were largely similar for female and male
embryos (Denicol et al., 2015).
In an earlier experiment, heifer calves produced from embryos exposed to CSF2
in vitro had similar birth weights as control embryos but, beginning at 4 mo of age, grew
faster than control calves (Kannampuzha-Francis et al., 2015). Similarly, there was no
effect of CSF2 on birthweight in the present experiment. Although CSF2 treatment
during the period of the morula and blastocyst stage did not have a significant effect on
136
postnatal phenotype, there was a developmental programming action of DKK1. Calves
derived from embryos treated with DKK1 were smaller at birth than calves from
embryos not exposed to DKK1. This result has several implications including the need
to understand how changes in molecular processes in the morula- and blastocyst-stage
embryo can modify development in a way that causes phenotype to be altered many
months later in postnatal life. One possibility is that DKK1 changes the embryonic
transcriptome or epigenome in ways that affect downstream cell differentiation.
Treatment of embryos with DKK1 causes small changes in the transcriptome at the
morula state (Denicol et al, 2015) as well as in trophectoderm and hypoblast formation
in one study (Denicol et al, 2014) although not in results presented above (Chapter 3).
From a practical perspective, these data provide additional evidence that
postnatal function of livestock species can potentially be modified by the environment
affecting the embryo during the preimplantation period. Such an idea is well supported
from the rodent literature (Fleming et al., 2015a, b).
Indeed, bovine embryos actively respond to the environment as early as 8-16 cell
stage of development by modifying their transcriptome (Cagnone and Sirard, 2014).
Perhaps, modification of postnatal phenotype for optimal livestock production should
consider not only genetic selection and provision of an optimal environment after birth
but should also consider how to alter the microenvironment of the preimplantation
embryo as well.
137
Table 4-1. Effect of exposure of embryos to the CSF2, DKK1 or the combination on embryonic development and pregnancy rate of cows receiving an embryoa
Treatment Embryonic development at day 7 Pregnancy rate (%)
CSF2 DKK1 Blastocysts/viable COC (%)
Blastocysts/cleaved zygote (%)
- - 26.9 ± 3.9 45.4 ± 5.2 40.8 ± 5.6 + - 33.0 ± 3.9 53.8 ± 4.5 34.7 ± 4.9 - + 25.0 ± 3.7 42.5 ±4.8 38.0 ± 5.1 + + 28.0 ± 4.3 46.5 ± 4.6 42.3 ± 4.9 Statistical significance (P) CSF2 0.18 0.20 0.85 CSF2 DKK1 0.36 0.29 0.64 DKK1 CSF2 x DKK1 0.58 0.65 0.30 CSF2 x DKK1 aData are least-squares means ± SEM from a total of 70 donors subjected to OPU on 1 to 4 occasions to yield 2339 cumulus-oocyte complexes (COC). Vehicle N=85; CSF2 N=104; DKK1 N=120; CSF2+DKK1 N=143.
138
Figure 4-1. Effect of addition of embryokines during day 5-7 after fertilization on birth
weight of the resultant calves. Data are least-squares means ± SEM from 31 calves after adjusting for calf sex and breed. Birth weight was affected by exposure to dickkopf-related protein 1 (DKK1) from day 5 to day 7 of culture (P=0.04) but not by colony-stimulating factor 2 (CSF2) or the CSF2 x DKK1 interaction.
139
CHAPTER 5 IDENTIFICATION OF POTENTIAL EMBRYOKINES IN THE BOVINE REPRODUCTIVE
TRACT
Introduction
The environment established by the mother for the preimplantation embryo plays
a key role in ensuring that development proceeds in a manner that optimizes pregnancy
success and postnatal development. Disruption of maternal physiology during the
preimplantation period can compromise embryonic survival. Examples include the effect
of establishing an abnormal ratio of estradiol to progesterone in mice (Yoshinaga and
Adams, 1966) and humans (Simon et al., 1995) and exposure of female embryo
transfer recipients to bisphenol A in mice (Xiao et al., 2011). Other conditions can
enhance maternal capacity for supporting development, as shown in cattle for treatment
with somatotropin (Moreira et al., 2002). The maternal environment during the
preimplantation period can also alter the developmental program of the embryo in a
manner that alters postnatal phenotype (review by Hansen et al., 2016). For example,
feeding a diet low in protein during the preimplantation period modified postnatal growth
and accumulation of body fat in rodents (Fleming et al., 2015).
The importance of the maternal environment for embryonic development is
illustrated by the consequences of embryo production in vitro. In cattle, for example,
embryos produced in vitro experience altered gene expression (Corcoran et al., 2006;
McHughes et al., 2009; Gad et al., 2012), metabolism (Khurana and Niemann, 2000),
lipid content (Crosier et al., 2000; Sudano et al., 2012), ultrastructure (Boni et al., 1999;
Rizos et al., 2002), DNA methylation (Niemann et al., 2010), competence to establish
pregnancy (Lonergan et al., 2007; Pontes et al., 2009) and properties of the resultant
offspring (Fernandez-Gonzalez et al., 2004; Farin et al., 2006; Ceelen et al., 2008).
140
Transfer of embryos produced in vitro to the oviduct can mitigate at least some of these
abnormalities (Enright et al., 2000; Lazzari et al., 2010; Gad et al., 2012), indicating the
importance of the absence of maternal signals as a cause of aberrant development.
An important mechanism by which the maternal oviduct and endometrium directs
embryonic development is through secretion of regulatory molecules called
embryokines. A number of growth factors can affect embryonic development in various
species. Among the most studied embryokines are CSF2 (Sjoblom et al., 1999; Loureiro
et al., 2009; Denicol et al., 2014), IGF1 (Lin et al., 2003; Jousan and Hansen, 2007;
Bonilla et al., 2011) and LIF (Kauma and Matt, 1995; Mohamed et al., 2004; Neira et al.,
2010).
In many cases, it is unknown whether molecules that affect embryonic
development in vitro are present in the reproductive tract at times coincident with
development of the preimplantation embryos. In addition, there are likely other
regulatory molecules produced by the reproductive tract that can act on the
preimplantation embryo. Indeed, the embryo is poised to respond to a plethora of
maternal regulatory molecules because of the wide range of growth factor and hormone
receptor genes that it expresses (Graf et al., 2014; Zuo et al., 2016).
The objective of the present study was to identify potential embryokines during
the first 7 days after ovulation using the cow as a model. It is during this period that the
bovine embryo develops from the zygote to the blastocyst stage, where it spends the
first 4-5 days in the oviduct and then moves into the uterine lumen (Betteridge and
Fléchon, 1988). The approach was to collect oviductal and endometrial tissue and
determine the relative amounts of expression of genes for 93 hormones, growth factors,
141
cytokines, chemokines, and WNT-related molecules that could potentially function as
embryokines.
Materials and Methods
Synchronization of the Estrous Cycle
The reproductive status of non-lactating Holstein cows was assessed by
transrectal ultrasonography and 20 cows with a detectable corpus luteum were
subjected to a hormonal protocol to synchronize ovulation. On day -18 (day of expected
ovulation = day 0), cows were injected, i.m., with 25 mg prostaglandin F2 (PGF,
Lutalyse®, Zoetis, Florham Park, NJ, USA) followed by 100 µg gonadorelin (GnRH;
Cystorelin®, Merial Inc., Duluth, GA, USA) on day -16. A second, identical injection of
GnRH was injected on day -9 and a progesterone-containing controlled internal drug
release device (CIDR®, Zoetis) was inserted intravaginally. At day -4, each cow was
administered 25 mg PGF, i.m., and the intravaginal device was removed. Another 25
mg PGF was injected at day -3 and 100 µg GnRH was injected i.m. at day -2, i.e. 24 h
after PGF. Transrectal ultrasonography of ovaries was performed on day -4, -1 and 0 to
confirm ovulation. A total of 15 cows were successfully synchronized and slaughtered at
either day 0 (n=4), 3 (n=4), 5 (n=3) or 7 (n=4) relative to the expected day of ovulation.
Slaughter was by captive-bolt stunning and exsanguination.
Collection of Oviductal and Endometrial Tissues and Uterine Flushings
Reproductive tracts were obtained immediately after slaughter and placed on ice.
Processing of all organs was completed within a maximum of 4 h from slaughter of the
first cow. Side of the reproductive tract was identified as being ipsilateral or contralateral
to the side of ovulation. Ovulation of cows slaughtered at day 0 was confirmed by
142
absence of a preovulatory follicle in 3 of 4 cows, and the presence of an ovulatory
fossa. For the remaining cow, ovaries were lost during processing and ovulation could
not be confirmed.
After dissecting the oviduct free from the mesosalpinx, the lower third of the
oviduct, corresponding to the isthmus, was used to cut transversal 1-mm sections while
the oviduct was gently stretched. Samples were snap frozen in liquid N2 for evaluation of
gene expression. Samples were stored in liquid N2 until transport to the laboratory and
storage at -80°C.
Uterine flushings and samples of endometrium were collected separately from
both uterine horns. The mesometrium was removed and each uterine horn was
clamped near the uterine body. The end near the uterotubal junction was opened with a
0.5 cm incision and 30 ml of Dulbeccos’s phosphate-buffered saline (DPBS) at room
temperature were flushed into the uterine horn from the opposite end using an 18 ga
needle. The fluid was propelled by massage along the uterine horn through the incision.
Recovered fluid was kept on ice; after centrifugation at 3000 x g for 15 min at 4 C, the
supernatant fraction was obtained and stored at -20°C.
After flushing, each uterine horn was opened with a longitudinal incision along
the curvature. Intercaruncular regions of endometrial tissue were harvested from the
middle section of uterine horns using a scalpel and tweezers. Some samples were snap
frozen, while others were frozen in optimal cutting temperature medium (O.C.T.®,
Sakura Finetek USA Inc., Torrance, CA, USA) on dry ice covered with 2 methylbutyrate
for immunofluorescence analyses. All samples were stored in liquid N2 until transport to
the laboratory where they were stored at -80°C.
143
RNA Extraction and Gene Expression
Snap-frozen samples of endometrium and oviduct were thawed and
homogenized in lysis buffer from Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA)
using a tissue homogenizer (Tissue Master 125, OMNI International, Kennesaw, GA,
USA) for 10 sec at speed 4. Homogenized tissue in lysis buffer was transferred to the
silica columns of the Qiagen RNeasy Mini kit and RNA was extracted by following
manufacturer instructions including DNase treatment.
Abundance of specific mRNA molecules for 93 genes potentially involved in
control of embryonic development was determined using the NanoString nCounter
analysis system (NanoString Technologies®, Seattle, WA, USA) (Geiss et al., 2008).
This analytical procedure consists of gene-specific 100-mer probe pairs (i.e. one probe
that captures the target gene, and one probe that serves as reporter) that are hybridized
to the sample in solution. The reporter probe carries a fluorescent signal, and only
probe pairs that hybridize to transcripts in the sample are immobilized and captured for
data collection. Probes were designed to quantify mRNA for 28 growth factors, 12
chemokines , 22 cytokines, 3 hormones, 19 WNT ligands and 9 WNT regulatory
molecules. In addition to the 93 regulatory molecule genes, expression of an
aminopeptidase (ANPEP) was also measured as an internal control because it has
been shown to be highly expressed in bovine endometrium at days 5 and 7 after
ovulation (Forde et al., 2009). Expression of a total of 6 housekeeping genes (ACTB,
RPL19, ERK1, GAPDH, SLC30A6, SUZ12) was also assessed.
The 100-probe set was designed and synthesized by NanoString Technologies®
by identifying an optimal sequence within the target transcript that met the criteria of
144
uniqueness in the genome and mutual independence from the other probes in the set.
In addition to the customized arrays of probes, internal controls were included in the
hybridization reaction. The level of expression of these mRNA was assessed in six
multiplexed hybridization reactions that occurred in solution, post-hybridization steps
handled on a custom liquid-handling robot. Subsequently, purified reactions were
subjected to the digital analyzer that automatically acquired images and collected data
by counting the number of times the reporter probe for each gene was detected.
Data were first normalized to external RNA spike-in controls and then normalized
to housekeeping genes (ACTB, ERK1, GAPDH, RPL19, SLC30A6, SUZ12) using two
normalization factors (i.e. one to adjust for spike-in controls and one for housekeeping
genes). Each normalization factor was calculated by for each sample dividing average
geometric mean of spike-in controls or housekeeping genes for all samples by the
geometric mean for an individual sample.
Genes were considered expressed if the number of reads was greater than two
standard deviations above the mean of negative controls (i.e., 10.4). For cases in which
transcript was detectable for some samples but not others, the value used for statistical
analysis for non-detectable samples was the minimum detection level (10.4).
Data were analyzed by least-squares analysis of variance using the GLM
procedure of SAS for Windows, version 9.4 (SAS Institute Inc, Cary, NC, USA). The
statistical model for expression in endometrium included the fixed effects of day, side,
and the day by side interaction, and the random effect of cow nested within day.
Orthogonal contrasts were performed to identify the pattern of variation over days,
including linear, quadratic and cubic effects of day. Two statistical models were used for
145
analysis of variance of gene expression in oviduct. Gene expression data for tissue
ipsilateral to the side of ovulation were analyzed with day as a fixed effect and the
random effect of cow nested within day. Day effects were separated into individual
degree-of-freedom comparisons using the same contrasts described for endometrial
tissue. In a separate analysis, side was included in the statistical model as a fixed effect
along with day and day by side interaction. In this case, only changes between day 0
and day 3 of the estrous cycle were evaluated because this is period that coincides with
embryonic development in the oviduct. Data are presented as least-squares means ±
standard error of the mean.
Immunofluorescence
Frozen tissue was processed on a Microm HM550 cryostatic microtome
(ThermoFisher Scientific Inc., Waltham, MA, USA) to obtain 4 µm sections that were
mounted on Superfrost plus® slides (Fisher Scientific, Suwanee, GA, USA) and kept at -
80°C. Slides were fixed in ice- cold acetone for 10 min and allowed to dry for 1 h at
room temperature. Re-hydration was performed using Tris-buffered saline (TBS; 20 mM
Tris-HCl, pH 7.5 containing 136.9 mM NaCl) for 20 min, followed by 1 h blocking with
the same buffer containing either 10% (v/v) goat serum (Millipore, Billerica, MA, USA)
for WNT5A, WNT7A and CSF2 or 10% (v/v) horse serum (Atlanta Biologicals, Flowery
Branch, GA, USA) for DKK1.
Primary antibodies included rabbit polyclonal antibodies against human WNT5A
and human WNT7A (Abcam, Cambridge, MA, USA; 98.4 and 98.6% predicted amino
acid sequence identity with bovine WNT5A and WNT7A, respectively), a mouse IgG1
monoclonal antibody against bovine CSF2 (GM-CSF 17.2 IgG1, Washington State
University Monoclonal Antibody Center, Pullman, WA, USA); and goat polyclonal anti-
146
human DKK1 antibody (R&D Systems Minneapolis, MN, USA; 90.5% predicted amino
acid sequence identity with bovine DKK1).
All antibodies were diluted with TBS containing 1% (v/v) goat serum, except for
anti-human DKK1, which was diluted in TBS. Concentrations were 5 µg/ml, 10 µg/ml, 10
µg/ml and 0.2 µg/ml for anti WNT5A, WNT7A, CSF2, and DKK1, respectively. As a
negative control, primary antibody was substituted with IgG of the species
corresponding to the primary antibody. Incubation with primary antibodies proceeded
overnight at 4°C. Sections were then washed three times for 5 min using TBS
containing 1% (v/v) goat serum, except for anti-human DKK1, which was washed with
TBS. Secondary antibodies were diluted to a concentration of 6.66 µg/ml and incubated
with tissue at room temperature for 1 h. A goat anti-rabbit antibody conjugated to Alexa
555 was used for WNT5A, a goat anti-rabbit antibody conjugated to Alexa 488 was
used for WNT7A, a goat anti-mouse antibody conjugated to Alexa 488 was used for
CSF2, and a rabbit anti goat antibody conjugated to Alexa 488 was used for DKK1. All
secondary antibodies were from Life Technologies (Carlsbad, CA, USA). Following
labeling with second antibody and washing of sections with TBS, sections were
incubated with 1 µg/ml Hoechst 33342 for 5 min to label nuclei.
Slides were mounted using SlowFade Gold antifade reagent (Life Technologies),
covered with a coverslip, and observed with a 40x objective using a Zeiss Axioplan 2
epifluorescence microscope (Zeiss, Göttingen, Germany) and Zeiss filter sets 02 (4,6-
diamidino-2-phenylindole), 03 (fluorescein isothiocyanate filter), and 04 (rhodamine).
Digital images were acquired using AxioVision software (Zeiss) and a high-resolution
black and white Zeiss AxioCam MRm digital camera.
147
Western Blotting for CSF2
All reagents were purchased from Fisher Scientific® unless otherwise stated.
Uterine flushings were thawed and 1 ml aliquots were precipitated with ice-cold
acetone (1:4 dilution, v:v) for 60 min at -20˚C. After centrifugation at 13,000 x g for 10
min, pellets were allowed to dry and then reconstituted with double distilled water.
Samples were mixed with Laemmli buffer, heated for 5 min at 95˚C, and stored at -80˚C
until analysis. Equal volumes were loaded into precast polyacrylamide gels (Mini-
PROTEAN TGX® 4-15% polyacrylamide; Bio-Rad, Hercules, CA, USA) and separated
by sodium dodecyl sulfate, polyacrylamide gel electrophoresis at 110 V for 60 min.
Proteins were then transferred to a nitrocellulose membrane (Hybond ECL®, GE
Healthcare Life Sciences, Pittsburgh, PA, USA) in a wet system using Tris-glycine
transfer buffer [0.06 % (w/v) Tris base, 2.88 % (w/v) glycine, 0.01 % (w/v) sodium
dodecyl sulfate, 20 % (v/v) methanol]. Protein transfer proceeded for 60 min at 65 V at
4°C. After 1 h incubation with blocking buffer (StartingBlock®, Thermo Scientific), blots
were probed with 1 µg/ml mouse IgG1 monoclonal antibody against bovine CSF2 (GM-
CSF 17.2 IgG1, VMRD) diluted in blocking buffer containing 0.2% (v/v) Tween 20 and
rocked overnight at 4°C. As a negative control, other blots were probed with an
equivalent concentration of mouse IgG1. After washing, membranes were incubated
with 0.1 µg/ml goat anti-mouse IRDye® 800CW conjugated anti-IgG antibody (LI-COR®
Biosciences, Lincoln, NE, USA) for 1 h in the dark at 4°C. Bands were visualized using
Odyssey® Infrared Imaging System (LI-COR® Biosciences).
148
Results
Expression of Putative Embryokines Expressed in Oviduct
Levels of expression of the 93 evaluated genes at each day of the estrous cycle
are shown in Table 5-1 and the 50-highest expressed on each day are presented in
Figure 5-1. Overall, there was wide variation in the magnitude of expression among
genes. Average reads varied from 13,625 to 16 for the 50 most-expressed genes. Of
the 93 genes evaluated, all were expressed in the oviduct ipsilateral to the side of
ovulation on day 0 and day 3 of the estrous cycle. By day 5, however, only 71 genes
were expressed. Those genes whose transcripts were not detectable were AMH,
BMP15, CCL4, DKK4, IFNB1, IL2, IL3, IL4, IL5, IL13, IL17A, IL21, SFRP5, TSHB,
WNT1, WNT3, WNT3A, WNT7B, WNT8A, WNT8B, WNT9B, and WNT10B. Transcripts
for these genes were also non-detectable at day 7. Moreover, 10 other genes were not
expressed at day 7 (FGF11, FGF1, GDF9, IL10, IL1B, INHBA, PGF, WNT7A, WNT10A,
WNT11). The 10 most highly-expressed genes in descending magnitude were CXCL12,
CTGF, IGF2, SFRP2, WNT5A, IK, CXCL10, CXCL3, HDGFRP2 and HDGF at day 0;
CXCL12, CTGF, IGF2, IK, IGF1, HDGF, WNT5A, CXCL3, CXCL10 and CCL14 at day
3; CTGF, GRO1, CXCL3, WNT5A, SFRP1 and IL8,. DKK3, CXCL12, IK, and HDGF at
day 5; and IK, CTGF, HDGF, CXCL12, HDGFRP2, CXCL3, GRO1, VEGFA, IGF2, and
VEGFB at day 7 after ovulation. Of these, CTGF, CXCL3, and IK were among the 10
most abundant transcripts at each day.
A total of 21 genes were significantly affected by day of the estrous cycle (Table
5-2). Of these genes, 11 genes were most highly expressed at estrus (CCL21, CTGF,
CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), one at day 3
149
(WNT4), 8 at day 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and
1 at day 7 (IK).
Further analysis was conducted to determine if gene expression was affected by
side of the oviduct relative to ovulation during the first three days of the estrous cycle
when the embryo is resident within the oviduct. There was an effect of side of the
oviduct for only 5 genes (DKK2, IL10, IL13, TGFB3, and WNT4). In each case,
expression was higher for the oviduct ipsilateral to the side of ovulation than for the
oviduct contralateral to the side of ovulation (Table 5-3). For 9 genes (BMP4, CXCL12,
CXCL16, FGF10, GRO1, HDGFRP2, IL34, SFRP2, and VEGFB), the effect of side of
ovulation depended on the day of the estrous cycle (Table 5-4). All but 3 of these genes
(BMP4, GRO1 and SFRP2) were more abundant in the oviduct ipsilateral to the side of
ovulation at day 0 whereas side had no effect at day 3. Expression of BMP4, GRO1 and
SFRP2 was higher for the ipsilateral side at day 0 but lower for the ipsilateral side at day
3.
Expression of Putative Embryokines Expressed in Endometrium
Data for all 93 evaluated genes are summarized in Table 5-5 and the transcript
abundance for the top 50 expressed genes at each day of the estrous cycle are
displayed in Figure 5-2. Overall, there was wide variation in the magnitude of gene
expression. For example, average reads varied from 26609 to 442 for the top 50 most
expressed genes across days.
All genes were expressed at day 0, 3 and 7 of the estrous cycle, but only 69 of
the 93 genes were expressed on day 5. Genes whose transcripts were not detected at
day 5 were AMH, BMP15, CCL21, CCL26, DKK4, FGF14, GDF9, IL2, IL3, IL4, IL5,
150
IL10, IL13, IL21, SFRP5, TSHB, WNT1, WNT2B, WNT3, WNT7B, WNT8A, WNT8B,
WNT9B,and WNT10B.
The 10 most highly expressed genes in descending magnitude were CTGF,
CXCL12, WNT5A, WNT6, IGF2, CXCL3, WIF1, HDGF, IK, and VEGFA at day 0;
WNT5A, CTGF, CXCL10, HDGF, CXCL3, IK, IGF2, CXCL12, VEGFA, and SFRP1 at
day 3; WNT5A, TDGF1, CXCL3, CTGF, SFRP1, GRO1, IK, HDGF, IGF1, and VEGFA
at day 5; and TDGF, WNT5A, CTGF, VEGFB, IK, HDGF, SFRP1, VEGFA, HDGFRP2
and WNT7A at day 7 after ovulation. A total of 5 genes (CTGF, HDGF, IK, VEGFA and
WNT5A) were among the ten highest expressed genes at each day of the estrous cycle.
day of the estrous cycle affected expression of 34 genes (Table 5-6). Of these,
10 were most highly expressed at day 0 (BMP7, CCL14, CCL21, CCL26, CTGF,
CXCL12, IGF2, IL33, SFRP2, and WIF1), 2 at day 3 (HDGF, IL15), 16 at day 5 (CSF2,
CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL6, IL8, SFRP1, SFRP4,
TDGF1, WNT16, and WNT5A) and six at day 7 (CXCL16, FGF13, HDGFRP2, VEGFB,
WNT7A and WNT11). Only one gene (IK) was differentially expressed between uterine
horns, being higher for endometrium ipsilateral to the side of ovulation than for
endometrium contralateral to the side of ovulation (900 ± 42 vs 788 ± 43). There were
also significant interactions between day and side for 6 genes (Table 5-7). Expression
of each of these 6 genes was higher for endometrium contralateral to the side of
ovulation at day 0 but expression was either higher for the contralateral side or not
different between sides at other days.
Immunolocalization of Selected Embryokines within Endometrium
Protein localization in the endometrium was evaluated for two known
embryokines (CSF2 and DKK1) and two putative embryokines that were highly
151
expressed in endometrium (WNT5A and WNT7A). Immunofluorescence was conducted
using tissue from the day of the estrous cycle at which gene expression was highest,
i.e, day 5 for CSF2, 7 for DKK1, 5 WNT5A, and 7 for WNT7A.
Both CSF2 (Figure 5-3) and WNT5A (Figure 5-3) were localized to the luminal
epithelium, glandular epithelium and stroma. DKK1 was localized to endometrial stromal
cells but was not detectable in glandular or luminal epithelium (Figure 5-3). In contrast
to the other proteins, immunoreactive WNT7A was localized to the nucleus in all three
major compartments of the endometrium. Nuclear immunolabeling was greater for
luminal and glandular epithelium than for stroma. In addition to nuclear localization,
immunoreactive WNT7A was also strongly localized to the apical domain of the luminal
epithelium only (Figure 5-4).
Accumulation of CSF2 in Uterine Flushings
Immunoreactive CSF2 was detected in uterine flushings at day 3, 5 and 7 of the
estrous cycle, and, faintly, on day 0 (Figure 5-5). On all days, there was a band of
immunoreactive protein of molecular weight (24,600). Additional lower and higher
molecular weight bands were identified at day 7. The major immunoreactive bands of
CSF2 in uterine flushings were of higher molecular weight than either of the two
immunoreactive bands identified in a preparation of recombinant CSF2 produced in
yeast (22,700 and 19,600).
Discussion
These results reveal that a large number of genes encoding for cell signaling
proteins are expressed by the oviduct and endometrium of the reproductive tract of the
cow during the first 7 days after ovulation. It is during this time when the bovine embryo
152
develops to the blastocyst stage, first in the oviduct and, after day 4-5, in the uterus
(Betteridge and Fléchon, 1988). Many of the proteins encoded by these genes are likely
to play important roles in development of the preimplantation embryo. Indeed, several
proteins whose genes were expressed by the oviduct and endometrium in this study
have been shown to modify embryonic development in the cow. These include activin A
(Trigal et al., 2011; Kannampuzha-Francis et al., 2016), BMP4 (La Rosa et al., 2011),
CSF2 (Loureiro et al., 2009; Denicol et al., 2014), CTGF (Kannampuzha-Francis et al.,
2016), DKK1 (Denicol et al., 2014), EGF (Sakagami et al., 2012), FGF2 (Fields et al.,
2011), HDGF (Gómez et al., 2014), IGF1 (Jousan and Hansen, 2007; Bonilla et al.,
2011), ILB1 (Paula-Lopes et al., 1998), LIF (Mohamed et al., 2004; Neira et al., 2010;
Mo et al., 2014), TGFβ (Neira et al., 2010), and WNT7A (Chapter 3). Other genes
expressed in the oviduct and endometrium that have been implicated in regulation of
preimplantation embryonic development in other species include NGF (Menino et al.,
1989), TGFA (Paria and Dey, 1990; Dardik et al., 1993) and WNT3A (Krivega et al.,
2015). Further studies on the effects of the proteins encoded by each of the 93 genes
expressed in the oviduct and endometrium can be useful to identify additional cell-
signaling molecules that act as embryokines.
Many of the cell signaling molecules encoded by the transcripts under
investigation may function to regulate physiology of the reproductive tract itself. The
angiogenic factor VEGFA was among the most 20 highly-expressed genes in the
oviduct and among the 10 highest expressed genes in the endometrium at each day of
the estrous cycle. Differential expression of genes involved in angiogenesis have been
related to capacity of heifers to become pregnant after embryo transfer (Ponsuksili et
153
al., 2014). In addition, various chemokine genes highly expressed in both oviduct
(including CXCL3, CXCL10, CXCL12, CCL14 and GRO1) and endometrium (CXCL3
and CXCL10). Chemokines participate in limiting bacterial infection in the reproductive
tract (Sheldon, 2015). Another highly-expressed gene in endometrium, WNT7A, is
involved in uterine gland morphogenesis (Dunlap et al., 2011).
Accumulation of transcripts does not necessarily mean that the protein is
synthesized and placed in a location where it can act on the embryo. This is particularly
a concern for genes expressed in oviduct because tissue sections analyzed included all
layers of the oviduct. Nonetheless, it is likely that accumulation of mRNA is indicative of
protein synthesis in many cases. Indeed, all the proteins examined
immunohistochemically - CSF2, DKK1, WNT5A, and WNT7A - were present in the
endometrium. Moreover, CSF2 was secreted into the uterine lumen as indicated by the
presence of immunoreactive protein in uterine flushings.
The main form of CSF2 detected in uterine flushes was of larger molecular
weight (24,600) than the two immunoreactive bands in recombinant bovine CSF2
(22,700 and 19,600). Although the recombinant CSF2 used was produced in yeast, and
is subject to posttranslational modifications, it is likely that CSF2 is more glycosylated.
Multiple forms of CSF2 have previously been reported in bovine uterine fluid (de Moraes
et al., 1999) and by lymphocytes (Cebon et al., 1990).
Interestingly, WNT7A was localized to the nucleus of cells of the three
endometrial compartments as well as to the apical domain of the luminal epithelium. It is
likely that WNT7A has a dual function within the reproductive tract since it can increase
the competence of bovine embryos to develop to the blastocyst stage (Chapter 3), and
154
regulate proliferation of endometrial epithelium in mouse (Dunlap et al., 2011). To the
best of our knowledge, nuclear localization of WNT has not been documented before.
Secretion of WNT requires palmitoylation (Willert et al., 2002) and it is possible that the
resultant increase in hydrophobicity could lead to localization to the nucleus.
There was a great deal of variation in transcript abundance among genes. It
would be overly simplistic, however, to assume that genes that are most highly
expressed are more important for embryonic development. For cell-signaling molecules,
active concentrations required for receptor binding are usually low, in the nanomolar
range (for example, Kannampuzha-Francis et al., 2016). Endometrial expression of two
well-characterized embryokines, CSF2 and DKK1, was low compared to many other
genes. Nonetheless, transcript abundance was sufficient to allow production of
detectable amounts of immunoreactive protein in the endometrium.
Examination of cyclic changes in gene expression in both oviduct and
endometrium is consistent for a role of ovarian steroids in regulation of some of the
genes examined. In oviduct, all 93 genes were expressed at day 0 and day 3 of the
estrous cycle but only 71 were expressed by day 5 and 61 by day 7. Of the genes
significantly affected by day of the estrous cycle, 11 were most highly expressed at day
0, when concentrations of estradiol were high and those of progesterone low; only one
was most highly expressed at day 7. For the endometrium, some genes were
upregulated at day 0 whereas another set was upregulated at day 7, when estradiol
concentrations would be low and progesterone concentrations elevated. While all genes
were expressed at day 0, 3 and 7 of the estrous cycle, only 69 of the 93 genes were
expressed on day 5. Of genes significantly affected by day of the estrous cycle, 10 were
155
most highly expressed at day 0 and another 22 at either day 5 of 7. In other studies on
changes in oviductal and endometrial gene expression during the estrous cycle of the
cow, there was a fraction of genes differentially expressed between estrus and diestrus
(Bauersachs et al., 2004, 2005; Mitko et al., 2008). Steroids play a key role on the
regulation of the reproductive tract. Exogenous supplementation of estradiol and
progesterone during early stages of the estrous cycle alters the transcriptome of oviduct
and endometrium (Groothuis et al., 2007; Forde et al., 2010).
Early studies indicated that side of the reproductive tract relative to ovulation had
an effect on secretion of proteins by oviduct and endometrium (Malayer et al., 1988;
Williams et al., 1992). Such a result is consistent with local regulation of the
reproductive tract by the preovulatory follicle or incipient corpus luteum. In contrast,
expression of few of the genes in the present experiment was affected by side of the
reproductive tract. Only 14 genes were differentially expressed between oviducts
ipsilateral and contralateral to ovulation, with the majority being upregulated in the
ipsilateral oviduct. Moreover, expression of only one gene in the endometrium differed
between uterine horns. Consistent with our findings was the general lack of effect of
side of the reproductive tract on the endometrial transcriptome (Bauersachs et al.,
2005). Earlier studies (Malayer et al., 1988; Williams et al., 1992) were based on
examination of protein secretion and an effect of side of the reproductive tract on post-
transcriptional mechanisms cannot be discounted.
Results indicate that the oviduct and endometrium express a wide range of cell-
signaling genes that have the potential to participate in regulation of development of the
preimplantation embryo. Expression of many of these genes varies with stage of the
156
estrous cycle, suggesting importance of both estradiol and progesterone in regulation of
gene expression.
157
Table 5-1. Least-squares means for expression of 93 genes in oviduct ipsilateral to the side of ovulation during the first seven days of the estrous cycle
Gene Normalized number of transcripts
day 0 day 3 day 5 day 7 AMH 28.5 30.6 10.4 10.4 ANPEP 332.2 146.8 224.9 508.7 BMP15 51.7 105.8 10.4 10.4 BMP2 109.1 35.7 55.0 17.9 BMP4 191.2 114.3 141.3 130.8 BMP7 148.0 82.4 151.1 57.0 CCL14 572.5 412.5 215.7 94.9 CCL21 192.4 99.7 24.0 36.9 CCL26 103.7 99.2 11.2 10.5 CCL4 30.5 26.6 10.4 10.4 CSF2 28.5 27.0 489.8 20.1 CTGF 3122.7 1973.0 13624.6 1019.9 CX3CL1 137.9 164.9 124.5 189.4 CXCL10 799.5 434.9 248.8 198.4 CXCL12 12385.4 3085.0 1010.0 773.8 CXCL16 183.5 75.5 125.7 65.7 CXCL3 794.6 442.0 9233.1 471.1 DKK1 33.8 27.0 20.2 10.5 DKK2 121.3 69.9 14.8 17.9 DKK3 244.4 157.9 1015.4 62.2 DKK4 121.4 38.7 10.4 10.4 EGF 52.1 36.3 17.7 18.8 FGF1 33.4 30.2 14.5 33.2 FGF10 172.5 70.0 83.9 41.2 FGF11 28.5 27.0 13.0 10.4 FGF12 55.3 32.1 23.9 10.4 FGF13 109.5 62.9 60.6 38.4 FGF14 77.0 47.6 39.5 23.5 FGF2 344.5 277.9 390.1 248.8 FIGF 68.2 65.9 12.5 121.3 GDF9 28.5 26.6 10.7 10.4 GRO1 202.0 78.7 10181.1 427.8 HDGF 681.6 640.8 826.8 948.9 HDGFRP2 772.8 273.8 377.1 489.1 HDGFRP3 190.4 162.5 132.3 151.2 HGF 160.2 69.9 166.2 37.9 IFNB1 75.4 62.7 10.4 10.4 IGF1 654.7 862.0 344.7 149.9 IGF2 1616.2 1043.4 654.7 318.3 IK 844.7 888.1 975.5 1314.4
158
Table 5-1. Continued
Gene Normalized number of transcripts
day 0 day 3 day 5 day 7
IL10 53.7 37.9 25.7 10.4 IL12A 28.5 50.2 58.5 11.6 IL12B 84.4 72.2 58.3 42.6 IL13 51.7 40.8 10.4 10.4 IL15 41.0 48.5 40.9 40.0 IL16 77.9 110.5 76.3 71.9 IL17A 51.7 44.5 10.4 10.4 IL18 226.8 205.8 114.1 78.4 IL1A 28.5 27.0 347.5 13.7 IL1B 52.1 26.6 52.4 10.4 IL2 51.7 62.7 10.4 10.4 IL21 28.5 44.9 10.4 10.4 IL3 28.5 26.6 10.4 10.4 IL33 545.0 359.9 114.2 77.4 IL34 120.8 37.9 46.4 16.9 IL4 28.5 26.6 10.4 10.4 IL5 28.5 44.5 10.4 10.4 IL6 31.7 60.1 98.2 13.7 IL7 98.2 52.9 13.0 11.6 IL8 28.5 44.5 1084.1 48.7 INHBA 53.3 30.0 34.0 10.4 NGF 36.6 67.8 11.0 16.8 PGF 61.4 35.6 29.5 10.4 SFRP1 528.2 363.7 1150.9 128.8 SFRP2 991.3 284.5 131.5 84.5 SFRP4 108.8 51.0 42.3 11.6 SFRP5 75.4 30.6 10.4 10.4 TDGF1 28.5 193.6 152.7 18.7 TGFB1 131.9 69.9 144.6 59.7 TGFB3 175.8 98.7 184.5 61.1 TSHB 28.5 26.6 10.4 10.4 VEGFA 628.2 403.3 462.4 394.1 VEGFB 472.9 312.5 302.3 308.3 WIF1 369.9 256.0 605.5 48.3 WNT1 51.7 44.5 10.4 10.4 WNT10A 28.5 48.5 10.7 10.4 WNT10B 28.5 26.6 10.4 10.4 WNT11 28.9 50.0 14.4 10.4 WNT16 37.8 76.8 79.2 77.8 WNT2 28.5 39.0 338.4 15.8 WNT2B 55.3 53.2 17.1 12.0 WNT3 28.5 26.6 10.4 10.4 WNT3A 28.5 71.5 10.4 10.4
159
Table 5-1. Continued
Gene
Normalized number of transcripts
day 0 day 0 day 0 day 0
WNT4 61.0 76.2 30.4 55.0 WNT5A 916.4 622.2 1341.3 273.9 WNT5B 28.5 28.5 28.8 49.3 WNT6 240.4 236.3 47.6 23.1 WNT7A 28.5 62.3 24.2 10.4 WNT7B 51.7 73.5 10.4 10.4 WNT8A 28.5 47.8 10.4 10.4 WNT8B 28.5 28.5 10.4 10.4 WNT9A 30.1 30.6 18.5 11.6 WNT9B 74.9 26.6 10.4 10.4
160
Table 5-2. Genes whose expression in the oviduct ipsilateral to the side of ovulation was affected by day of the estrous cycle within the first 7 days after ovulation a
Gene
Normalized number of transcripts P-value
day 0 day 3 day 5 day 7 linear quadratic cubic
BMP7 148 ± 61 82 ± 43 151 ± 61 57 ± 61 0.011 - -
CCL21 192 ± 57 100 ± 41 24 ± 57 37 ± 57 0.076 - -
CTGF 3123 ± 1760 1973 ± 1244 13625 ± 1760 1020 ± 1760 - 0.013 0.002
CXCL10 799 ± 193 435 ± 136 249 ± 193 198 ± 193 0.058 - -
CXCL16 184 ± 33 76 ± 23 126 ± 33 66 ± 33 0.080 - 0.082
DKK3 244 ± 355 158 ± 251 1015 ± 355 62 ± 355 - - 0.096
FGF10 173 ± 33 70 ± 23 84 ± 33 41 ± 33 0.040 - -
HGF 160 ± 52 70 ± 37 166 ± 52 38 ± 52 - - 0.093
IK 845 ± 150 888 ± 106 976 ± 150 1314 ± 150 0.065 - -
IL18 227 ± 59 206 ± 42 114 ± 59 78 ± 59 0.084 - -
IL33 545 ± 69 360 ± 49 114 ± 69 77 ± 69 0.002 - -
IL34 121 ± 33 38 ± 23 46 ± 33 17 ± 33 0.082 - -
IL6 32 ± 29 60 ± 21 98 ± 29 14 ± 29 - 0.086 -
PGF 61 ± 18 36 ± 13 29 ± 18 10 ± 18 0.092 - -
SFRP1 528 ± 350 364 ± 248 1151 ± 350 129 ± 350 - 0.092 -
SFRP2 991 ± 216 284 ± 153 131 ± 216 85 ± 216 0.024 - -
TGFB1 132 ± 30 70 ± 21 145 ± 30 60 ± 30 - - 0.046
WIF1 370 ± 77 256 ± 55 605 ± 77 48 ± 77 - 0.022 0.004
WNT2 28 ± 108 39 ± 77 338 ± 108 16 ± 108 - - 0.076
WNT4 61 ± 16 76 ± 11 30 ± 16 55 ± 16 - - 0.077
WNT5A 916 ± 355 622 ± 251 1341 ± 355 274 ± 355 - - 0.092 a Data are least-squares means ± SEM. -) indicates P > 0.1
161
Table 5-3. Genes whose expression in the oviduct was differentially expressed between sides ipsilateral and contralateral to the side of ovulation within the first three days after ovulationa
Gene
Normalized number of transcripts
P-value Ipsilateral Contralateral
DKK2 96 ± 24 28 ± 19 0.052 IL10 46 ± 12 17 ± 10 0.093 IL13 46 ± 12 17 ± 10 0.082 TGFB3 137 ± 22 61 ± 18 0.025 WNT4 69 ± 08 41 ± 07 0.024 a Data are least-squares means ± SEM .
162
Table 5-4. Genes differentially expressed in oviduct ipsi and contralateral to the side of ovulation that vary during the first 3 days after ovulationa
Gene
Normalized number of transcripts
P-value
day 0 day 3
Ipsilateral Contralateral Ipsilateral Contralateral
BMP4 191 ± 30 93 ± 21 114 ± 21 196 ± 21 0.004 CXCL12 12385 ± 3463 1988 ± 2449 3085 ± 2449 4124 ± 2449 0.063 CXCL16 184 ± 22 74 ± 15 76 ± 15 75 ± 15 0.010 FGF10 173 ± 31 56 ± 22 70 ± 22 73 ± 22 0.037 GRO1 202 ± 61 74 ± 43 79 ± 43 254 ± 43 0.010 HDGFRP2 773 ± 140 327 ± 99 274 ± 99 511 ± 99 0.011 IL34 121 ± 26 25 ± 18 38 ± 18 16 ± 18 0.095 SFRP2 991 ± 257 280 ± 181 284 ± 181 469 ± 181 0.052 VEGFB 473 ± 60 253 ± 42 313 ± 42 390 ± 42 0.010 a Data are least-squares means ± SEM for effect of day by side interaction.
163
Table 5-5. Least-squares means for expression of 93 genes in endometrium during the first seven days of the estrous cycle averaged from both sides of the reproductive tract.
Gene Normalized number of transcripts
day 0 day 3 day 5 day 7
AMH 26.1 13.0 10.4 12.2 ANPEP 136.0 188.1 1945.9 14294.3 BMP15 59.5 13.0 10.4 19.4 BMP2 107.1 21.1 25.9 37.5 BMP4 224.2 104.9 178.0 93.0 BMP7 184.0 129.8 81.5 27.6 CCL14 226.6 170.4 137.7 89.7 CCL21 78.0 17.0 10.4 12.2 CCL26 40.1 17.0 10.4 12.2 CCL4 59.5 17.0 10.6 12.2 CSF2 42.7 13.0 85.4 12.2 CTGF 3309.5 2297.3 2038.2 1461.7 CX3CL1 97.6 65.9 214.6 25.0 CXCL10 243.8 1178.2 234.3 91.2 CXCL12 2079.0 701.1 603.9 212.8 CXCL16 106.8 139.7 167.0 349.3 CXCL3 950.9 895.2 3559.6 98.4 DKK1 49.0 19.4 71.4 82.0 DKK2 36.2 13.0 10.4 12.2 DKK3 513.6 335.0 630.0 189.6 DKK4 161.0 17.0 10.4 15.8 EGF 30.2 24.2 15.6 16.1 FGF1 33.2 51.1 88.7 61.9 FGF10 169.1 62.1 67.3 42.3 FGF11 25.9 19.8 12.5 32.4 FGF12 53.2 25.8 67.1 49.6 FGF13 77.3 41.4 30.4 105.6 FGF14 46.8 20.9 10.4 12.2 FGF2 186.4 95.0 272.9 62.6 FIGF 79.3 14.6 11.2 16.5 GDF9 25.9 13.0 10.4 12.2 GRO1 474.1 171.2 985.6 31.0 HDGF 825.0 1103.9 827.1 742.9 HDGFRP2 451.7 373.8 369.6 472.2 HDGFRP3 162.2 173.7 172.2 140.9 HGF 137.3 65.1 165.2 55.3 IFNB1 177.2 17.0 11.3 22.8 IGF1 390.7 305.6 759.4 415.3 IGF2 1015.4 709.8 606.2 424.1 IK 768.2 842.1 842.8 923.5 IL10 45.3 24.8 10.4 12.2
164
Table 5-5. Continued
Gene Normalized number of transcripts
day 0 day 3 day 5 day 7
IL12A 88.4 20.9 20.5 13.3 IL12B 100.2 52.4 49.3 56.6 IL13 59.5 20.9 10.4 12.2 IL15 53.0 107.0 53.1 19.0 IL16 59.7 47.8 56.7 35.1 IL17A 76.4 20.9 73.9 12.2 IL18 172.0 125.4 107.4 56.1 IL1A 45.3 17.0 46.1 12.5 IL1B 33.2 13.6 332.6 12.2 IL2 42.7 13.0 10.4 12.2 IL21 42.7 13.0 10.4 12.2 IL3 25.9 13.0 10.4 12.2 IL33 178.2 109.1 99.2 37.8 IL34 39.8 15.6 21.2 19.0 IL4 25.9 20.9 10.4 12.2 IL5 59.5 20.9 10.4 12.2 IL6 28.6 13.0 177.3 19.3 IL7 76.4 17.0 11.0 12.2 IL8 46.7 16.5 487.2 15.8 INHBA 49.7 18.4 13.1 12.3 NGF 28.4 13.0 19.6 232.3 PGF 36.0 71.4 95.6 72.3 SFRP1 513.2 553.4 1515.5 676.8 SFRP2 195.4 27.5 22.1 21.7 SFRP4 46.5 26.5 169.4 53.1 SFRP5 50.7 13.0 10.4 12.7 TDGF1 158.6 286.9 3653.5 26609.5 TGFB1 196.3 89.7 184.1 61.4 TGFB3 135.5 125.8 125.2 23.8 TSHB 25.9 13.0 10.4 12.2 VEGFA 542.6 699.8 694.4 648.8 VEGFB 442.4 396.3 379.9 1160.3 WIF1 831.7 202.4 121.6 129.3 WNT1 93.2 20.9 10.4 19.3 WNT10A 59.7 29.1 15.9 19.3 WNT10B 25.9 20.9 10.4 15.8 WNT11 80.7 14.5 44.5 213.9 WNT16 128.5 81.0 177.5 83.0 WNT2 190.5 108.9 165.8 27.7 WNT2B 26.9 15.9 10.4 12.2 WNT3 25.9 13.0 10.4 12.2 WNT3A 76.4 13.0 10.5 40.6 WNT4 30.3 24.4 26.5 12.2
165
Table 5-5. Continued
Gene Normalized number of transcripts
day 0 day 3 day 5 day 7
WNT5A 1624.9 3998.4 4184.9 2083.4 WNT5B 36.5 15.7 28.0 21.8 WNT6 1150.7 79.7 106.9 89.3 WNT7A 36.2 161.2 191.5 442.8 WNT7B 59.5 20.9 10.4 19.3 WNT8A 25.9 20.9 10.4 15.8 WNT8B 127.0 13.0 10.4 12.2 WNT9A 28.6 14.2 12.5 18.5 WNT9B 59.5 13.0 10.4 12.2
166
Table 5-6. Genes whose expression in the endometrium was affected by day of the estrous cycle within the first 7 days after ovulation a
Gene
Normalized number of transcripts P-values
day 0 day 3 day 5 day 7 linear quadratic cubic BMP7 184 ± 27 130 ± 25 82 ± 27 28 ± 27 0.00 - - CCL14 227 ± 49 170 ± 46 138 ± 49 90 ± 49 0.06 - - CCL21 78 ± 23 17 ± 22 10 ± 23 12 ± 23 0.06 - - CCL26 40 ± 11 17 ± 11 10 ± 11 12 ± 11 0.09 - - CSF2 43 ± 32 13 ± 30 85 ± 32 12 ± 32 - - 0.09 CTGF 3310±705 2297±652 2038±705 1462 ± 705 0.08 - - CX3CL1 98 ± 68 66 ± 63 215 ± 68 25 ± 68 - - 0.09 CXCL3 951 ± 986 895 ± 913 3560±986 98 ± 986 - 0.09 0.05 CXCL12 2079±686 701 ± 635 604 ± 686 213 ± 686 0.08 - - CXCL16 107 ± 28 140 ± 26 167 ± 28 349 ± 28 <.0001 0.01 - FGF1 33 ± 15 51 ± 13 89 ± 15 62 ± 15 0.07 - - FGF2 186 ± 68 95 ± 63 273 ± 68 63 ± 68 - - 0.04 FGF13 77 ± 8 41 ± 8 30 ± 8 106 ± 8 0.06 <.0001 - GRO1 474 ± 295 171 ± 273 986 ± 295 31 ± 295 - - 0.03 HDGF 825 ± 62 1104 ± 58 827 ± 62 743 ± 62 0.07 0.01 0.01 HDGFRP2 452 ± 38 374 ± 36 370 ± 38 472 ± 38 - 0.03 - HGF 137 ± 37 65 ± 34 165 ± 37 55 ± 37 - - 0.03 IGF1 391 ± 133 306 ± 123 759 ± 133 415 ± 133 - - 0.03 IGF2 1015±212 710 ± 196 606 ± 212 424 ± 212 0.06 - - IL1B 33 ± 114 14 ± 105 333 ± 114 12 ± 114 - - 0.06 IL8 47 ± 163 17 ± 151 487 ± 163 16 ± 163 - - 0.05 IL15 53 ± 14 107 ± 14 53 ± 14 19 ± 14 0.02 0.004 0.04 IL16 60 ± 8 48 ± 8 57 ± 8 35 ± 8 - - - IL33 178 ± 44 109 ± 41 99 ± 44 38 ± 44 0.04 - - SFRP1 513 ± 188 553 ± 174 1516±188 677 ± 188 0.10 0.03 0.003 SFRP2 195 ± 68 28 ± 63 22 ± 68 22 ± 68 0.10 - - SFRP4 47 ± 52 27 ± 48 169 ± 52 53 ± 52 - - 0.07 TDGF1 159 ± 980 287 ± 907 3654±980 26610±980 <.0001 <.0001 0.00 VEGFB 442 ± 71 396 ± 66 380 ± 71 1160 ± 71 <.0001 <.0001 0.02 WIF1 832 ± 111 202 ± 102 122 ± 111 129 ± 111 0.00 0.01 - WNT5A 1625±620 3998±574 4185±620 2083 ± 620 - 0.001 - WNT7A 36 ± 22 161 ± 20 192 ± 22 443 ± 22 <.0001 0.007 0.00 WNT11 81 ± 39 15 ± 36 45 ± 39 214 ± 39 0.02 0.01 - WNT16 129 ± 39 81 ± 36 178 ± 39 83 ± 39 - - 0.06 a Data are least squares means ± SEM. -) indicate P > 0.1
167
Table 5-7. Genes whose expression was affected by the interaction between day of the estrous cycle and side of the reproductive tract relative to ovulationa
Gene
Normalized number of transcripts
P-value day 0 day 3 day 5 day 7
Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral
DKK3 222 ± 149 805 ± 129
291 ± 129 379 ± 129
814 ± 129 447 ± 149
202 ± 129 178 ± 149
0.01
IL16 33 ± 13 86 ± 11 42 ± 11 53 ± 11 64 ± 11 50 ± 13 40 ± 11 31 ± 13 0.04
IL34 18 ± 12 62 ± 10 18 ± 10 13 ± 10 25 ± 10 17 ± 12 17 ± 10 21 ± 12 0.09
NGF 16 ± 21 41 ± 18 16 ± 18 10 ± 18 19 ± 18 20 ± 21 184 ± 18 281 ± 21 0.06
WNT2 51 ± 52 330 ± 45 108 ± 45 110 ± 45 184 ± 45 148 ± 52 34 ± 45 21 ± 52 0.01
WNT5B 13 ± 12 61 ± 11 18 ± 11 13 ± 11 31 ± 11 26 ± 12 25 ± 11 19 ± 12 0.07 a Data are least-squares means ± SEM
168
Figure 5-1. Expression of the top 50 expressed genes in the oviduct at days 0, 3, 5 and
7 after ovulation. Data are least-squares means.
169
Figure 5-2. Expression of the top 50 expressed genes in the endometrium at days 0, 3, 5 and 7 after ovulation. Data are least-squares means.
170
Figure 5-3. Immunolocalization of CSF2, DKK1 and WNT5A in endometrium. DNA was labeled with Hoescht (blue). A) Representative images showing CSF2 (green) localized to luminal epithelium (LE), glandular epithelium (GE) and stroma. B) Representative images showing DKK1 (green) localized to stroma. C) Representative images showing WNT5A (red) localized to LE, GE and stroma.
171
Figure 5-4. Immunolocalization of WNT7A in endometrium. Representative images
showing WNT7A (green) localized to luminal epithelium (LE), glandular epithelium (GE) and stroma. DNA was labeled with Hoescht (blue). Scale bar 100 µm.
172
Figure 5-5. Detection of CSF2 in uterine fluid by Western blotting. Lanes samples of
uterine flushings from individual cows at day 0 (n=4), day 3 (n=4), day 5 (n=3) and day 7 (n=4) after ovulation. The location of molecular weight standards at 25 and 20 kDa is shown to the left of the blot while the migration distance of two bands in recombinant bovine CSF2 are shown by the arrows to the right of the blot.
173
CHAPTER 6 GENERAL DISCUSSION
The fate of the new embryo is problematic. In lactating dairy cows, less than 40%
of zygotes survive the embryonic period (Diskin and Morris, 2008; Hansen, 2011). The
embryo’s experience during the preimplantation period can also have long-term
consequences that extend into postnatal life (Sinclair et al., 2010; Hansen, 2015).
Intrinsic errors in embryonic development and alterations in the maternal environment
are determinants of poor competence of the embryo for development. Before the
initiation of this dissertation, there was evidence that WNT signaling regulates a number
of developmental processes but its role during preimplantation development was
unclear. The overall objective of the dissertation research was to unravel the role of
WNT signaling during preimplantation development including WNT of embryonic and
maternal origin. In addition, it was of interest to evaluate whether WNT signaling during
the preimplantation period can alter the developmental program of the embryo to
change the characteristics of the resultant calf. Results reported here lead to the
conclusion that WNT signaling is an important regulator of embryonic development
during the preimplantation period but that such signaling is distinctive in important
respects. Not only is the canonical signaling pathway unusual, with no evidence of
nuclear accumulation of is β-catenin, the most important sources of embryo-regulatory
WNT are the maternal system because inhibition of embryonic WNT secretion had no
effect on development.
The observations that 1) endogenous WNTs do not play a key role on blastocyst
formation while exogenous WNTs improve development to the blastocyst stage; 2)
several WNT-related molecules are expressed in the reproductive tract at the time of
174
preimplantation development; and 3) exogenous manipulation of WNT signaling
changes the phenotype of animals after birth; are interpreted to mean that WNT
signaling represents an important system used by the mother to regulate embryonic
development. Even maternal WNT are dispensable for blastocyst formation, as
evidenced by offspring produced by in vitro fertilization in the absence of WNT signaling
molecules. However, modulation of development by WNT is likely to have other effects
on the embryo that include actions that result in modification of the phenotype of the
offspring.
Perhaps the biggest implication of the research described here is that exogenous
regulation of WNT signaling may represent an opportunity to optimize in vitro embryo
development. Embryos produced in vitro have several abnormal features compared to
embryos produced in vivo including differences in the transcriptome (Corcoran et al.,
2006; McHughes et al., 2009; Gad et al., 2012), metabolism (Khurana and Niemann,
2000), lipid content (Crosier et al., 2000; Sudano et al., 2012), ultrastructure (Boni et al.,
1999; Rizos et al., 2002), and epigenome (Niemann et al., 2010). Moreover, the
pregnancy rate of embryos produced in vitro is lower than for embryos produced in vivo
(Lonergan et al., 2007; Pontes et al., 2009) and properties of the resultant offspring also
differ (Fernandez-Gonzalez et al., 2004; Farin et al., 2006; Ceelen et al., 2008). Results
from Chapter 2 and 3 indicate that addition of either WNT11 or WNT7A to culture
medium increased the proportion of embryos becoming a blastocyst. Any physiological
role of WNT7A, in particular, reflects actions of maternally-derived WNTs because it is
not expressed in the embryo (Chapter 2), and is highly expressed in the endometrium
(Chapter 5). Moreover, exposure of embryos to DKK1 programmed the embryo to
175
express a different phenotype after birth (Chapter 4). Therefore, there is a real need to
investigate the maternal contribution to regulation of WNT signaling in the
preimplantation embryo. It is critical, moreover, to evaluate post-natal characteristics
even in absence of visible effects during early stages of development.
An additional important implication of the current research is that dysregulation
of maternal secretion of WNTs into the reproductive tract (decreased secretion of
embryotrophic WNT like WNT7A or increased secretion of WNT that activate pathways
similar to that of AMBMP) could be a cause of infertility in cattle. Deregulation of WNT-
related molecules is associated with sub fertility in cattle (Cerri et al., 2012; Minten et
al., 2013). In particular, endometrial expression of DKK1 at day 17 of the estrous cycle
was lower for lactating versus non-lactating cows (Cerri et al., 2012). Lactation is often
considered as inducing subfertility in dairy cows (reviewed by Sartori et al., 2010;
Hansen, 2011). Similarly, expression of DKK1 was highest in fertile heifers, intermediate
in infertile heifers and lowest in sub-fertile heifers on day 14 of the estrous cycle (Minten
et al., 2013). Perhaps methods can be devised to regulate WNT signaling within the
reproductive tract to improve fertility in cattle. WNT signaling within the reproductive
tract is regulated by steroids in sheep (Satterfield et al., 2008), human (Tulac et al.,
2006) and rats (Katayama et al., 2006). In cattle, however, regulatory mechanisms for
WNTs and other secreted molecules of this pathway in the reproductive tract remain
unknown. Understanding these processes is pertinent to be able to manipulate the
maternal environment and ensure optimal conditions to the embryo.
Although some of the cell signaling molecules encoded by genes detected in
Chapter 5 function to regulate physiology of the reproductive tract itself, others play
176
important roles on regulation of the preimplantation embryo as was observed after
exposure of embryos to WNT7A and WNT11 (Chapter 2 and 3). The interplay between
ligands and regulatory molecules, in addition to the variety of outcomes upon ligand
receptor interaction, suggest the need to define the balance of WNT related molecules
secreted by maternal tissues. Consequences of maternal environment on embryonic
WNT signaling probably depend on the array of maternally-derived WNT-related
molecules as well as receptor and co-receptor availability within the embryo. Individual
WNTs preferentially stimulate canonical or non-canonical signaling depending upon
ability to bind different receptors. In bovine embryos non-canonical WNT signaling
improved embryonic development, as a higher proportion of embryos became
blastocysts after exposure to WNT11 which activated WNT/PCP pathway via
phosphorylation of JNK (Chapter 2), and WNT7A that did not regulate intracellular β-
catenin (Chapter 3). Furthermore, increase of cytoplasmic β-catenin by GSK3 inhibitor
and the WNT mimetic AMBMP caused a reduction in the proportion of embryos that
developed to the blastocyst stage (Chapter 3). Nevertheless, the complex nature of
WNT signaling pathway represents a real limitation in developing understanding and
entails the necessity for completing a series of studies to progressively shed light on the
aspects of preimplantation development driven by maternally-derived molecules.
A striking finding of the research presented here is that the typical downstream
WNT signaling mediated by nuclear β-catenin is not fully functional during
preimplantation. Conversely, WNT signaling relies on non-nuclear β-catenin as well as
β-catenin independent pathways. Despite not being widely discussed, the absence of β-
catenin in the nucleus of preimplantation embryos may be a common condition across
177
mammals, as it has been observed in mouse, pig and human embryos (Kemler et al.
2004; Lim et al. 2013; Krivega et al. 2015). The finding that β-catenin does not
translocate to the nucleus in the bovine embryo does not mean that WNTs are not
involved in regulation of embryonic development. In addition to canonical signaling,
there are a variety of β-catenin independent pathways (Filmus et al. 2008; Chien et al.
2009; van Amerongen and Nusse, 2009; Gao, 2012), as well as a signaling mediated by
membrane-bound β-catenin (Lyashenko et al. 2011; Kim et al. 2013; Krivega et al.
2015).
Treatment of embryos with DKK1 increased subsequent pregnancy rates
following embryo transfer (Denicol et al., 2014). Our results showed no beneficial effect
of DKK1 on embryonic development or post-transfer survival (Chapter 4), but the failure
to observe a positive effect could be as a result of fundamental differences in the
conditions between the two studies. In particular, the study described in Chapter 4 was
conducted using an embryo culture medium containing serum and serum could possibly
mask the effects of DKK1 because it contains an undefined variety of signaling
molecules. Indeed, effects of CSF2 on development to the blastocyst stage were
prevented by addition of serum to culture medium (de Moraes and Hansen, 1997).
Further, fetal bovine serum could also conceivable contain DKK1(Kaiser et al. 2008).
Nonetheless, calves born from embryos cultured with DKK1 were lighter at birth than
embryos cultured in absence of exogenous DKK1. Thus, actions of WNT signaling on
development of the preimplantation embryo can cause long-term changes affecting
postnatal phenotype even in the presence of serum.
178
Looking forward, we can build on the insights developed in this dissertation in
many ways but particularly in understanding the maternal contribution to regulation of
embryonic WNT signaling. Doing so may represent an opportunity to increase fertility of
cattle and optimize culture conditions of bovine embryos.
179
LIST OF REFERENCES
Abe H, Yamashita S, Satoh T and Hoshi H (2002) Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. Molecular Reproduction and Development 61:57–66.
Abe K, Takeichi M, Hawkins K, Mohamet L, Ritson S, Merry CLR, Ward CM, Soncin F, Mohamet L, Ritson S et al. (2013) E-cadherin is required for the proper activation of the Lifr/Gp130 signaling pathway in mouse embryonic stem cells. Stem Cells 6:13–19.
Adler PN (2002) Planar signaling and morphogenesis in Drosophila. Developmental Cell 2:525–535.
Ajduk A, Biswas Shivhare S and Zernicka-Goetz M (2014) The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo. Developmental Biology 392:133–140.
Amerongen R V, Mikels A & Nusse R (2008) Alternative Wnt signaling is initiated by distinct receptors. Cell Biology 1:1–6.
Aparicio IM, Fair T & Lonergan P (2000) Identification and regulation of glycogen synthase kinase-3 during bovine embryo development. Reproduction 140:83–92.
Arman E, Haffner-Krausz R, Chen Y, Heath JK and Lonai P (1998) Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. PNAS 95:5082–5087.
Artus J, Piliszek A and Hadjantonakis A-K (2011) The primitive endoderm lineage of the mouse blastocyst: Sequential transcription factor activation and regulation of differentiation by Sox17. Developmental Biology Program 350:393–404.
Axelrod JD, Miller JR, Shulman JM, Moon RT and Perrimon N (1998) Differential recruitment of dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes and Development 12:2610–2622.
Bachvarova R, De Leon V, Johnson A, Kaplan G and Paynton B V. (1985) Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. Developmental Biology 108:325–331.
Bafico A, Liu G, Yaniv A, Gazit A and Aaronson SA (2001) Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nature Cell Biology 3:683–686.
Barcroft LC, Offenberg H, Thomsen P and Watson AJ (2003) Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Developmental Biology 256:342–354.
180
Barrow JR, Thomas KR, Boussadia-zahui O, Moore R, Kemler R, Capecchi MR and Mcmahon AP (2003) Ectodermal Wnt3 beta cantenin signaling is required for the establishment and maintenance of the apical ectodermal ridge. Molecular and Cellular Biology 17:394–409.
Bauersachs S, Rehfeld S, Ulbrich SE, Mallok S, Prelle K, Wenigerkind H, Einspanier R, Blum H and Wolf E (2004) Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle. Journal of Molecular Endocrinology 32:449–466.
Bauersachs S, Ulbrich SE, Gross K, Schmidt SEM, Meyer HHD, Einspanier R, Wenigerkind H, Vermehren M, Blum H, Sinowatz F et al. (2005) Gene expression profiling of bovine endometrium during the oestrous cycle: Detection of molecular pathways involved in functional changes. Journal of Molecular Endocrinology 34:889–908.
Beck F, Erler T, Russell A and James R (1995) Expression of Cdx-2 in the mouse embryo and placenta: Possible role in patterning of the extra-embryonic membranes. Developmental Dynamics 204:219–227.
Becker DL and Davies CS (1995) Role of gap junctions in the development of the preimplantation mouse embryo. Microscopy Research and Technique 31:364–374.
Bell CE, Calder MD and Watson AJ (2008) Genomic RNA profiling and the programme controlling preimplantation mammalian development. Molecular Human Reproduction 14:691–701.
Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M and Pfeffer PL (2011) Trophectoderm lineage determination in cattle. Developmental Cell 20:244–255.
Berge D, Kurek D, Blauwkamp T, Koole W, Maas A, Siu RK and Nusse R (2014) Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells. Nat Cell Biol. 13:1070–1075.
Betteridge K and Fléchon J (1988) The anatomy and physiology of the pre-attachment bovine embryos. Theriogenology 29:155–187.
Betts DH, MacPhee DJ, Kidder GM and Watson AJ (1997) Ouabain sensitivity and expression of Na/K-ATPaseα and β-subunit isoform genes during bovine early development. Molecular Reproduction and Development 46:114–126.
Biechele S, Cockburn K, Lanner F, Cox BJ and Rossant J (2013) Porcn-dependent Wnt signaling is not required prior to mouse gastrulation. Development 140:2961–2971.
Biechele S, Cockburn K, Lanner F, Cox BJ and Rossant J (2013) Porcn-dependent Wnt signaling is not required prior to mouse gastrulation. Development 140:2961–2971.
181
Block J, Hansen PJ, Loureiro B and Bonilla L (2011) Improving post-transfer survival of bovine embryos produced in vitro: Actions of insulin-like growth factor-1, colony stimulating factor-2 and hyaluronan. Theriogenology 76:1602–1609.
Block J, Wrenzycki C, Niemann H, Herrmann D and Hansen PJ (2008) Effects of insulin-like growth factor-1 on cellular and molecular characteristics of bovine blastocysts produced in vitro. Molecular Reproduction and Development 75:895–903.
Boni R, Tosti E, Roviello S and Dale B (1999) Intercellular communication in in vivo- and in vitro-produced bovine embryos. Biology of Reproduction 61:1050–1055.
Bonilla AQS, Ozawa M and Hansen PJ (2011) Timing and dependence upon mitogen-activated protein kinase signaling for pro-developmental actions of insulin-like growth factor 1 on the preimplantation bovine embryo. Growth Hormone & IGF Research 21:107–111.
Borland RM, Biggers JD and Lechene CP (1977) Fluid transport by rabbit preimplantation blastocysts in vitro. Reproduction 51:131–135.
Braude P, Bolton V and Moore S (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332:459–461.
Brembeck FH, Rosário M & Birchmeier W (2006) Balancing cell adhesion and Wnt signaling, the key role of β-catenin. Current Opinion in Genetics and Development 16:51–59.
Brodland GW (2002) The Differential Interfacial Tension Hypothesis (DITH): a comprehensive theory for the self-rearrangement of embryonic cells and tissues. Journal of Biomechanical Engineering 124:188–197.
Bultman SJ, Gebuhr TC, Pan H, Svoboda P, Schultz RM and Magnuson T (2006) Maternal BRG1 regulates zygotic genome activation in the mouse. Genes and Development 20:1744–1754.
Burton A and Torres-Padilla M-E (2014) Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis. Nature Reviews. Molecular Cell Biology 15: 722–734.
Cadigan KM & Nusse R (1997) Wnt signalling: a common theme in animal development. Genes & Development 11:3286–3305.
Cagnone G and Sirard M (2014) The impact of exposure to serum lipids during in vitro culture on the transcriptome of bovine blastocysts. Theriogenology 81:712–722.
Cai KQ, Capo-Chichi, Callinice D Malgorzata ER, Dong-Hua Y and Xiang-Xu2 X (2008) Dynamic GATA6 expression in primitive endoderm formation and maturation in early mouse embryogenesis. Dev Dyn 237:2820–2829.
Camargo LSA, Boite MC, Wohlres-Viana S, Mota GB, Serapiao RV, Sa WF, Viana JHM and Nogueira LAG (2011) Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro. Cryobiology 63:256–262.
182
Camilli TC and Weeraratna AT (2010) Striking the target in Wnt-y conditions: Intervening in Wnt signaling during cancer progression. Biochemical Pharmacology 80:702–711.
Caneparo L, Huang Y, Staudt N, Tada M, Ahrendt R, Kazanskaya O, Niehrs C and Houart C (2007) Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/β-catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek. Genes and Development 21:465–480.
Cao Q and Richter JD (2002) Dissolution of the maskin ± eIF4E complex by cytoplasmic polyadenylation and poly ( A ) -binding protein controls cyclin B1 mRNA translation and oocyte maturation. EMBO Journal 21.
Cao Y (2013) Regulation of germ layer formation by pluripotency factors during embryogenesis. Cell & Bioscience 3:15-18.
Carroll TJ, Park JS, Hayashi S, Majumdar A and McMahon AP (2005) Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Developmental Cell 9:283–292.
Cebon J, Nicola N, Ward M, Gardner I, Dempsey P, Layton J, Duhrsen U, Burgess AW, Nice E and Morstyn G (1990) Granulocyte-macrophage colony stimulating factor from human lymphocytes. Journal of Biological Chemistry 265:4483–4491.
Ceelen M, van Weissenbruch MM, Vermeiden JPW, van Leeuwen FE and Delemarre-van De Waal HA (2008) Cardiometabolic differences in children born after in vitro fertilization: follow-up study. J Clin Endocrinol Metab 93:1682–1688.
Cerri RL, Thompson IM, Kim IH, Ealy AD, Hansen PJ, Staples CR, Li JL, Santos JEP and Thatcher WW (2012) Effects of lactation and pregnancy on gene expression of endometrium of Holstein cows at day 17 of the estrous cycle or pregnancy. Journal of Dairy Science 95:5657–5675.
Cha S-W, Tadjuidje E, Tao Q, Wylie C & Heasman J (2008) Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation. Development 135:3719–3729.
Chan SW, Lim CJ, Chong YF, Pobbati A V., Huang C and Hong W (2011) Hippo pathway-independent restriction of TAZ and YAP by angiomotin. Journal of Biological Chemistry 286:7018–7026.
Chazaud C, Yamanaka Y, Pawson T and Rossant J (2006) Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Developmental Cell 10:615–624.
Chen M, Qian C, Bi LL, Zhao F, Zhang GY, Wang ZQ, Gan XD & Wang YG (2014) Enrichment of cardiac differentiation by a large starting number of embryonic stem cells in embryoid bodies is mediated by the Wnt11-JNK pathway. Biotechnology Letters 37:475–481.
183
Chien AJ, Conrad WH & Moon RT (2009) A Wnt survival guide: from flies to human disease. The Journal of Investigative Dermatology 129 1614–1627.
Clevers H, Loh KM and Nusse R (2014) Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346: 1248012.
Coffey ET (2014) Nuclear and cytosolic JNK signalling in neurons. Nature Reviews. Neuroscience 15:285–299.
Cole MF, Johnstone SE, Newman JJ, Kagey MH and Young RA (2008) Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. Genes and Development 22:746–755.
Corcoran D, Fair T, Park S, Rizos D, Patel O V., Smith GW, Coussens PM, Ireland JJ, Boland MP, Evans ACO et al. (2006) Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos. Reproduction 131:651–660.
Crosier AE, Farin PW, Dykstra MJ, Alexander JE and Farin CE (2000) Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro. Biology of Reproduction 62:1459–1465.
Dardik A, Doherty AS and Schultz RM (1993) Protein secretion by the mouse blastocyst-stimulatory effect on secretion into the blastocoele by transforming growth factor-α. Molecular Reproduction and Development 34:396–401.
de Moraes AA and Hansen. PJ (1997) Granulocyte-macrophage colony-stimulating factor promotes development of in vitro produced bovine embryos. Biology of Reproduction 57:1060–1065.
De Moraes AAS, Paula-Lopes FF, Chegini N and Hansen PJ (1999) Localization of granulocyte-macrophage colony-stimulating factor in the bovine reproductive tract. Journal of Reproductive Immunology 42:135–145.
De Sousa P, Juneja SC, Caveney S, Houghton FD, Davies TC, Reaume G, Rossant J and Kidder GM (1997) Normal development of preimplantation mouse embryos deficient in gap junctional coupling. Journal of Cell Science 110:1751–1758.
Dejmek J, Säfholm A, Kamp Nielsen C, Andersson T and Leandersson K (2006) Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells. Molecular and Cellular Biology 26:6024–6036.
Denicol AC, Dobbs KB, McLean KM, Carambula SF, Loureiro B & Hansen PJ (2013) Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage. Scientific Reports 3:1266.
Denicol AC, Block J, Kelley DE, Pohler KG, Dobbs KB, Mortensen CJ, Ortega MS and Hansen PJ (2014) The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo. FASEB Journal 3975–3986.
184
Denicol AC, Le BCS, Dobbs KB and Mingoti GZ (2015) Influence of sex on basal and Dickkopf-1 regulated gene expression in the bovine morula. 1–19.
Dietrich JE and Hiiragi T (2007) Stochastic patterning in the mouse pre-implantation embryo. Development 134:4219–4231.
Dobbs K, Khan F, Sakatani M, Moss J, Ozawa M, Ealy A & Hansen PJ (2013) Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2. Biology of Reproduction 89:141.
Ducibella T and Anderson E (1975) Cell shape and membrane changes in the eight-cell mouse embryo: Prerequisites for morphogenesis of the blastocyst. Developmental Biology 47:45–58.
Ducibella T and Anderson E (1979) The effects of calcium deficiency on the formation of the zonula occludens and blastocoel in the mouse embryo. Developmental Biology 73:46–58.
Ducibella T, Ukena T, Karnovsky M and Anderson E (1977) Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo. Journal of Cell Biology 74:153–167.
Dunlap K a, Filant J, Hayashi K, Rucker 3rd EB, Song G, Deng JM, Behringer RR, DeMayo FJ, Lydon J, Jeong JW et al. (2011) Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice. Biol Reprod 85:386–396.
Dziadek M (1979) Cell differentiation in isolated inner cell masses of mouse blastocysts in vitro: onset of specific gene expression. Journal of Embryology and Experimental Morphology 53:367–379.
Enders A, Given R and Schlafke S (1978) Differentiation and migration of endoderm in the rat and mouse at implantation. Anat Rec 190:65–77.
Enright BP, Lonergan P, Fair T, Ward FA, Yang X and Boland MP (2000) Culture of in vitro produced bovine zygotes in vitro vs in vivo: Implications for early embryo development and quality. Theriogenology 54:659–673.
Fair T (2010) Mammalian oocyte development: Checkpoints for competence. Reproduction, Fertility and Development 22:13–20.
Farin PW, Piedrahita JA and Farin CE (2006) Errors in development of fetuses and placentas from in vitro-produced bovine embryos. 65:178–191.
Feldman B, Poueymirou W, Papaioannou V, DeChiara T and Goldfarb M (1995) Requirement of FGF-4 for postimplantation mouse development. Science 267:246–469.
Feng YL and Gordon JW (1997) Removal of cytoplasm from one-celled mouse embryos induces early blastocyst formation. Journal of Experimental Zoology 277:345–352.
185
Ferguson JD, Galligan DT and Thomsen N (1994) Principal descriptors of body condition score in Holstein cows. Journal of Dairy Science 77:2695–2703.
Fernandez-Gonzalez R, Moreira P, Bilbao A, Jimenez A, Perez-crespo M, Ramírez MA, Rodríguez De Fonseca F, Pintado B, Guitierrez-Adan A and Gutie A (2004) Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. PNAS 101:5880–5885.
Fields SD, Hansen PJ and Ealy AD (2011) Fibroblast growth factor requirements for in vitro development of bovine embryos. Theriogenology 75:1466–1475.
Fierro-González JC, White MD, Silva JC and Plachta N (2013) Cadherin-dependent filopodia control preimplantation embryo compaction. Nature Cell Biology 15:1424–1433.
Filmus J, Capurro M & Rast J (2008) Glypicans. Genome Biology 9:224.
FitzHarris G, Marangos P and Carroll J (2003) Cell cycle-dependent regulation of structure of endoplasmic reticulum and inositol 1,4,5- trisphosphate-induced Ca2+ release in mouse oocytes and embryos. Molecular Biology of the Cell 14:288–301.
Flaherty MP & Dawn B (2008) Noncanonical Wnt11 signaling and cardiomyogenic differentiation. Trends in Cardiovascular Medicine 18:260–268.
Fleming TP and Pickering SJ (1985) Maturation and polarization of the endocytotic system in outside blastomeres during mouse preimplantation development. Journal of Embryology and Experimental Morphology 89:175–208.
Fleming TP, Sheth B & Fesenko I (2001) Cell adhesion in the preimplantation mammalian embryo and its role in trophectoderm differentiation and blastocyst morphogenesis. Front Biosci 6:1000–1007.
Fleming TP, Velazquez MA and Eckert JJ (2015a) Embryos, DOHaD and David Barker. Developmental Origins of Health and Disease 6:377–383.
Fleming TP, Watkins AJ, Sun C, Velazquez MA, Neil R and Eckert JJ (2015b) Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo’s potential , affecting adult health. Reprod Fertil Dev. 27:684–692.
Forde N, Carter F, Fair T, Crowe M a, Evans a CO, Spencer TE, Bazer FW, McBride R, Boland MP, O’Gaora P et al. (2009) Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle. Biology of Reproduction 81:784–794.
Forde N, Spencer TE, Bazer FW, Song G, Roche JF and Lonergan P (2010) Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium. Physiological Genomics 41:53–62.
186
Fox CA and Wickens M (1990) Poly(A) removal during oocyte maturation: A default reaction selectively prevented by specific sequences in the 3’ UTR of certain maternal mRNAs. Genes and Development 4:2287–2298.
Frankenberg S, Gerbe F, Bessonnard S, Belville C, Pouchin P, Bardot O and Chazaud C (2011) Primitive Endoderm Differentiates via a Three-Step Mechanism Involving Nanog and RTK Signaling. Developmental Cell 21:1005–1013.
Gad A, Hoelker M, Besenfelder U, Havlicek V, Cinar U, Rings F, Held E, Dufort I, Sirard M-A, Schellander K et al. (2012) Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions. Biology of Reproduction 87:100.
Gao B (2012) Wnt regulation of planar cell polarity (PCP). Current Topics in Developmental Biology 101:263–295.
Gardner RL (2001) Specification of embryonic axes begins before cleavage in normal mouse development. Development 128:839–847.
Garriock RJ and Krieg PA (2007) Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus. Developmental Biology 304:127–140.
Geetha-Loganathan P, Nimmagadda S, Fu K & Richman JM (2014) Avian facial morphogenesis is regulated by JNK/PCP wingless-related (WNT) signaling. The Journal of Biological Chemistry 289:24153–24167.
Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP, Ferree S, George RD, Grogan T et al. (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnology 26:317–325.
Goissis MD and Cibelli JB (2014) Functional characterization of CDX2 during bovine preimplantation development in vitro. Molecular Reproduction and Development 81:962–970.
Goldin SN and Papaioannou VE (2003) Paracrine action of FGF4 during periimplantation development maintains trophectoderm and primitive endoderm. Genesis 36:40–47.
Gómez E, Correia-Álvarez E, Caamaño JN, Díez C, Carrocera S, Peynot N, Martín D, Giraud-Delville C, Duranthon V, Sandra O et al. (2014) Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture. Reproduction 148:353–365.
Goossens K, Van Poucke M, Van Soom A, Vandesompele J, Van Zeveren A & Peelman LJ (2005) Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Developmental Biology 5:27.
Graf A, Krebs S, Zakhartchenko V, Schwalb B, Blum H and Wolf E (2014a) Fine mapping of genome activation in bovine embryos by RNA sequencing. PNAS 111:1–6.
187
Graf A, Krebs S, Heininen-brown M, Zakhartchenko V, Blum H and Wolf E (2014b) Genome activation in bovine embryos : Review of the literature and new insights
from RNA sequencing experiments ଝ. Animal Reproduction Science 149:46–58.
Graham CF & Lehtonen E (1979) Formation and consequences of cell patterns in preimplantation mouse development. Embryology 49:277–294.
Gray RS, Roszko I and Solnica-Krezel L (2011) Planar cell polarity: coordinating morphogenetic cell behaviors with embryonic polarity. Developmental Cell 21:120–133.
Groothuis PG, Dassen HHNM, Romano A and Punyadeera C (2007) Estrogen and the endometrium: Lessons learned from gene expression profiling in rodents and human. Human Reproduction Update 13:405–417.
Guo G, Huss M, Tong GQ, Wang C, Li Sun L, Clarke ND and Robson P (2010) Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Developmental Cell 18:675–685.
Guo X, Day TF, Jiang X, Garrett-Beal L, Topol L & Yang Y (2004) Wnt/β-catenin signaling is sufficient and necessary for synovial joint formation. Genes and Development 18:2404–2417.
Haegel H, Larue L, Ohsugi M, Fedorov L, Herrenknecht K, Kemler R, Immunbiologie M & Freiburg D- (1995) Lack of β -catenin affects mouse development at gastrulation. 3537:3529–3537.
Hansen PJ (2011) Challenges to fertility in dairy cattle : from ovulation to the fetal stage of pregnancy. Rev Bras Reprod Anim 35:229–238.
Hansen PJ, Dobbs KB, Denicol AC and Siqueira LGB (2016) Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development. Cell and Tissue Research 363:237–247.
Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Mower SA, Neely B, Shuey LS, Stokes JE and Trimmer SA (1995) Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 43:141–152.
Hasler JF, McCauley AD, Lathrop WF and Foote RH (1987) Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program. Theriogenology 27:139–168.
Hawkins K, Mohamet L, Ritson S, Merry CLR & Ward CM (2012) E-cadherin and, in its absence, N-cadherin promotes nanog expression in mouse embryonic stem cells via STAT3 Phosphorylation. Stem Cells 30:1842–1851.
Hayashi K, Burghardt RC, Bazer FW and Spencer TE (2007) WNTs in the ovine uterus: potential regulation of periimplantation ovine conceptus development. Endocrinology 148:3496–3506.
188
He T, Sparks AB, Rago C, Hermeking H, Zawel L, Costa LT, Morin PJ, Vogelstein B and Kinzler KW (1998) Identification of c- MYC as a target of the APC pathway. Science 281:1509–1512.
Hillman N, Sherman MI and Graham C (1972) The effect of spatial arrangement on cell determination during mouse development. Embryol. Exp. Morph 28:263–278.
Hirate Y, Cockburn K, Rossant J and Sasaki H (2012) Tead4 is constitutively nuclear, while nuclear vs. cytoplasmic Yap distribution is regulated in preimplantation mouse embryos. PNAS 109 e3389-90.
Hirate Y, Hirahara S, Inoue KI, Suzuki A, Alarcon VB, Akimoto K, Hirai T, Hara T, Adachi M, Chida K et al. (2013) Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos. Biophysical Chemistry 23:1181–1194.
Ho SY and Keller TH (2015) The use of porcupine inhibitors to target Wnt-driven cancers. Bioorganic & Medicinal Chemistry Letters 25:5472–5476.
Hoffman BD and Yap AS (2015) Towards a dynamic understanding of cadherin-based mechanobiology. Trends in Cell Biology 25:803–814.
Home P, Ray S, Dutta D, Bronshteyn I, Larson M and Paul S (2009) GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression. Journal of Biological Chemistry 284:28729–28737.
Hsieh JC, Kodjabachian L, Rebbert ML, Rattner a, Smallwood PM, Samos CH, Nusse R, Dawid IB and Nathans J (1999) A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature 398:431–436.
Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C & Birchmeier W (2000) Requirement for β-catenin in anterior-posterior axis formation in mice. Cell Biology 148:567–578.
Hyttle P, Sinowatz F & Vejlsted M (2010) Domestic Animal Embryology. Saunders, Elsevier.
Ikeya M and Takada S (2001) Wnt-3a is required for somite specification along the anteroposterior axis of the mouse embryo and for regulation of cdx-1 expression. Mechanisms of Development 103:27–33.
Jiang Z, Sun J, Dong H, Luo O, Zheng X, Obergfell C, Tang Y, Bi J, O’Neill R, Ruan Y et al. (2014) Transcriptional profiles of bovine in vivo pre-implantation development. BMC Genomics 15:756.
Johnson MH and McConnell JML (2004) Lineage allocation and cell polarity during mouse embryogenesis. Seminars in Cell and Developmental Biology 15:583–597.
Jousan FD and Hansen PJ (2007) Insulin-like growth factor-I promotes resistance of bovine preimplantation embryos to heat shock through actions independent of its anti-apoptotic actions requiring PI3K signaling. Molecular Reproduction and Development 74:189–196.
189
Kanka J (2003) Gene expression and chromatin structure in the pre-implantation embryo. Theriogenology 59:3–19.
Kannampuzha-Francis J, Denicol AC, Loureiro B, Kaniyamattam K, Ortega MS and Hansen PJ (2015) Exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle. Molecular Reproduction and Development 82:892–897.
Kannampuzha-Francis J, Tribulo P and Hansen. PJ (2016) Actions of activin A , connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo. Reproduction, Fertility and Development.
Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, Osteen K, Taylor RN, Lessey BA, Giudice LC et al. (2016) Global Gene Profiling in Human Endometrium during the Window of Implantation. :2119–2138.
Kauma SW and Matt DW (1995) Coculture cells that express leukemia inhibitory factor (LIF) enhance mouse blastocyst development in vitro. Assist Reprod Genet 12:153–156.
Kawano Y and Kypta R (2003) Secreted antagonists of the Wnt signalling pathway. Journal of Cell Science 116:2627–2634.
Kazanskaya O, Glinka A and Niehrs C (2000) The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning. Development 127:4981–4992.
Kemler R, Babinet C, Eisen H and Jacob F (1977) Surface antigen in early differentiation. Proc Natl Acad Sci USA 74:4449―52.
Kemler R, Hierholzer A, Kanzler B, Kuppig S, Hansen K, Taketo MM, de Vries WN, Knowles BB & Solter D (2004) Stabilization of β-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast. Development 131:5817–5824.
Kemp C, Willems E, Abdo S, Lambiv L and Leyns L (2005) Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development. Developmental Dynamics 233:1064–1075.
Khurana NK and Niemann H (2000) Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo. Biology of Reproduction 62 847–856.
Kielman, MF, Rindapää M, Gaspar C, van Poppel N, Breukel C, van Leeuwen S, Taketo MM, Roberts S, Smits R and Fodde R (2002) Apc modulates embryonic stem-cell differentiation by controlling the dosage of β-catenin signaling. Nature genetics 32:594-605.
Kim N-G, Koh E, Chen X and Gumbiner BM (2011) E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components. PNAS 108:11930–11935.
190
Killick R, Ribe EM, Al-Shawi R, Malik B, Hooper C, Fernandes C, Dobson R, Nolan PM, Lourdusamy a, Furney S et al. (2014) Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway. Molecular Psychiatry 19:88–98.
Kim H, Wu J, Ye S, Tai C-I, Zhou X, Yan H, Li P, Pera M & Ying Q-L (2013) Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewa. Nature Communications 4.
King GJ, Atkinson BA and Robertson HA (1981) Development of the intercaruncular areas during early gestation and establishment of the bovine placenta. Journal of Reproduction and Fertility :469–474.
Kleber M and Sommer L (2004) Wnt signaling and the regulation of stem cell function. Current Opinion in Cell Biology 16:681–687.
Klein TJ and Mlodzik M (2005) Planar cell polarization: an emerging model points in the right direction. Annual Review of Cell and Developmental Biology 21:155–176.
Kohn AD & Moon RT (2005) Wnt and calcium signaling: β-catenin-independent pathways. Cell Calcium 38:439–446.
Koutsourakis M, Langeveld a, Patient R, Beddington R and Grosveld F (1999) The transcription factor GATA6 is essential for early extraembryonic development. Development 126:723–732.
Krasnow RE and Adler PN (1994) A single frizzled protein has a dual function in tissue polarity. Development 120:1883–1893.
Krause U, Ryan DM, Clough BH and Gregory C a (2014) An unexpected role for a Wnt-inhibitor: Dickkopf-1 triggers a novel cancer survival mechanism through modulation of aldehyde-dehydrogenase-1 activity. Cell Death & Disease 5 e1093.
Krawetz R & Kelly GM (2008) Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm. Cellular Signalling 20:506–517.
Kremenevskaja N, von Wasielewski R, Rao a S, Schöfl C, Andersson T and Brabant G (2005) Wnt-5a has tumor suppressor activity in thyroid carcinoma. Oncogene 24:2144–2154.
Krivega M, Essahib W & Van de Velde H (2015) WNT3 and membrane-associated β-
catenin regulate trophectoderm lineage differentiation in human blastocysts. Molecular Human Reproduction 711–722.
Kues WA, Sudheer S, Herrmann D, Carnwath JW, Havlicek V, Besenfelder U, Lehrach H and Adjaye J (2008) Genome-wide expression profiling reveals distinct clusters of transcriptional regulation during bovine preimplantation development in vivo. 1–6.
191
Kühl M, Sheldahl LC, Park M, Miller JR & Moon RT (2000) The Wnt / Ca 2+ pathway - a new vertebrate Wnt signaling pathway takes shape. Trends in Genetics 16:279–283.
Kuijk EW, Du Puy L, Van Tol HT a, Oei CHY, Haagsman HP, Colenbrander B and Roelen B a J (2008) Differences in early lineage segregation between mammals. Developmental Dynamics 237) The Wnt / Ca 2+ pathway - a new vertebrate Wnt
signaling pathway takes shape. 918–927.
Kuijk EW, van Tol LT, Van de Velde H, Wubbolts R, Welling M, Geijsen N and Roelen BJ (2012) The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos. Development 139) The Wnt / Ca 2+ pathway - a new vertebrate Wnt signaling pathway takes shape.
871–882.
Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S and Smith A (2007) FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134:2895–2902.
Kwong WY, Wild a E, Roberts P, Willis a C and Fleming TP (2000) Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127:4195–4202.
La Rosa I, Camargo LSA, Pereira MM, Fernandez-Martin R, Paz DA and Salamone DF (2011) Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos. Reproductive Biology and Endocrinology 9:18.
Lappas M (2014) GSK3β is increased in adipose tissue and skeletal muscle from women with gestational diabetes where it regulates the inflammatory response. PLoS ONE 9:1–23.
Lazzari G, Colleoni S, Lagutina I, Crotti G and Turini P (2010) Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species. Theriogenology 73:748–757.
Lee DR, Lee JE, Yoon HS, Roh SI and Kim MK (2001) Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner. Journal of Experimental Zoology 290:61–71.
Lee K-B, Wee G, Zhang K, Folger JK, Knott JG and Smith GW (2014) Functional role of the bovine oocyte-specific protein JY-1 in meiotic maturation, cumulus expansion, and subsequent embryonic development. Biology of Reproduction 90:69.
Lee SM, Tole S, Grove E and McMahon a P (2000) A local Wnt-3a signal is required for development of the mammalian hippocampus. Development. 127:457–467.
192
Leung CY and Zernicka-Goetz M (2013) Angiomotin prevents pluripotent lineage differentiation in mouse embryos via Hippo pathway-dependent and -independent mechanisms. Nature Communications 4:2251.
Lhomond G, McClay DR, Gache C & Croce JC (2012) Frizzled1/2/7 signaling directs β-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis. Development 139:816–825.
Li C, Xiao J, Hormi K, Borok Z and Minoo P (2002) Wnt5a participates in distal lung morphogenesis. Developmental Biology 248:68–81.
Li J, Liu W-M, Cao Y-J, Peng S, Zhang Y and Duan E-K (2008) Roles of Dickkopf-1 and its receptor Kremen1 during embryonic implantation in mice. Fertility and Sterility 90:1470–1479.
Li L, Lu X and Dean J (2013) The maternal to zygotic transition in mammals. Molecular Aspects of Medicine 34:919–938.
Li R, Wang C, Tong J, Su Y, Lin Y, Zhou X & Ye L (2014) WNT6 promotes the migration and differentiation of human dental pulp cells partly through c-Jun N-terminal kinase signaling pathway. Journal of Endodontics 40:943–948.
Lim KT, Gupta MK, Lee SH, Jung YH, Han DW & Lee HT (2013) Possible involvement of Wnt/β-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts. Theriogenology 79:284–290.
Lin S-CJ, Wani M a, Whitsett J a and Wells JM (2010) Klf5 regulates lineage formation in the pre-implantation mouse embryo. Development 137 3953–3963.
Lin T, Yen J-M, Gong K-B, Hsu T-T and Chen L-R (2003) IGF-I/IGFBP-I increases blastocyst formation and total cell number in mouse embryo culture and facilitates the establishment of a stem-cell line. BMC Cell Biology 4:14.
Liu J, Wu X, Mitchell B, Kintner C, Ding S & Schultz PG (2005) A small-molecule agonist of the Wnt signaling pathway. Angewandte Chemie 44:1987–1990.
Liu P, Wakamiya M, Shea MJ, Albrecht U, Behringer RR and Bradley A (1999) Requirement for Wnt3 in vertebrate axis formation. Nature Genetics 22:361–365.
Liu S, Zhang E, Yang M & Lu L (2014) Overexpression of Wnt11 promotes chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in synergism with TGF-β. Molecular and Cellular Biochemistry 390:123–131.
Liu T, Lee YN, Malbon CC and Wang HY (2002) Activation of the β-catenin/Lef-Tcf pathway is obligate for formation of primitive endoderm by mouse F9 totipotent teratocarcinoma cells in response to retinoic acid. Journal of Biological Chemistry 277:30887–30891.
Liu Z and Foote RH (1997) Effects of amino acids and alpha-amanitin on bovine embryo development in a simple protein-free medium. Molecular Reproduction and Development 46:278–285.
193
Lloyd S, Fleming TP and Collins JE (2003) Expression of Wnt genes during mouse preimplantation development. Gene Expression Patterns 3:309–312.
Logan CY & Nusse R (2004) The Wnt signaling pathway in development and disease. Annual Review of Cell and Developmental Biology 20:781–810.
Lonergan P, Woods A, Fair T, Carter F, Rizos D, Ward F, Quinn K and Evans A (2007) Effect of embryo source and recipient progesterone environment on embryo development in cattle. Reproduction, Fertility and Development 19:861–868.
Loureiro B, Bonilla L, Block J, Fear J, Bonilla A and Hansen PJ (2009) Colony-Stimulating factor 2 (CSF-2) improves development and posttransfer survival of bovine embryos produced in vitro. Endocrinology 150:5046–5054.
Loureiro B, Block J, Favoreto MG, Carambula S, Pennington K a., Ealy AD and Hansen PJ (2011a) Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression. Reproduction 141:617–624.
Loureiro B, Oliveira LJ, Favoreto MG and Hansen PJ (2011b) Colony-stimulating factor 2 inhibits induction of apoptosis in the bovine preimplantation embryo. American Journal of Reproductive Immunology 65:578–588.
Lu C-W, Yabuuchi A, Chen L, Viswanathan S, Kim K and Daley. GQ (2008) Ras-Mitogen Activated Protein Kinase Signaling Promotes Trophectoderm Formation from Embryonic Stem Cells and Murine Embryos. Nature Genetics 40:921–926.
Lyashenko N, Winter M, Migliorini D, Biechele T, Moon RT & Hartmann C (2011) Differential requirement for the dual functions of β-catenin in embryonic stem cell self-renewal and germ layer formation. Nature Cell Biology 13:753–761.
Ma J, Flemr M, Strnad H, Svoboda P and Schultz RM (2013) Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse. Biol Reprod 88:11.
MacDonald BT, Tamai K & He X (2009) Wnt/β-Catenin signaling: components, mechanisms, and diseases. Developmental Cell 17:9–26.
Maître J-L, Berthoumieux H, Krens SFG, Salbreux G, Jülicher F, Paluch E, Heisenberg C-PC-P, Maitre J-L, Berthoumieux H, Krens SFG et al. (2012) Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. Science 338:253–256.
Maître J-L, Niwayama R, Turlier H, Nédélec F and Hiiragi T (2015) Pulsatile cell-autonomous contractility drives compaction in the mouse embryo. Nature Cell Biology 17:849–855.
Majumdar A, Vainio S, Kispert A, McMahon J and McMahon AP (2003) Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development. Development 130:3175–3185.
194
Malayer JR, Hansen PJ and Buhi WC (1988) Effect of day of the oestrous cycle, side of the reproductive tract and heat shock on in-vitro protein secretion by bovine endometrium. Journal of Reproduction and Fertility 84:567–578.
Malcuit C, Kurokawa M and Fissore RA (2006) Calcium oscillations and mammalian egg activation. Journal of Cellular Physiology 206:565–573.
Mamo S, Mehta JP, Forde N, McGettigan P and Lonergan P (2012) Conceptus-Endometrium Crosstalk During Maternal Recognition of Pregnancy in Cattle. Biology of Reproduction 87:6–6.
Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, Delius H, Hoppe D, Stannek P, Walter C et al. (2002) Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417:664–667.
Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka a and Niehrs C (2001) LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411:321–325.
Marikawa Y and Alarcon VB (2012) Creation of trophectoderm, the first epithelium, in mouse preimplantation development. Mouse Development 165–184.
McHughes CE, Springer GK, Spate LD, Li R, Woods R, Green MP, Korte SW, Murphy CN, Green JA and Prather RS (2009) Identification and quantification of differentially represented transcripts in in vitro and in vivo derived preimplantation bovine embryos. Molecular Reproduction and Development 76:48–60.
McMahon AP and Bradleyt A (1990) The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62:1073–1085.
Medvedev S, Yang J, Hecht NB and Schultz RM (2008) CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation. Developmental Biology 321:205–215.
Meilhac SM, Adams RJ, Morris S, Danckaert A, Le Garrec JF and Zernicka-Goetz M (2009) Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst. Developmental Biology 331:210–221.
Memili E and First NL (2000) Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote 8:87–96.
Menino AR, Williams JS, Gardiner CS and John TL (1989) Mouse embryo development in media containing growth factors. Animal Reproduction Science 18:125–137.
Mesquita FS, Pugliesi G, Scolari SC, França MR, Ramos RS, Oliveira M, Papa PC,
Bressan FF, Meirelles F V., Silva LA et al. (2014) Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows. Theriogenology 81:861–869.
195
Messerschmidt DM and Kemler R (2010) Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism. Developmental Biology 344:129–137.
Metcalf D (2013) Granulocyte-macrophage colony-stimulating factors. In Control of Animal Cell Proliferation 2:109–132.
Miller C and Sassoon DA (1998) Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract. Development 125:3201–3211.
Minten M a., Bilby TR, Bruno RGS, Allen CC, Madsen C a., Wang Z, Sawyer JE, Tibary A, Neibergs HL, Geary TW et al. (2013) Effects of Fertility on Gene Expression and Function of the Bovine Endometrium. PLoS ONE 8 e69444.
Mitko K, Ulbrich SE, Wenigerkind H, Sinowatz F, Blum H, Wolf E and Bauersachs S (2008) Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle. Reproduction 135:225–240.
Miyagi C, Yamashita S, Ohba Y, Yoshizaki H, Matsuda M and Hirano T (2004) STATS noncell-autonomously controls planar cell polarity during zebrafish convergence and extension. Journal of Cell Biology 166:975–981.
Miyazaki T, Kuo T-C, Dharmarajan A, Atlas SJ and Wallach EE (1989) In vivo administration of allopurinol affects ovulation and early embryonic developemtn in rabbits. American Journal of Obstetrics and Gynecology 161:1709–1714.
Mo X, Wu G, Yuan D, Jia B, Liu C, Zhu S and Hou Y (2014) Leukemia inhibitory factor enhances bovine oocyte maturation and early embryo development. Molecular Reproduction and Development 81:608–618.
Mohamed OA, Dufort D, Clarke HJ, Obstetrics D, Clarke CHJ, Dufort D, Room F, Hospital V, West PA and Ha QCC (2004) Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biology of Reproduction 71:417–424.
Monkley SJ, Delaney SJ, Pennisi DJ, Christiansen JH and Wainwright BJ (1996) Targeted disruption of the Wnt2 gene results in placentation defects. Development 122:3343–3353.
Moreira F, Badinga L, Burnley C and Thatcher WW (2002) Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows. Theriogenology 57:1371–1387.
Morosan-Puopolo G, Balakrishnan-Renuka A, Yusuf F, Chen J, Dai F, Zoidl G, Lüdtke TH-W, Kispert A, Theiss C, Abdelsabour-Khalaf M et al. (2014) Wnt11 is required for oriented migration of dermogenic progenitor cells from the dorsomedial lip of the avian dermomyotome. PloS One 9 e92679.
196
Morrisey EE, Tang Z, Sigrist K, Lu MM, Jiang F, Ip HS and Parmacek MS (1998) GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes and Development 12:3579–3590.
Neira JA, Tainturier D, Peña MA and Martal J (2010) Effect of the association of IGF-I, IGF-II, bFGF, TGFβ1, GM-CSF, and LIF on the development of bovine embryos produced in vitro. Theriogenology 73:595–604.
Newcomb R and Rowson LEA (1975) Conception rate after uterine transfer of cow eggs, in relation to synchronization of oestrus and age of eggs. J Reprod Fert 43:539–541.
Newport J and Kirschner M (1982) A major developmental transition in early xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell 30:675–686.
Nichols J and Smith A (2009) Naive and Primed Pluripotent States. Cell Stem Cell 4:487–492.
Niemann H and Wrenzycki C (2000) Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology 53:21–34.
Niemann H, Carnwath JW, Herrmann D, Wieczorek G, Lemme E, Lucas-Hahn A and Olek S (2010) DNA methylation patterns reflect epigenetic reprogramming in bovine embryos. Cellular Reprogramming 12:33–42.
Nishioka N, Inoue K ichi, Adachi K, Kiyonari H, Ota M, Ralston A, Yabuta N, Hirahara S, Stephenson RO, Ogonuki N et al. (2009) The hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Developmental Cell 16:398–410.
Nishioka N, Yamamoto S, Kiyonari H, Sato H, Sawada A, Ota M, Nakao K and Sasaki H (2007) Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mechanisms of Development 5:270–283.
Niswander L and Martin GR (1992) Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114:755–768.
Niwa H (2007) Open conformation chromatin and pluripotency. Genes and Development 21:2671–2676.
Niwa H, Miyazaki J and Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics 24:372–376.
Nusse R (2001) Making head or tail of Dickkopf. Nature 411:255–256.
Nusse R and Varmus HE (1982) Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 31:99–109.
197
Nusse RC, Fuerer W, Ching K, Harnish C, Logan A, Zeng D, Ten B, and Y. Kalani (2008) Wnt signaling and stem cell control. In Cold Spring Harbor symposia on quantitative biology 73:59-66
Nüsslein-Volhard C and Wieschaus E (1980) Mutations affecting segment number and polarity in Drosophila. Nature 287:795–801.
Ogawa K, Nishinakamura R, Iwamatsu Y, Shimosato D and Niwa H (2006) Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochemical and Biophysical Research Communications 343:159–166.
Orsulic S, Huber O, Aberle H, Arnold S & Kemler R (1999) E-cadherin binding prevents β -catenin nuclear localization and β -catenin / LEF- 1-mediated transactivation. 1245:1237–1245.
Ortega MS, Rocha-Frigoni NAS, Mingoti GZ, Roth Z & Hansen PJ (2016) Modification of embryonic resistance to heat shock in cattle by melatonin and genetic variation in HSPA1L. Journal of Dairy Science 99:1–13.
Ozawa M & Hansen PJ (2011) A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting. Fertility and Sterility 95:799–802.
Ozawa M, Sakatani M, Yao J, Shanker S, Yu F, Yamashita R, Wakabayashi S, Nakai K, Dobbs KB, Sudano MJ et al. (2012) Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst. BMC Developmental Biology 12:33.
Pandur P, Lasche M, Leonard ME & Kuhl M (2002) Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature 418:636–641.
Paramasivam M, Sarkeshik A, Yates JR, Fernandes MJG and McCollum D (2011) Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor. Molecular Biology of the Cell 22:3725–3733.
Paria BC and Dey SK (1990) Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proceedings of the National Academy of Sciences 87:4756–4760.
Parr B and McMahon P (1998) Sexually dimorphic development of the mammalian reproductive tract requires Wnt-7a. Nature 395:707–710.
Parr BA, Cornish VA, Cybulsky MI and Mcmahon AP (2001) Wnt7b Regulates Placental Development in Mice. 332:324–332.
Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH & First NL (1986) Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25:591–600.
198
Pasternak C, Spudich JA and Elson EL (1989) Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature 341:549–551.
Paula-Lopes FF, de Moraes AA, Edwards JL, Justice JE and Hansen PJ (1998) Regulation of preimplantation development of bovine embryos by interleukin-1β. Biology of Reproduction 59:1406–1412.
Paynton B V., Rempel R and Bachvarova R (1988) Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Developmental Biology 129:304–314.
Peng S, Li J, Miao C, Jia L, Hu Z, Zhao P, Li J, Zhang Y, Chen Q and Duan E (2008) Dickkopf-1 secreted by decidual cells promotes trophoblast cell invasion during murine placentation. Reproduction 135:367–375.
Perez-Moreno M, Jamora C & Fuchs E (2003) Sticky Business : Orchestrating Cellular Signals at Adherens Junctions. 112:535–548.
Piotrowska K and Zernicka-Goetz M (2001) Role for sperm in spatial patterning of the early mouse embryo. Nature 409:517–521.
Piotrowska K, Wianny F, Pedersen R a and Zernicka-Goetz M (2001) Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development. Development 128:3739–3748.
Plachta N, Bollenbach T, Pease S, Fraser SE and Pantazis P (2011) Oct4 kinetics predict cell lineage patterning in the early mammalian embryo. Nat Cell Biol 13:117–123.
Plusa B, Piliszek A, Frankenberg S, Artus J and Hadjantonakis A-K (2008) Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst. Development 135:3081–3091.
Ponsuksili S, Tesfaye D, Schellander K, Hoelker M, Hadlich F, Schwerin M and Wimmers K (2014) Differential expression of miRNAs and theirtarget mRNAs in endometria prior to maternal recognition of pregnancy associates with endometrial receptivity for in vivo- and in vitro-produced bovine embryos. Biology of Reproduction 91:135–135.
Pontes JHF, Nonato-Junior I, Sanches B V, Ereno-Junior JC, Uvo S, Barreiros TRR, Oliveira JA, Hasler JF and Seneda MM (2009) Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows. Theriogenology 71:690–697.
Potireddy S, Vassena R, Patel BG and Latham KE (2006) Analysis of polysomal mRNA populations of mouse oocytes and zygotes: Dynamic changes in maternal mRNA utilization and function. Developmental Biology 298:155–166.
199
Proffitt DK, Madan B, Ke Z, Pendharkar V, Ding L, Lee MA, Hannoush RN and Virshup DM (2013) Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer. Cancer Research 73:502–507.
Qin L, Liu C, Ni Y-G, Shi Y-N, Ao B-X, Liu Z and Liao D-F (2016) Wnt5a/Ror2 suppresses the proliferation of smooth muscle cell via Pkc signaling pathway. Journal of Hypertension 34:49–50.
Railo A, Nagy II, Kilpeläinen P & Vainio S (2008) Wnt-11 signaling leads to down-regulation of the Wnt/β-catenin, JNK/AP-1 and NF-κB pathways and promotes viability in the CHO-K1 cells. Experimental Cell Research 314:2389–2399.
Ralston A, Cox BJ, Nishioka N, Sasaki H, Chea E, Rugg-Gunn P, Guo G, Robson P, Draper JS and Rossant J (2010) Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2. Development 137:395–403.
Rayon T, Menchero S, Nieto A, Xenopoulos P, Crespo M, Cockburn K, Cañon S, Sasaki H, Hadjantonakis A-K, de la Pompa JL et al. (2014) Notch and Hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst Teresa. Dev Cell 30:410–422.
Reyes JM and Ross PJ (2016) Cytoplasmic polyadenylation in mammalian oocyte maturation. Wiley Interdisciplinary Reviews: RNA 7:71–89.
Rijsewijk F, Schuermann M, Wagenaar E, Parren P, Weigel D and Nusse R (1987) The Drosophila homology of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless. Cell 50:649–657.
Rizos D (2002) Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biology of Reproduction 68:236–243.
Rizos D, Ward F, Duffy PAT, Boland MP and Lonergan P (2002) Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: Implications for blastocyst yield and blastocyst quality. Molecular Reproduction and Development 61:234–248.
Robitaille J, MacDonald MLE, Kaykas A, Sheldahl LC, Zeisler J, Dubé M-P, Zhang L-H, Singaraja RR, Guernsey DL, Zheng B et al. (2002) Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nature Genetics 32:326–330.
Rossant J (2008) Stem Cells and Early Lineage Development. Cell 132:527–531.
Rossant J and Tam PPL (2009) Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 136:701–713.
Rossant J, Chazaud C and Yamanaka Y (2003) Lineage allocation and asymmetries in the early mouse embryo. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 358:1341–1348
200
Rudloff S & Kemler R (2012) Differential requirements for β-catenin during mouse development. 3721:3711–3721.
Sakagami N, Umeki H, Nishino O, Uchiyama H, Ichikawa K, Takeshita K, Kaneko E and Akiyama K (2012) Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese back cows. Reproduction and Development 58:140–146.
Sakurai N, Takahashi K, Emura N, Fujii T, Hirayama H, Kageyama S, Hashizume T and Sawai K (2016) The necessity of OCT-4 and CDX2 for early development and gene expression involved in differentiation of inner cell mass and trophectoderm lineages in bovine embryos. Cellular Reprogramming 18:309-318.
Samarage CR, White MD, Álvarez YD, Fierro-González JC, Henon Y, Jesudason EC, Bissiere S, Fouras A and Plachta N (2015) Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo. Developmental Cell 34:435–447.
Sartori R, Bastos M and Wiltbank M (2010) Factors affecting fertilisation and early embryo quality in single- and superovulated dairy cattle. Reprod Fertil Dev 22:151–158.
Sato N, Meijer L, Skaltsounis L, Greengard P & Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Medicine 10:55–63.
Sawa H (2012) Control of cell polarity and asymmetric division in C. elegans. Current Topics in Developmental Biology 101:55–76.
Schier AF (2007) The maternal-zygotic transition: death and birth of RNAs. Science 316:406–407.
Schmidt C, McGonnell IM, Allen S, Otto A & Patel K (2007) Wnt6 controls amniote neural crest induction through the non-canonical signaling pathway. Developmental Dynamics 236:2502–2511.
Schreck I, Al-Rawi M, Mingot JM, Scholl C, Diefenbacher ME, O’ Donnell P, Bohmann D & Weiss C (2011) C-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK. Biochemical and Biophysical Research Communications 407:735–740.
Seifert JRK and Mlodzik M (2007) Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility. Nature Reviews Genetics 8:126–138.
Sheldahl LC, Park M, Malbon CC and Moon RT (1999) Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Current Biology 9:695–698.
Sheldon IM (2015) Genes and environmental factors that influence disease resistance to microbes in the female reproductive tract of dairy cattle. Reprod Fertil Dev 27:72–81.
201
Shi W, Xu G, Wang C, Sperber SM, Chen Y, Zhou Q, Deng Y and Zhao H (2015) Heat Shock 70-kDa Protein 5 (Hspa5) Is Essential for Pronephros Formation by Mediating Retinoic Acid Signaling. Journal of Biological Chemistry 290:577–589.
Shih J and Keller R (1992) Cell motility driving mediolateral intercalation in explants of Xenopus laevis. Development 116:901–914.
Shimizu H, Julius MA, Giarré M, Zheng Z, Brown AM & Kitajewski J (1997) Transformation by Wnt family proteins correlates with regulation of β-catenin. Cell Growth & Differentiation 8:1349–1358.
Silhankova M and Korswagen HC (2007) Migration of neuronal cells along the anterior-posterior body axis of C. elegans: Wnts are in control. Current Opinion in Genetics and Development 17:320–325.
Simon C, Cano F, Valbuena D, Remohi J and Pellicer A (1995) Clinical evidence of detrimental effect on uterine receptivity of high serum oestradiol concentrations in high and normal responder patients. Human Reproduction 10:2432–2437.
Siqueira LGB and Hansen PJ (2016) Sex differences in response of the bovine embryo to colony-stimulating factor 2. Reproduction 152:645–654.
Sirard M (2012) Factors affecting oocyte and embryo transcriptomes. Reproduction in Domestic Animals 47:148–155.
Sjoblom C, Wikland M and Robertson SA (1999) Granulocyte – macrophage colony-stimulating factor promotes human blastocyst development in vitro. Human Reproduction 14:3069–3076.
Slusarski DC, Corces VG and Moon RT (1997) Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling. Nature 390:410–413.
Sokol SY (2011) Maintaining embryonic stem cell pluripotency with Wnt signaling. Development 138:4341–4350.
Sozen B, Can A & Demir N (2014) Cell fate regulation during preimplantation development : A view of adhesion-linked molecular interactions. Developmental Biology 395:73–83.
Später D, Hill TP, O’sullivan RJ, Gruber M, Conner D a and Hartmann C (2006) Wnt9a signaling is required for joint integrity and regulation of Ihh during chondrogenesis. Development 133:3039–3049.
Stark K, Vainio S, Vassileva G and McMahon a P (1994) Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature 372:679–683.
202
Stephenson RO, Yamanaka Y and Rossant J (2010) Disorganized epithelial polarity and excess trophectoderm cell fate in preimplantation embryos lacking E-cadherin. Development 137:3383–3391.
Stitzel ML and Seydoux G (2007) Regulation of the oocyte-to-zygote transition. Science 316:407–408.
Stringfellow D and Givens M (2010) Manual of the International Embryo Transfer Society: A Procedural Guide and General Information for the Use of Embryo Transfer Technology Emphasizing Sanitary Procedures. III International Embryo Transfer Society.
Strumpf D, Mao CA, Yamanaka Y, Ralston A, Chawengsaksophak K, Beck F and Rossant J (2005) Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132:2093–2102.
Sudano MJ, Santos VG, Tata A, Ferreira CR, Paschoal DM, Eberlin MN, Landim-Alvarenga FD and Machado R (2012) Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts. Biology of Reproduction 87:130.
Tadros W & Lipshitz HD (2009) The maternal-to-zygotic transition : a play in two acts. Development 136:3033–3042.
Tahinci E, Thorne C a, Franklin JL, Salic A, Christian KM, Lee L a, Coffey RJ & Lee E (2007) Lrp6 is required for convergent extension during Xenopus gastrulation. Development 134:4095–4106.
Takada R, Satomi Y, Kurata T, Ueno N, Norioka S, Kondoh H, Takao T, Takada S. (2006) Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Developmental cell 11:791-801.
Tarkowski AK and Wróblewska J (1967) Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. Journal of Embryology and Experimental Morphology 18:155–180.
Tesar PJ, Chenoweth JG, Brook F a, Davies TJ, Evans EP, Mack DL, Gardner RL and McKay RD (2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448:196–199.
Tetsu O & McCormick F (1999) β-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398:422–426.
Trigal B, Gómez E, Díez C, Caamaño JN, Martín D, Carrocera S and Muñoz M (2011) In vitro development of bovine embryos cultured with activin A. Theriogenology 75:584–588.
203
Tulac S, Nayak NR, Kao LC, Van Waes M, Huang J, Lobo S, Germeyer A, Lessey BA, Taylor RN, Suchanek E et al. (2003) Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium. The Journal of Clinical Endocrinology & Metabolism 88:3860–3866.
Uysal-Onganer P & Kypta RM (2012) Wnt11 in 2011 - the regulation and function of a non-canonical Wnt. Acta Physiologica 204:52–64.
Vainio S, Heikkilä M, Kispert a, Chin N and McMahon a P (1999) Female development in mammals is regulated by Wnt-4 signalling. Nature 397:405–409.
van Amerongen R & Nusse R (2009) Towards an integrated view of Wnt signaling in development. Development 136:3205–3214.
van Amerongen R and Berns A (2006) Knockout mouse models to study Wnt signal transduction. Trends in Genetics 22:678–689.
Van Langendonckt A, Donnay I, Schuurbiers N, Auquier P, Carolan C, Massip A and Dessy F (1997) Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medium. Journal of Reproduction and Fertility 109:87–93.
Varnum SM and Michael Wormington W (1990) Deadenylation of maternal mRNAs during Xenopus opcyte maturation does not require specific cis-sequences: A default mechanism for translational control. Genes and Development 4:2278–2286.
Veeman MT, Axelrod JD & Moon RT (2003) A second canon: functions and mechanisms of β-catenin-independent Wnt signaling. Developmental Cell 5:367–377.
Vendrell V, Vázquez-Echeverría C, López-Hernández I, Alonso BD, Martinez S, Pujades C and Schimmang T (2013) Roles of Wnt8a during formation and patterning of the mouse inner ear. Mechanisms of Development 130:160–168.
Verheijen MHG, Wolthuis RMF, Bos JL and Defize LHK (1999) The Ras/Erk pathway induces primitive endoderm but prevents parietal endoderm differentiation of F9 embryonal carcinoma cells. Journal of Biological Chemistry 274:1487–1494.
Vertino A, Taylor-Jones J, Longo K, Bearden E, Lane T, McGehee R, McDougald O and Peterson C (2005) Wnt10b deficiency promotes coexpression of myogenic and asipogenic programs in myoblast. Molecular Biology of the Cell 16:2039–2048.
Vestweber D, Ocklind C, Gossler A, Odin P, Öbrink B and Kemler R (1985) Comparison of two cell-adhesion molecules, uvomorulin and cell-CAM 105. Experimental Cell Research 157:451–461.
Voronina E and Wessel GM (2003) The regulation of oocyte maturation. Current Topics in Developmental Biology 58:53–110.
Wallingford JB (2005) Vertebrate Gastrulation : Polarity Genes Control the The PCP signaling cascade controls polarized cell behaviors in various. Current Biology 15:414–416.
204
Wang H and Dey SK (2006) Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 7:185–199.
Wang J, Zhang K, Wang J, Wu X, Liu X, Li B, Zhu Y, Yu Y, Cheng Q, Hu Z et al. (2015) Underexpression of LKB1 tumor suppressor is associated with enhanced Wnt signaling and malignant characteristics of human intrahepatic cholangiocarcinoma. Oncotarget 6:18905.
Wang Q, Lu J, Zhang S, Wang S, Wang W, Wang B, Wang F, Chen Q, Duan E, Leitges M et al. (2013) Wnt6 is essential for stromal cell proliferation during decidualization in mice. Biology of Reproduction 88:5.
Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C, Wilkins A, Perry VH, Sheth B, Kwong WY et al. (2008) Adaptive responses by mouse early embryos to maternal diet protect fetal growth but predispose to adult onset disease. Biology of Reproduction 78:299–306.
Westfall TA, Brimeyer R, Twedt J, Gladon J, Olberding A, Furutani-Seiki M and Slusarski DC (2003) Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/β-catenin activity. Journal of Cell Biology 162:889–898.
Wicklow E, Blij S, Frum T, Hirate Y, Lang R a, Sasaki H and Ralston A (2014) HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genetics 10.
Willert J, Epping M, Pollack JR, Brown PO and Nusse R (2002) A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC Developmental Biology 2:8.
Willert K, Brown JD, Danenberg E, Duncan a W, Weissman IL, Reya T, Yates JR and Nusse R (2003) Wnt proteins are lipid-modi ed and can act as stem cell growth factors. Nature 423:448–452.
Williams BL, Gwazdauskas FC and Pearson RE (1992) The effect of day of the estrous cycle, location of ovulatory, structure, and progesterone on in vitro bovine,endometrial secretions. Journal of Dairy Science 75:2112–2118.
Wolff B, Sanglier JJ & Wang Y (1997) Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo- cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem Biol 4:139–147.
Wray J, Kalkan T, Gomez-Lopez S, Eckardt D, Cook A, Kemler R & Smith A (2011) Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nature Cell Biology 13:838–845.
Wrenzycki C, Herrmann D, Carnwath J and Niemann H (1996) Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. Journal of Reproduction and Fertility 108:17–24.
205
Xiao S, Diao H, Smith MA, Song X and Ye X (2011) Preimplantation exposure to bisphenol A (BPA) affects embryo transport, preimplantation embryo development, and uterine receptivity in mice. Reproductive Toxicology 32:434–441.
Xie H, Tranguch S, Jia X, Zhang H, Das SK, Dey SK, Kuo CJ and Wang H (2008) Inactivation of nuclear Wnt-β-catenin signaling limits blastocyst competency for implantation. Development 135:717–727.
Yagi R, Kohn MJ, Karavanova I, Kaneko KJ, Vullhorst D, DePamphilis ML and Buonanno A (2007) Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 134:3827–3836.
Yamanaka Y, Ralston A, Stephenson RO and Rossant J (2006) Cell and molecular regulation of the mouse blastocyst. Developmental Dynamics 235:2301–2314.
Yan L, Yang M, Guo H, Yang L, Wu J, Li R, Liu P, Lian Y, Zheng X, Yan J et al. (2013) Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nature Structural & Molecular Biology 20:1131–1139.
Yang Y (2003) Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development 130:1003–1015.
Ying Q, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P and Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–524.
Yoshinaga K and Adams CE (1966) Delayed implantation in the spayed, progesterone treated adult mouse. Journal of Reproduction and Fertility 12:593–595.
Young LE, Sinclair KD and Wilmut I (1998) Large offspring syndrome in cattle and sheep. Reviews of Reproduction 3:155–163.
Yuan J, Zhang J, Wong BW, Si X, Wong J, Yang D & Luo H (2005) Inhibition of glycogen synthase kinase 3β suppresses coxsackievirus-induced cytopathic effect and apoptosis via stabilization of β-catenin. Cell Death and Differentiation 12:1097–1106.
Zeke A, Misheva M, Remenyi A & Bogoyevitch MA (2016) JNK Signaling : Regulation and Functions Based on Complex Protein- Protein Partnerships. Microbiology and Molecular Biology Reviews 80:793–835.
Zernicka-Goetz M, Morris S a and Bruce AW (2009) Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo. Nature Reviews. Genetics 10:467–477.
Zhao B, Li L, Tumaneng K, Wang CY and Guan KL (2010) A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP. Genes and Development 24:72–85.
206
Zhao B, Zhao B, Wei X, Wei X, Li W, Li W, Udan RS, Udan RS, Yang Q, Yang Q et al. (2007) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes & Development 21:2747–2761.
Zheng W, Gorre N, Shen Y, Noda T, Ogawa W, Lundin E and Liu K (2010) Maternal phosphatidylinositol 3-kinase signalling is crucial for embryonic genome activation and preimplantation embryogenesis. EMBO Rep 11:890–895.
Zinovyeva AY, Yamamoto Y, Sawa H and Forrester WC (2008) Complex network of Wnt signaling regulates neuronal migrations during Caenorhabditis elegans development. Genetics 179:1357–1371.
Zuo Y, Su G, Wang S, Yang L, Liao M, Wei Z, Bai C and Li G (2016) Exploring timing activation of functional pathway based on differential co-expression analysis in preimplantation embryogenesis. Oncotarget 7:74120–74131.
207
BIOGRAPHICAL SKETCH
Paula Tribulo was born and raised in Cordoba, Argentina. She started learning
about reproductive biology very early in life because her father is a veterinarian working
mainly in bovine reproduction. After graduating from veterinary school from The Catholic
University of Cordoba, she pursued a post-graduate degree program in “Specialization
in Bovine Reproduction” in Cordoba, Argentina. Following graduation, she moved to
Saskatoon, Canada to do an internship funded by an award from the Canadian
Government. Upon completion of the internship, she went home to get married, and
started her graduate career. Paula and her husband, Marcos Zenobi, moved to
Saskatoon where the University of Saskatchewan awarded her a Dean’s Scholarship to
pursue a Master of Science working in reproductive physiology under the supervision of
Dr. Greg Adams. After graduation in 2012, Paula, Marcos and their newborn son,
Lautaro, moved to Gainesville to begin a doctoral program with Dr. Peter Hansen, in the
Animal Molecular and Cellular Biology Graduate Program at University of Florida.
After completing her Doctor of Philosophy degree, Paula will continue to work as
a postdoctoral scientist with Dr. Hansen for a year. Paula’s long-term goals are to return
to Argentina to become a professor and scientist in her country.