Advanced Glycobiology May 2016

1

Glossary of Terms for Fertilization. Implantation, Placentation Accessory Gland (male reproductive): Prostate, seminal vesicle and Cowper’s (bulbourethral) gland, that contribute to seminal fluid during ejaculation. Amniotic Fluid: Fluid bathing the developing embryo. Acrosome: Sperm organelle derived from Golgi. Alpha agglutinin: Mating type protein in yeast. Anisogamy: Gametes differ in size. ART: Assisted Reproduction Technology. Atresia: Degeneration of ovarian follicles resulting in depletion of follicles with eggs in females. Beta-Lactoglobulin: Major whey protein in cow and sheep’s milk. Beta Defensin 126: Sialylated epiddymal defensing with mucin-like domain. Bindin: Sperm protein in sea urchin sperm. CA-125: (MUC 16) Mucin found on immune cells and secreted by ovarian cells, both normal and cancerous. Capacitation: Changes in sperm triggered by exposure to female tract factors resulting in membrane reorganization motility and signal transduction. Chauvinist gene: Gene only expressed in the male germ line e.g. protamines, DEF126, PH20. Corpus luteum: (yellow body) Temporary endocrine structure formed by ovulated follicle in female ovary. Cortical Reaction: Degranulation reaction releasing glycoproteins and enzymes that block polyspermy upon fertilization. Cumulus Oophorus: Mass of cumulus cells and secretions associated with the ovulated egg. The cells secrete hormones (progesterone) and have been suggested to be involved in nutrition of sperm, selection of sperm with normal morphology, induction of acrosome reaction and support of oocyte maturation. Cytotrophoblast: Placental cells attached to the placenta, forming the disk shaped placenta and the villi. Decidua: Lining of the endometrium which is shed each month. Endometrium: Inner mucus membrane of mammalian of uterus. Epididymis: Organ next to testes that is involved in sperm maturation. Fallopian Tubes (Salpynx/oviduct): Upper portion of the female reproductive tract. Feto-Embryonic Protection System: Concept of molecular networks that shield the fetus and embryo from maternal immune rejection. Follicle (Ovarian): Structure holding female germ cells. Follicle stimulating hormone (FSH): FSH is synthesized and secreted by the gonadotropic cells of the anterior pituitary gland,[1] and regulates the development, growth, pubertal maturation, and reproductive processes of the body. Fucoidan: Sulphated fucose polymer. FUT5: Fucosyl transferase on sperm membrane that acts only as lectin. Germ line/ Soma: Cells that generate gametes and cells that form the perishable body.

Advanced Glycobiology May 2016

2

Glycodelin (Placental Protein 14, PP14/ Progestagen associated endometrial protein, PAEP): 180 AA lipocalin protein heavily glycosylated 17.5% by weight) with hoimology to β lactoglobulin. Glycodelin A: Isoform with anti-fertilization properties secreted by female reproductive tract. Immune suppressive, pro-apoptotic for immune cells and involved in implantation. Glycodelin S: Non-sialylated. Fucose-rich glycoform, found in seminal plasma. Glycodelin C: Deglycosylated version of Glycodelin F (taken up by cumulus cells and partially deglycosylted), stimulate sperm-ZP binding Glycodelin F: Follicular fluid Gdn, binds spermatozoa in head region and inhiibits acrosome reaction. Granulosa Cells: Cells of the ovarian follicle that give rise to cumulus oophorus, produce sex steroids and growth factors. Hemizona Assay: Use of bisected matching halves of an ovum to test ZP binding by capacitated sperm in vitro. One of the halves can be exposed to test substance while the other provides the control. Hyperactivation: Characteristic change in motility of sperm after capacitation. ICSI: Intra-cytoplasmic sperm injection, allows fewrtilization of eggs by immotile sperm, used in humans since 1992. Isogamy: Gametes are of same size, ancestral state still in existence in some algae and yeast. IVF: In vitro fertilization, involving the overlay of an ovum with millions of sperm. Interstitial trophoblast: “Free floating” invasive throphoblast (fetal placental) cells that invade the decidua and interact with NK cells of the mother. Luteinizing hormone (LH): Hormone produced by gonadotropic cells in the anterior pituitary that triggers ovulation in females, stimulates testosterone production by Leydig cells in males. Myometrium: Muscular layer of the uterus. Ovastacin: A metalloproteinase similar to hatching enzymes from arthropods released by cortical granules, cleaves ZP2. Perivitelline Space: Space between egg membrane and zone pellucida. PH20/SPAM1: Sperm Adhesion molecule 1, HYAL1, hyauronidase 1. Polycystic Ovary Syndrome (PCOS): Condition associated with increased secretion of luteinizing hormone, androgen and insulin. SialylLewisX: Tetra saccharide with α2,3 linked Sia and α1,3 linked fucose. Sperm Chromatin Structural Assay (SCSA): Assay for quantifying DNA breaks in sperm. Syncytiotrophoblast: Outermost layer of placenta (trophoblast) consisting of multinucleated cells (syncytia). Trophoblast: Outer layer of blastocyst which gives rise to placenta. Utero-Tubal Junction: Transition between uterus and lower fallopian tubes, Vestment: Extracellular matrix and cumulus cells surrounding the ovulated ovum. VERL: Mollusk egg coat receptor for sperm lysine. Window of Implantation: Eight days after luteinizing hormone surge (ovulation) Zona pellucida: (“transparent zone”) Extra-cellular matrix of the mature egg.

5/12/16&

1&

Advanced Glycobiology

Lecture 14 Fertilization, Implantation, Placentation

May 12, 2016 Pascal Gagneux

Division, Reproduction, Aging? Caulobacter crescentus

Ackermann & Stearns Science (2003)

Fig. 1. (A) Life cycle of C. crescentus.

Reproduction Asexual

Parthenogenesis Sexual

Aphid fundatrix giving birth to already gravid offspring

Female Whiptail lizards laying unfertilized eggs Most vertebrates

Advantages of Sex

•  Purging of deleterious mutations.!•  Maintenance of genetic variation. !•  Evolutionary Evasion of pathogens and parasites (Red Queen). !

Disadvantages / Cost of Sex

•  Need for males and female!•  Loss of co-adapted allele complexes !•  Conflict between sexes !•  Genomic conflicts, e.g. meiotic drive. !

Micro and Macro Gametes

•  Why are same size gametes so rare?!

5/12/16&

2&

Requirement for Gamete Recognition

•  Costs of Outbreeding and Inbreeding !

Joly, E. Biology Direct (2011)

The existence of species rests on a metastable equilibrium between inbreeding and outbreeding. !Swanson & Vacquier. Nat. Rev. Genetics (2002)

Rapid Evolution of Recognition

Ene, J.V. and Bennett, R.J. Nat. Rev. Microbiol. (2014)

Asexual, Parasexual, Sexual External vs Internal Fertilization

•  How to find each other? !•  How to survive in another individual’s body? !

External vs Internal Gestation

•  Laying eggs and incubation. !•  Laying developing eggs (brood parasites, Honey Guide)!

•  Ovo-vivipary (Tsetse Fly)!

Implantation

•  Embryo -Endometrial recognition (opportunity for quality control).!

5/12/16&

3&

Swanson et al. Mol. Biol Evol. (2011)

The Molecular Basis of Sex

Yeast alpha aggluinin

Abalone VERL

Human ZP

Abalone VERL with N-Glycosylation sites

Placentation

•  Immunological Paradox of Mammalian Pregnancy !

Peter Medawar!

Immunological Paradox of Mammalian Pregnancy ! Victim of parental glycan mismatch!

1956, Obstetrics and Gynecology

•  Male germ cell must navigate to the egg !- survive and evade female immune response !- retain functional potential of acrosome !- retain integrity of haploid genome !

Species Population Individual Gamete

•  Correct species !

•  Not a pathogen!

•  High fitness mate ! •  Correct diploid alleles!

•  Compatible genome!

•  Acrosome-intact!

•  Genetically intact!

•  Female must protect against incompatible sperm !!

Immune paradox of insemination! !

Darszon et al. Physiol. Rev. (2010)

Male Gametes!

5/12/16&

4&

Lectins on Sperm

Nicolson & Yanagimachi Science (1972)!

The Mammalian Sperm Glycome!

loosely associated glycoprotein(e.g. glycodelin S)

gangliosides

polysialic acid

heparan sulfate

gangliosides

GPI anchored glycoprotein

GPI anchored glycoprotein

beta defensin

beta defensin

membrane glycoprotein

Tecle & Gagneux 2015 Mol. Repr. Develop.

Spermatogenesis!

Tecle & Gagneux 2015 Mol. Repr. Develop. Tecle & Gagneux 2015 Mol. Repr. Develop.

Synthesis of the Mammalian Sperm Glycome!

Schröter, S, et al. Human Repr. Update . (1999)

Sperm Glycocalyx

SPAG11!

CD52!

Schröter, S, et al. Human Repr. Update . (1999)

Sperm Glycocalyx

5/12/16&

5&

Sugar nucleotide: CMP-Neu5Ac Glycosyl transferases: ST6Gal I

Bellannée et al. 2013 Cell Tissue Res.

Testis

Head1 Head2

Corpus Cauda

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Picmol/ 100ug protein

CMP-Neu5Ac

Ma et al. in press

The Epididymis, an extra-cellular golgi?!

Ma et al. in prep.

Ma et al. in press

Gain of Sialic Acids“SAMP”

Self Associated Molecular Pattern

Tollner, T. L. et al. 2012. Nat. Rev. Urol.

DEFB126 highly sialylated glycoprotein CD-52, PH20,CD55, CD59, Prion GP sialylated GPI anchored gycopeptide Gangliosides (mono-. di- & trisialylated)

20 glycosylation sites/ DEFB126, heavily sialylated

Acquisition of a special, highly sialylated glycocalyx during epididymal transit:!

DEFB126!

Theodore L. Tollner, Charles L. Bevins & Gary N. CherrNature Reviews Urology 9, 365-375 (July 2012)

DEF126 deletion mutation!

Xin et al. Scientific Reports (2016)

Lectin binding of human spermassociates with DEFB126 mutationand serves as a potential biomarkerfor subfertility, yet frequency isaround 45%!

Opportunities for Sperm competition and Cryptic Female Choice

Swelling

Vaginal secretions

Cervical cryptsCervical mucins

Utero-tubal junction

OviductLength ofoviduct

~15cm

~15cm

30 cm

6km

60 µm 100 X106

100s

CAPACITATION

sprint

Uterine cavity: leukocytes antibodies complement

1

Immunological Paradox of Mammalian Fertilization !

modified from Hafner et al. 2013. Mucosal Immunology

Leukocytic Reaction

Sialic acid binding Ig Superfamily Lectins

Siglecs2 7 9

Female Reproductive Immunity !

5/12/16&

6&

Functions of the Mammalian Sperm Glycome !

Tecle & Gagneux 2015 Mol. Repr. Develop.Andersch-Björkman et al., Molecular & Cellular Proteomics (2007)

Cervical Mucins during cycle and HA during pregnancy

Three gel-forming (MUC5B, MUC5AC, and MUC6) and twotransmembrane mucins (MUC16 and MUC1) were identified.

Brunelli et al., FASEB J.(2007)

Atomic Force Microscopy

Immune Modulation by the sperm glycocalyx !

Pang et al. JBC (2007)

bisecting type N-glycans

Sperm hyaluronidase!

Theodore L. Tollner, Charles L. Bevins & Gary N. CherrNature Reviews Urology 9, 365-375 (July 2012)

Gamete glycocalyces

•  Egg and Sperm have special glycoclyces. !•  Egg Zone Pelucida!

Okabe, M Development (2013)

Yauger, B, Boggs, NA, and Dean J. Reproduction (2011)

Egg Glycocalyx

5/12/16&

7&

Pang et al. Science (2011)

Sialyl Lewis X on ZP N-glycans UT Junction and Calmegin KO

Nakanishi et al. Biol Reprod. (2004)!

Yamaguchi et al. Biol Reprod. (2009)!

UT Junction and ADAM3 KO

Liu et al. Reprod Biol Endocrinol. (2003)

Egg Glycocalyx: dynamic cortical granules

LCA, Lens culinaris agglutinin ABL2 mAb

Okabe, M Asian J Andrology. (2015)

A new view of mammalian fertilization

Okabe, M Development (2013)

5/12/16&

8&

Okabe, M Asian J Andrology. (2015)

double fusion

Okabe, M Asian J Andrology. (2015)

Izumo-taisha!出雲大社!

Japanese Shrine !Juno!

Roman Goddess!

Bianchi, E. et al. Nature (2014)

Solid protein hand shake

Seppälä et al. Human Repr. Update (2007)!

Glycodelin Glycoforms Immunosuppressive Anti-fertilizing

With many Glycan Falvors! Immunological Paradox of Mammalian Fertilization !

5/12/16&

9&

Early stages of human development from fertilization to blastocyst formation

Mouse Blastocyst!stained for Neu5Gc!

sialic acid ! Genbacev et al. Science (2003)!

Glycan-mediated implantation !

Ilekis. Potential placental molecular therapeutic targets. Am J Obstet Gynecol (2016)!

Making a home !

Red-Horse et al. Development (2005)

Home Invasion!!

Vascular Remodelling!

Red-Horse et al. Development (2005)

Anchoring and Invading!

Red-Horse et al. Development (2005)

5/12/16&

10&

Syncytin Glycoprotein capture

Brinkman-Van der Linden et al. Glycobiology. (2007)

Siglec 6 on Human Placenta

Placentation

Brinkman-Van der Linden et al. Glycobiology. (2007)

Preecclampsia: Pregnancy Associated Hypertension

Parham & Moffet Nature REV Immunol. (2013)

Rumer et al. Reproductive Sciences. (2012)

Placentation preecclampsia upregulation of Siglec 6

Gubbels et al. Molecular Cancer (2010)

Placentation MUC16 as NK cell suppressor

MUC16 interacts with Siglec 9 on NK cells

5/12/16&

11&

Glycobiology of Human Reproduction - !How Sugarcoating Solves Difficult Interactions!