AN ANNUAL CELEBRATION OF RESEARCH IN REPRODUCTIVE SCIENCES

ANIMAL REPRODUCTION AND BIOTECHNOLOGY LABORATORY

THE 14th ANNUAL

Rocky Mountain Reproductive Sciences Symposium

2021 CONFERENCE THEME:

Human Implantation:How much do we know?

APRIL 16, 20218:45 A.M.– 4:15 P.M.

VIRTUAL CONFERENCE

Hosted by CSU's Animal Reproduction & Biotechnology Laboratory and sponsored by

the Society for the Study of Reproduction

arbl.colostate.edu | ssr.org

Program Table of Contents………………………………..…….……..1About the Symposium……………………….……….………..………..2Program Agenda……………………..………………………..…….……..3Student Platform Session Abstracts………………..……...........9Poster Session Abstracts………….…...................................18Acknowledgements…………………….………….…....................41

RMRSS 2021PROGRAM CONTENTS

1

RMRSS 2021

Now in its 14th year, the Rocky Mountain Reproductive Sciences Symposium brings together a diverse group of scientists to discuss advances in both human and animal reproduction that deepen our understanding of reproductive physiology. It’s a one-day event focused on student training, not only to significantly improve communication and cross-fertilization of research ideas, but also to share resources and expertise across human and animal models.

Hosted virtually this year by the Society for the Study of Reproduction and CSU’s Animal Reproduction & Biotechnology Laboratory, the day's events feature student abstract platform presentations, poster sessions, and keynote lectures by leaders in the field of reproductive physiology. Attendees include post-baccalaureate trainees, faculty, private clinicians, and other research scientists. The symposium has led to the establishment of new collaborations between institutes to advance the field of reproductive sciences and is a great platform for student and fellow training.

2021 Keynote Speakers

“Using stem cells to model peri-implantation immune events in humans”Danny Schust, MDDavid G. Hall professor of obstetrics and gynecology and director of the Division of Reproductive Endocrinology and Infertility, University of Missouri School of Medicine

“In vitro culture of human embryos beyond implantation” Ye Yuan, PhDResearch Director, Colorado Center for Reproductive Medicine

2

8:50 AM Opening Remarks: Dr. Thomas (Tod) Hansen, ARBL, Colorado State University

9:00 AM Keynote Lecture: Using stem cells to model peri-implantation immune events in humans. Dr. Danny Schust, MD. David G. Hall Professor of Obstetrics and Gynecology and Director of the Division of Reproductive Endocrinology and Infertility, University of Missouri School of Medicine-Intros & first questions: Rachel West & Karli Swenson

10:15 AM Trainee Oral Platform Presentations, Part I-10 min talk with 5 min questions-Intros & first questions: Evelyn Llerena Cari & Carolina Gonzalez-Berrios

10:15-10:30 AM – Hanah Georges, Colorado State University1. BVDV Fetal Immunotolerance; A Unique Story of Epigenetics AndOsteoimmunology

10:30-10:45 AM – Gwendolynn Hummel, University of Wyoming2. Feed Intake Restriction Decreases Microbiome Diversity in The MaternalRumen and Placenta, But Not the Vagina, In Late Gestation

10:45-11:00 AM – Shaihla Khan, Colorado Center for Reproductive Medicine3. Impaired Mitochondrial Quality Control Pathways Undermine the Quality ofAged Mouse and Human Oocytes

11:00-11:15 AM – Deirdre Logsdon, Colorado Center for Reproductive Medicine4. Maternal Physiology and Blastocyst Morphological Grade Correlate with anInherent Difference in Human Peri-Implantation Developmental Potential

11:15 AM Break/Lunch (Lunch can extend into the poster sessions where/when convenient).

3

RMRSS 2021PROGRAM AGENDA

11:30 AM- 1:00 PM - Poster SessionPoster presentations will be pre-recorded and sent to SSR’s Virtual Conference Company. These will be managed in one of 4 virtual rooms (with 5-6 presentations per room). After playing the 5-minute poster presentation video, the presenter will be available online to address any questions for 5 minutes. Attendees can move from room to room to view student poster presentations of interest.

Virtual Room 1: Developmental origins of health and disease-Intros & first questions: Hanah Georges & Gwendolynn Hummel

11:30-11:45 AM - Betelhem Ashebo, University of Colorado9. Increased Lactate Production and Mass in The Perirenal Adipose Tissue ofFetal Sheep with Prolonged Hypoxemia

11:45 AM-12:00 PM - Cameron Lynch, Colorado State University 10. Uncovering the Importance of Placental SLC2A3 (GLUT3) On Fetal Growthand Physiology at Mid-Gestation Via RNA Interference in Sheep

12:00-12:15 PM - Sarah Weir, University of Wyoming11. Impacts of Fetal Sex on Relative Percent of Specific Phyla Colonizing theBovine Placenta

12:15-12:30 PM - Rachel West, Colorado Center for Reproductive Medicine12. The Human Endogenous Retrovirus ERVW-1 Promotes Trophoblast StemCell Proliferation and Differentiation Towards Syncytiotrophoblast

12:30-12:45 PM - Joseph Westrich, Colorado State University13. Establishment of An Immune-Competent Animal Model of Subclinical ZikaVirus Infection and Its Impact at The Maternal-Fetal Interface

4

RMRSS 2021PROGRAM AGENDA

Virtual Room 2: Gamete and Early Embryo-Intros & first questions: Dr. Agata Parsons & Shaihla Kahn

11:30-11:45 AM - Giovana Di Donato Catandi, College of Veterinary Medicine and Biomedical Sciences, Colorado State University (CSU)14. Mare Diet Supplementation Alters Oocyte Lipid Content andDevelopmental Competence

11:45 AM-12:00 PM - Kyle Fresa, Colorado State University15. PEPCK Activity is Essential During Early Embryo Development

12:00-12:15 PM - Carolina Gonzalez Berrios, Colorado State University,16. Elucidating the Mechanism of Early Embryo Mortality in Holstein-FriesianCows

12:15-12:30 PM - Deirdre Logsdon, CO Center for Reproductive Medicine17. Prolonged Exposure of Human Blastocysts to Hyaluronan-Enriched TransferMedia Has No Effect on Peri-Implantation Stage Embryo Development DuringIn Vitro Culture

12:30-12:45 PM - Riley Thompson, College of Veterinary Medicine and Biomedical Sciences, Colorado State University18. Generation of Organoids Derived from Agricultural and Companion AnimalReproductive Tissue: Preliminary Findings

12:45-1:00 PM - Sung Woo Kim, Animal Genetic Resources Research Center, National Institute of Animal Science, RDA19. Multiple IVF method using single frozen straw in bovine species

5

RMRSS 2021PROGRAM AGENDA

Virtual Room 3: Reproductive Endocrinology/Neuroendocrinology-Intros & first questions: Dr. Brenda Alexander

11:30-11:45 AM - Katherine Agster, University of Wyoming, Laramie20. The Effect of Medroxyprogesterone Acetate on Neural Activity In TheVentral Tegmental Area Of Reindeer Bulls

11:45 AM-12:00 PM - Amy Desaulniers, University of Central Missouri21. Stimulation of GnRHR-II Partially Rescues Steroidogenesis DespiteSuppressed Luteinizing Hormone Secretion in Neonatal Boars

12:00-12:15 PM - Jessica Lederman, Colorado State University22. Estrus Suppression in The Mare Through the Use of Altrenogest ReleasingIntravaginal Rings

12:15-12:30 PM - Nanette Santoro, University of CO School of Medicine23. Gonadotropin Response to Insulin and Lipid Infusion Reproduces theReprometabolic Syndrome of Obesity in Eumenorrheic Lean Women: ARandomized Crossover Trial

12:30-12:45 PM - Rosemary McDonald, University of Colorado 24. Cell Type Specific Effects of Hyperlipidemia and Hyperinsulinemia,Characteristic of Reprometabolic Syndrome, on Pituitary Function

Virtual Room 4: Reproductive Endocrinology/Ovarian Signaling-Intros & first questions: Dr. Pat Burns

11:30-11:45 AM - Thanh Ha Luu, University of Colorado 25. Can IV Administration of Rfsh Correct the HypogonadotropicHypogonadism State Seen in Obese Women?

11:45 AM-12:00 PM - Amanda Christensen, University of Wyoming26. Hormonal Impact on Citrullination and Peptidyl Arginine DeiminaseActivity in Female Reproductive Tract 6

RMRSS 2021PROGRAM AGENDA

12:00-12:15 PM Samuel Gebermedhn, Colorado State University27. Targeted Loading of Heat Stress-Associated Mirnas Into ExtracellularVesicles: Potential Application to Modulate Response to Heat Stress in BovineGranulosa Cells

12:15-12:30 PM - Nico Menjivar, Colorado State University28. The Role of Granulosa Cell Derived Extracellular Vesicles in Bovine OocytesExposed to Thermal Stress During In Vitro Maturation

12:30-12:45 PM - Anika Shelrud, University of Northern Colorado29. The Effects of Omega-3 Fatty Acids on Cytokine-Induced Regulated CellDeath in Bovine Luteal Microvascular Endothelial Cells”

12:45-1:00 PM - Corrine Monaco, University of Nebraska Medical Center 30. Fibroblasts of the Bovine Corpus Luteum Release Prostaglandin F2α Into theMicroenvironment in Response to Pro-Inflammatory Cytokines

1:00 PM Trainee Oral Platform Presentations, Part II -10 min talk with 5 min questions-Intros & first questions: Dr. Samuel Gabermedhn & Dr. Riley Thompson

1:00-1:15 PM – Michael Nash, University of Colorado, Anschutz Medical Campus5. Maternal Western-style Diet Drives Glycolytic Programming inHematopoietic Progenitor Cells and Underlies a Pro-fibrotic Liver Response inNon-human Primate Offspring

1:15-1:30 PM – Asma Omar, Colorado State University6. Maternal High-Fat Diet Increases Fetal Muscle Fat Metabolism and FattyAcid Transporter Expression in An Ovine Model

7

RMRSS 2021PROGRAM AGENDA

1:30-1:45 PM – Agata Parsons Aubone, Colorado State University Preliminary 7. Analysis of Androgen Receptor Knockdown Placentas in Sheep

1:45-2:00 PM – Amelia Tanner, Colorado State University8. Chorionic Somatomammotropin RNA Interference Reduces Global NutrientUptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction

2:10 PM Keynote Lecture: In vitro culture of human embryos beyond implantation. Dr. Ye Yuan, Research Director Colorado Center for Reproductive Medicine-Intros & first questions: Dr. Deirdre Logsdon & Rosie McDonald

3:10 PM Closing comments by Tod Hansen and Committee and opening of informal virtual room discussions

Virtual Room 1: Reproductive Endocrinology

Virtual Room 2: Gamete and Early Embryo

Virtual Room 3: Maternal fetal medicine

Virtual Room 4: Developmental origins of health and disease

Virtual Room 5: Male/Female reproductive pathology

Virtual Room 6: Reproductive Oncology

Virtual Room 7: Covid-19 and reproduction

4:10 PM 2021 RMRSS closes

8

RMRSS 2021PROGRAM AGENDA

RMRSS 2021STUDENT PLATFORM SESSION ABSTRACTS

9

1. BVDV fetal immunotolerance; a unique story of epigenetics andosteoimmunology. First Author: Hanah Georges h.georges@colorstate.eduColorado State UniversityPre-Doctoral Platform

Maternal infection with Bovine Viral Diarrhea Virus (BVDV) has life-long negative effects on progeny. Despite preventative measures, BVDV continues to plague the cattle industry, costing $1.5 billion annually and producing persistently infected (PI) calves that remain the primary reservoirs of the virus. Previous studies concluded that attenuation of the PI fetal immune system was caused by a lack of T-cell response in the fetus, resulting in an inability for T-cells and B-cells to mature properly. In this study, it was hypothesized that T-cell activation and signaling genes were epigenetically altered after fetal infection. Splenic tissue from PI and control fetuses were collected on day 245 of gestation, 170 days post-maternal infection. DNA was isolated for reduced representation bisulfite sequencing and protein isolated for proteomics. Methylation sequencing files were bioinformatically analyzed using the methylKit R package while proteomics was performed using Scaffold. Within set parameters, 2,641 regions were differentially methylated: 1,951 hypermethylated and 691 hypomethylated regions in PI fetuses compared to controls. Results revealed hypermethylation of nuclear factor of activated T cells (NFAT) 1 and 4, which is likely to shift the Th cell differentiation from Th1 to Th2 cells. An increase in NFAT2 due to hypomethylation would promote anergy of T-cells, further exacerbating the shift from Th1 to Th2 cells. This shift of Th cells is associated with T-cell receptor hyper-reactivity and lymphoproliferative disorder. Additionally, the hypomethylation of ORAI and calmodulin may contribute to the Th2 hyper-reactivity by increasing the amount of calcium transported into a cell upon T-cell activation. The proteomic analysis revealed 12 significantly different proteins in PI vs. control animals. Increased proteins (TMA7, PEPD, NUDC, SNRPF) in PI vs. controls are associated with protein processing, most likely contributing to viral replication. Decreased proteins are mostly associated with lymphocyte migration and development, possibly indicating impaired immune development due to early fetal infection (THY1, HNRPC, CSRP1, VAPB, CSTB, AKAP2, CNN2). Of particular interest, THY1, decreased in protein expression and hypermethylated, is important for cell adhesion and communication in the immune system, for T-cell activation, and for osteoblast formation. Additionally, VAPB, which was also decreased in PI fetuses, is critical for osteoclast differentiation and is regulated by the NFAT pathway. Together, the decrease in THY1 and VAPB through direct methylation and methylated regulatory pathways may contribute to the altered bone formation in PI animals seen clinically in several PI infections. Osteoclast and osteoblasts are also critical immune cells, being hematopoietic cells in the same monocytic family as monocytes, macrophages, and dendritic cells, they have recently been identified as T-cell modulators and antigen presenting cells. In addition to the decrease in osteoclast and osteoblast proteins contributing to altered bone development, it is likely that this decrease would also have detrimental effects on T-cell development and activation. The observed epigenetic modification of critical T-cell genes and the decreased expression of proteins critical for immune and bone development may help explain the inability of postnatal PI calves to fight secondary infections efficiently, contributing to performance loss and continued BVDV viral shedding.

10

2. Feed intake restriction decreases microbiome diversity in the maternal rumen and placenta,but not the vagina, in late gestation.First Author Gwendolynn Hummel ghummel@uwyo.edu, University of Wyoming

Pre-Doctoral Platform

The mature maternal rumen microbiome offers a potentially rich source of microbes for the seeding of the reproductive and placental microbiomes, although the impacts of feed restriction upon these microbial communities remains unknown. We hypothesize the maternal rumen microbiome is altered by feed restriction, and these microbial changes are reflected in the placental, but not vaginal, microbiome in late gestation. Our objectives were to isolate and identify microbes from rumen fluid (RF) and the vagina (VAG) of control (CON; n=8) and 70% feed intake restricted (FR; n=7) beef cows 10 days prior to their expected calving date, and compare these microbial communities to those of the placenta and vagina. Experimental protocols were approved by the University of Wyoming International Care and Use Committee. After 16S rRNA sequencing of the hypervariable V4 region, all analyses was performed in QIIME2. Each P-value was adjusted for false discovery rate, and significance considered when q ≤ 0.05. Feed intake restriction decreased the richness (q = 0.03) and non-phylogenetic diversity (q = 0.05) of the FR rumen compared to CON dams. However, FR did not affect the VAG microbiome (q ≥ 0.11). Interestingly, when comparing the RF and VAG microbiome, samples from FR cows did not differ across these sample types (q ≥ 0.08) but CON samples did differ in evenness, richness, and Bray-Curtis (q ≤ 0.05), potentially indicating a more robust microbial relationship between these maternal microbiomes. All tissues sampled from the expelled bovine placenta, including the intercotyledonary membrane (ICM), cotyledon (COT), and allantois (AL), differed in non-phylogenetic beta-diversity between CON and FR pregnancies (q ≤ 0.05). A less diverse placental microbiome, particularly within the AL, was present in FR pregnancies, where similarities existed between the AL and COT in FR pregnancies in unweighted UniFrac (q = 0.11) that were not observed in CON pregnancies (q = 0.02). Additionally, the ICM and COT differed in weighted UniFrac (q ≤ 0.04) in CON placentae where these FR tissues did not differ (q = 0.12). This lack of diversity continued when the FR AL was compared to the RF, which tended toward similarity in weighted and unweighted UniFrac (q = 0.07). The CON AL and RF were different under both metrics (q ≤ 0.02). However, neither the RF or VAG of either treatment differed from the placenta under Bray-Curtis or Jaccard distances (q ≥ 0.12). These findings indicate that, although the relationships between the maternal rumen and reproductive microbiomes remain generally stable, FR may be capable of altering the diversity and ecology of the placental microbiome. Overall, this may indicate that the fetal gut and other reproductive microbiomes may be impacted by the plane of maternal nutrition.

11

3. Impaired Mitochondrial Quality Control Pathways Undermine the Quality of Aged Mouseand Human OocytesFirst Author: Shaihla Khan, skhan@flcolo.com, Colorado Center for Reproductive MedicinePost-Doctoral Platform

The quantity and competence of oocytes decreases rapidly as women age. Studies have shown that mitochondrial metabolism is critical for oocyte and subsequent embryo quality. Therefore, the objective of this study was to investigate the mechanisms involved in loss of mitochondrial quality in aged mouse and human oocytes. We first evaluated expression of proteins involved in mitochondrial upregulated protein response (UPR mt) and mitophagy in in-vitro matured metaphase II (MII) oocytes collected from young (2 months) and aged (13.5 months) outbred CF1 mice. We observed a significant decrease in the expression of UPR mt proteins HSPD1 and LONP1, as well as mitophagy protein PRKN and its phosphorylation (p-PRKN) between young and aged oocytes. These results suggest that the expression of proteins critical for UPR mt and mitophagy pathways are compromised in aged oocytes. To test if mitochondrially targeted antioxidant Mitoquinone (MQ) would relieve age associated mitochondrial stress, young and aged oocytes were matured in vitro with or without MQ . MQ did not rescue protein expression of either HSPD1 or LONP1. However, we observed a significant increase in p-PRKN and PRKN protein expression in aged oocytes treated with MQ compared to aged oocytes. Aging mammalian oocytes are characterized with increased reactive oxygen species (ROS) levels. Since mitochondria are important sources of ROS, we next investigated if oocytes challenged with high oxygen environment would exhibit altered mitochondrial quality control proteins comparable to aged oocytes. Although we did not observe differences in UPR mt protein expression, we observed decreased p-PRKN in oxidatively stressed (20% O2) mouse oocytes compared to oocytes that were cultured under normal oxygen environment (6.5% O2). This decreased p-PRKN level also corresponded with an increase in mtDNA copy number, indicating that impaired mitophagy due to reduced p-PRKN could lead to an increase in mitochondrial numbers to combat stress. Interestingly, we could rescue p-PRKN expression and mtDNA copy number by treating the challenged oocytes with MQ. Examination of oocyte developmental potential showed a downward trend in hatching and blastocyst development of oxidatively stressed oocytes compared to the control group. However, treating stressed oocytes with MQ resulted in a significant increase in hatching and blastocyst development percentage. Our results also showed an increase in the inner cell mass percentage with MQ treatment of stressed oocytes compared to control group, indicating improvement in embryo quality as well. Consistent with our results in mice, oocytes from advanced maternal age (AMA) women (> 37 years) compared to young women (< 32 years) also exhibited a significant decrease in p-PKRN and PRKN. Our findings suggest that aged human oocytes may have similar mitophagy deficiency as aged and oxidatively challenged mouse oocytes. If true, treatment of aged human oocytes with MQ would not only rescue mitophagy via PRKN activity, but may also improve human blastocyst development and quality. All experiments were performed at least three times. Results were analyzed using with either Students t-test or one-way ANOVA with Tukeys post hoc test to determine significance with 95% confidence interval.

12

4. Maternal physiology and blastocyst morphological grade correlate with an inherentdifference in human peri-implantation developmental potentialFirst Author: Deirdre Logsdon, DLogsdon@ColoCRM.com, Colorado Center for ReproductiveMedicine

Pre-Doctoral Platform

Earlier mouse studies from our laboratory suggest that the extended culture of embryos during the peri-implantation period in vitro can be used to predict their fetal developmental potential. Extended embryo culture (EEC) also offers a unique opportunity to study the peri-implantation development of human embryos for experiments aimed to improve human in vitro fertilization (IVF) procedures. Our group has been using this system to culture human embryos for up to an additional seven days to study implantation for various projects over the past three years. Since similar extended culture parameters were measured for these projects, we performed a retrospective analysis of a total of 653 human embryos to determine what maternal factors correlate with peri-implantation developmental outcomes. Frozen day five or day six human blastocysts donated for research (WIRB study no. 1179872) were warmed, dezonated, and cultured in vitro up to EEC day seven following an established protocol (Deglincerti et al., Nature 2016). The trophectoderm outgrowth area, total cell number, epiblast cell number, and human chorionic gonadotropin (hCG) production in spent media were measured at various time points during EEC. Patient information including maternal BMI, age, infertility diagnosis, FSH, LH, FSH: LH ratio, estradiol, and anti-müllerian hormone (AMH) levels on menstrual cycle day 3, and an antral follicle count (AFC) determined by transvaginal ultrasound were used in our analysis. Additionally, blastocysts with the highest morphological grade that typically yield live birth rates above 65% (Good Quality) were compared with those of lower morphological grade that typically yield less than 55% live birth rate (Fair Quality), based on our historical data. We then used a spearman correlation coefficient to assess the strength of the association between the aforementioned maternal factors and extended culture parameters. A chi-squared analysis was used to compare binomial outcomes with categorical predictors. T tests or ANOVA’s were used for comparing two or multiple continuous variables from categorical predictors respectively. We determined that several maternal physiological influences during oocyte development (age, BMI, and FSH: LH, AMH, AFC) were significantly correlated with extended culture outcomes (p<0.05). Embryos with high morphological grade had significantly increased outgrowth areas on average compared to those with low morphological grade. (Good Quality: 0.17 ± 0.02; Fair Quality: 0.12 ± 0.02, p<0.05). Maternal age was correlated with hindered extended culture measurements in both the non-donor fertility patients as well as in healthy donors. When comparing embryos with the highest morphological grade only, advanced maternal age (35+ years old) still contributed to decreased hCG production (p<0.05) and hindered epiblast formation compared to those from younger patients (<35 years old, p<0.01). In conclusion, we show that extended culture of human blastocysts may be valuable in predicting post-transfer developmental potential. Additionally, maternal physiology during oocyte development including age, BMI, and ovarian reserve tests significantly correlates with the peri-implantation developmental potential of human embryos.

13

5. Maternal Western-style Diet Drives Glycolytic Programming in Hematopoietic ProgenitorCells and Underlies a Pro-fibrotic Liver Response in Non-human Primate Offspring.First Author: Michael Nash, Michael.nash@cuanschutz.edu, Anschutz Medical CampusPre-Doctoral Platform

Introduction: Early life exposures are key determinants of newborn health and may underlie inflammation-mediated diseases in later life including non-alcoholic fatty liver disease (NAFLD). Hematopoietic stem and progenitor cells (HSPCs) develop in the fetal liver then migrate to the bone marrow (BM), seeding both tissues with hematopoietic cells that will last a lifetime. However, the impact of maternal WSD (mWSD) on fetal HSPC development and post-natal function of HSPCs and macrophage (Mj) is unclear.Methods: We studied HSPC from the BM and liver of early third trimester non-human primate (NHP) fetuses exposed to mWSD, and BM HSPCs and BM derived Mj (BMDM) from 3-year-old (3YO) NHP exposed to mWSD and weaned onto a chow diet (CD) at 7 months of age. We utilized fluorescence lifetime imaging (FLIM) of NADH and FAD to measure glycolysis and oxidative phosphorylation (oxphos) in HSPCs, and metabolome profiling in bone marrow and liver immune cells. Bulk RNA-sequencing was used to measure HSPC gene expression and a nanostring panel of immune response genes was used to assess BMDM gene expression. Ingenuity pathway analysis was used to identify pathways and regulators in HSPCs and BMDM. Hepatic collagen content was measured using second harmonic generation. Data were analyzed by t-test.Results: FLIM and metabolite profiling demonstrated that BM and liver HSPCs from mWSD fetuses have increased glycolysis and decreased oxphos, and a similar signature was found in BM HSPCs from mWSD 3YO. Bulk RNA-sequencing of mWSD fetal BM HSPCs showed gene expression consistent with activation of inflammatory pathways. The top predicted regulators were CEBPA and CD38, which both regulate hematopoiesis and response to inflammatory signals. Gene expression in BMDM from mWSD 3YO demonstrated increased NFKB activation, increased glycolysis, decreased oxphos, impaired anti-inflammatory response to IL4, and a pro-inflammatory response to LPS. Finally, livers from fetal and 3YO mWSD offspring had increased collagen deposition, a feature of fibrosis and NAFLD.Conclusions: Our findings demonstrate that mWSD drives fetal metabolic programming at the level of HSPCs and Mj, which likely mediates the development of a pro-inflammatory, pro-fibrotic disease pattern in the fetal liver that persists at 3YO. These results offer new insight into how mWSD drives early immune cell-driven liver damage and fibrosis in NAFLD.

14

6. Maternal high-fat diet increases fetal muscle fat metabolism and fatty acid transporterexpression in an ovine modelFirst Author: Asma Omar, asmaomar@colostate.edu, Colorado State UniversityPre-Doctoral Platform

Excessive maternal dietary fat consumption during pregnancy may be linked to adverse effects on offspring health, including greater risk of developing metabolic syndrome later in life. Metabolic syndrome is generally considered to be a preventable condition, but the extent to which it is “programmed” during fetal development remains unclear. The aim of this study was to determine the effect of a maternal high-fat diet (MHFD) during pregnancy on fetal muscle oxidative metabolism and related protein and mRNA expressions in an ovine model. Methods: White-faced ewes were fed either a control diet (Show-rite NewCo Lamb Feed-17% protein, 5% Fat) or a high-fat diet (Show-rite NewCo Lamb Feed+ 6% Rumen-protected Fat) from 2-3 weeks before pregnancy until mid-gestation (75 days), when a C-section was performed to collect the placenta and fetal tissues for analysis. Results: MHFD tended to increase fetal body and organ weights, but only significantly increased fetal body length and liver mass (P < 0.05). MHFD increased mRNA expression of placental (cotyledon) fatty acid transport protein-1 (FATP-1) and peroxisome proliferator activated receptor gamma, suggesting an upregulation of placental fatty acid metabolism and transport. Fetal muscle fatty acid oxidation capacity was greater in animals from MHFD pregnancies, with no effect on pyruvate oxidation. This was associated with greater fetal muscle mRNA and protein expression of FATP4, while mRNA expression glucose transporters (GLUT1 and GLUT3) decreased. Muscle expression of insulin signaling enzymes reflected a mild decreases in insulin sensitivity, but these did not reach statistical significance. Conclusions: These studies indicate that MHFD induces an increase in placental and fetal muscle fatty acid transport and oxidation capacity, and favors lower blood glucose uptake compared to controls. Whether these shifts in fetal metabolism predispose offspring from MHFD pregnancies to elevated blood sugar and Type 2 diabetes later in life merits further investigation.

15

7. Preliminary analysis of Androgen Receptor knockdown placentas in sheepFirst Author: Agata Parsons Aubone, aubone@colostate.edu Colorado StateUniviversityPre-Doctoral PlatformNumerous pathologies of pregnancy originate from placental dysfunction. It is essential to understandthe functions of key genes in the placenta in order to discern the etiology of placental pathologies. Anumber of pregnancy complications or disorders are associated with abnormal androgen levels and/orsignaling. Little is known about the role of testosterone and androgen signaling in placentaldevelopment, despite the fact that testosterone rises in maternal circulation during pregnancy and iselevated in a number of placenta-induced pregnancy disorders including gestational diabetes andpreeclampsia. Our overall goal is to determine the role of androgen receptor (AR) signaling in placentalgene expression. Previous work in our laboratory revealed the presence of AR in sheep placentascollected from early, mid, and late gestation, and its binding to vascular endothelial growth factor Apromoter. We therefore hypothesized that AR signaling regulates placental angiogenesis. Here wereport on our preliminary findings in which placental-specific gene targeting was used to knockdownAR in sheep. Three different shRNA constructs were designed (AR4, AR6, and AR7). This is a reporton the effect of shRNA construct AR4. Briefly, day 9 ovine blastocysts were flushed and then infectedwith either pLKO.3G lentiviral shRNA construct or a scramble control. Transabdominal ultrasound wasperformed on gestational Day 65 in 3 scramble control and 3 AR4 infected ewes. Ultrasound findingsrevealed compromised pregnancies which featured smaller placentomes, less amniotic fluid andsmaller fetuses in the AR4 treatment group compared to the control group. Fetectomies wereperformed a day later on gestational Day 66. Preliminary analysis revealed reduced placental weight,reduced placentome numbers, and reduced fetal length and weight, in 2 out of 3 AR4 infected fetusescompared to the scramble control fetuses. Experiments are currently underway to determine the effectof AR knockdown on expression of placental angiogenic factors. Early observations indicate ARsignaling is necessary for proper placental development and fetal growth.

Funding: USDA-NIFA-Grant 2019-67015-29000

16

8. Chorionic somatomammotropin RNA interference reduces global nutrient uptake andumbilical blood flow resulting in intrauterine growth restriction.First Author: Amelia Tanner, amelia.tanner@colostate.edu Colorado State UniversityPre-Doctoral Platform

Previously we have described two distinct phenotypes in response to chorionic somatomammotropin (CSH) RNA interference (RNAi) near term: 1) pregnancies with intrauterine growth restriction (IUGR), and 2) pregnancies in which fetal and placental size were not impacted. To better understand the biological functions of CSH in both phenotypes, we employed steady-state techniques to measure changes in blood flow and nutrient uptake near term (130 days of gestational age; dGA). In the absence of IUGR, the in vivo physiological ramifications of CSH RNAi included perturbed uterine blood flow and placental glucose metabolism. In the current study, we describe the in vivo consequences of CSH RNAi in pregnancies with IUGR. The trophectoderm of hatched blastocysts (9 dGA) were infected with a lentivirus expressing either a scrambled control (NTS) or CSH-specific shRNA (tg6), prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with maternal and fetal vascular catheters. At 130 dGA, pregnancies underwent steady-state metabolic studies with the 3H2O transplacental diffusion technique. Uterine (maternal), umbilical (fetal), and uteroplacental (placental) uptake rates of oxygen, glucose, lactate, and amino acids were calculated using the Fick principle. Tissues were subsequently harvested. Data resulting from scrambled control (NTS; n=4) and CSH RNAi (tg6; n=4) pregnancies were compared by Student’s T-test. CSH RNAi reduced (P≤0.05) fetal and uterine weights by 30% and 43% respectively. CSH RNAi tended (P≤0.10) to reduce (35%) fetal liver weights. Fetal biceps femoris, soleus, flexor digitorum superficialis, transverse abdominus, and extensor digitorum longus muscle weights were all reduced (P ≤ 0.05) in CSH RNAi fetuses. Umbilical blood flow (mL/min) was suppressed (P≤0.05) by 36% at 90 dGA and by 40% at 130 dGA in CSH RNAi pregnancies. This ultimately resulted in reduced (P≤0.01) umbilical IGF1 concentrations, as well as reductions (P≤0.05) in the umbilical uptakes of oxygen, glucose, lactate (mmol/min) and several amino acids (µmol/min) in CSH RNAi pregnancies. CSH RNAi also reduced (P≤0.05) uterine uptakes of oxygen, glucose, and many individual amino acids including 7 essential and 5 nonessential amino acids. Uteroplacental glucose uptakes were also reduced (P≤0.05) by CSH RNAi. In the present study, CSH RNAi reduced umbilical blood flow and global nutrient uptake which ultimately led to reduced fetal and uterine weights. These data suggest that CSH is not only important for uterine blood flow and uteroplacental glucose uptake, but it also facilitates adequate umbilical blood flow necessary for the uptakes of oxygen, oxidative substrates, and hormones necessary to support fetal growth. Supported by NIH HD093701.

17

18

RMRSS 2021POSTER SESSION ABSTRACTS

9. Increased lactate production and mass in the perirenal adipose tissue of fetal sheep withprolonged hypoxemiaFirst Author: Betelhem Ashebo, betelhem.ashebo@ucdenver.edu Univ of ColoradoUndergraduate Poster

Introduction: The major period of proliferation and differentiation (adipogenesis) of pre-adipocytes is during fetal development. Thus, gestation is a critical window where adipose mass can be programmed as supported by studies in humans and animal models showing “catch-up” growth and adipose tissue expansion after in utero nutrient and growth restriction. The objective of this study was to test the effects of hypoxemia, a feature of pregnancies with fetal growth restriction, on fetal adipose tissue development. Our hypothesis was that prolonged exposure to intrauterine hypoxemia would increase perirenal adipose tissue (PRAT) mass because of increased nutrient utilization and lipid synthesis.

Methods: We used pregnant sheep to produce hypoxemia (HOX) via maternal intratracheal nitrogen gas insufflation for 10 days, between 0.8 to 0.9 gestation, compared to ewes receiving intratracheal compressed air (CON). At the end of the study, fetal blood and PRAT samples were collected from CON (n=7) and HOX (n=10) fetuses. Gene expression, protein expression, lipid content, and assays of mitochondrial function were measured. Data were analyzed by t-test.

Results: HOX fetuses had a 20% decrease in fetal arterial pO2 and no change in fetal weight. PRAT mass, absolute and relative to fetal weight, was increased by 30% in HOX fetuses (P<0.005). Expression of the glucose transporter gene (GLUT4) was unexpectedly decreased in HOX PRAT, while expression of lactate dehydrogenase (LDHA) and the lactate transporter (MCT1) were increased (P<0.05). There was no change in other glycolytic (GLUT1, PFK1), lipogenic (FASN, SREBP1C), or adipogenic genes (PPARG1, PPARG1, LEP, UCP1). Triglyceride content (per g of PRAT) and mitochondrial DNA content, a measure of mitochondrial capacity, were similar between groups. Expression of PDK1 and PDK2, pyruvate dehydrogenase kinase genes, tended to be increased (P<0.15). Phosphorylation of pyruvate dehydrogenase (PDH) protein, the target of PDK’s, was increased (P=0.07). In HOX compared to CON fetuses, plasma glucose concentrations were similar, yet lactate, pyruvate, and norepinephrine concentrations were increased, and insulin was decreased. In CON and HOX fetuses, PRAT mass was associated with plasma lactate concentrations (r2=0.36, P<0.01) and LDHA expression (r2=0.41, P<0.01).

Conclusions: Our results demonstrate increased PRAT mass, limited pyruvate oxidation, and increased lactate production in HOX fetuses, in the absence of increased triglycerides and lipogenic genes. We speculate that increased lactate production may provide an alternate fuel source for adipocytes or that lactate may antagonize lipolysis, as it does in human and rodent adipocytes. Additional studies are needed to test this and further characterize adipocyte size and type (multilocular versus unilocular lipid droplets). Together, this will provide new insight into the mechanisms for how hypoxemia primes adipose tissue expansion in utero.

19

10. Uncovering the importance of placental SLC2A3 (GLUT3) on fetal growth and physiology atmid-gestation via RNA interference in sheepFirst Author: Cameron Lynch, Cameron.lynch@colostate.eduMasters Student Poster

Glucose is the predominant energy substrate for fetal oxidative processes and growth. In order for glucose to be transported from maternal to fetal circulation in the ruminant placenta, it must be sequentially transported by SLC2A1 (GLUT1) on the maternal-fetal syncytial layer, then by SLC2A3 (GLUT3) on the apical trophoblast membrane, and again by SLC2A1 on the basolateral membrane of the trophoblast. In intrauterine growth restricted (IUGR) pregnancies, transport of glucose, amino acids, and oxygen can be deficient. Any deficiency in SLC2A3 could impact trophoblast glucose uptake and transfer to the fetus, thus potentially altering placental development and consequently setting the stage for IUGR. It was our objective to use placenta-specific RNA interference (RNAi) to diminish SLC2A3, and determine the impact at mid-gestation (75 dGA) in sheep. Single hatched blastocysts were harvested from donor ewes and the trophectoderm was infected with lentiviral constructs expressing either a scramble control (SC) or SLC2A3-specific (GLUT3-RNAi) short-hairpin RNA, and then surgically transferred into a synchronized recipient ewe. The resulting pregnancies underwent ultrasound Doppler velocimetry and fetal measurements at 70 dGA, and a terminal surgery at 75 dGA for collection of uterine and umbilical arterial and venous blood, fetal and placental measurements, and tissue samples. Due to a lack of fetal sex x treatment interactions, statistical comparisons between SC (n=6) and GLUT3-RNAi (n=6) pregnancies were made by Student’s T-Test. At 70 dGA, while umbilical artery velocimetry was not impacted, binocular distance (p=0.07), femur length (p=0.01) and tibia length (p=0.03) were reduced in GLUT3-RNAi pregnancies. These results were confirmed at 75 dGA, as GLUT3-RNAi fetuses had reduced fetal weight (p=0.08), head circumference (p=0.05), femur length (p=0.03), and tibia length (p=0.05). While there were no significant reductions in maternal glucose or insulin concentrations in the GLUT3-RNAi pregnancies, reductions in umbilical artery insulin (44%; p=0.10), umbilical vein glucose (42%; p=0.002) and umbilical artery glucose (46%; p=0.02) concentrations were observed. By contrast, umbilical vein chorionic somatomammotropin (CSH) concentrations were increased by 70% (p=0.03) in the GLUT3-RNAi pregnancies. GLUT3-RNAi resulted in a 37% reduction (p=0.05) in placental SLC2A3 protein concentration, whereas SLC2A1 concentration was increased 38% (p=0.09). Apparent attempts at compensation for SLC2A3-deficiency, by increasing SLC2A1 and CSH, did not prevent fetal hypoglycemia and impacts on fetal development. While it has been suggested that SLC2A3 (GLUT3) is predominantly important in late gestation, our data indicate that SLC2A3 is important for normal fetal development during the first-half of gestation. Due to its location on the apical (maternal facing) trophoblast membrane, coupled with our results, may suggest that SLC2A3 is the rate-limiting glucose transporter, at least during the first-half of gestation. Supported by NIH HD094952.

20

11. Impacts of Fetal Sex on Relative Percent of Specific Phyla Colonizing the BovinePlacenta First Author: Sarah Weir, sweir1@uwyo.edu, Univ of WyomingOther: Poster

Lack of research into the potential effects of fetal sex hormones during development on the placental microbiome flora prompted our interest in the subject. We hypothesized that gonadal hormones produced by the developing fetus in utero would influence the growth of colonizing flora in the placenta. Our objective was to characterize the relationship between fetal sex and the placental microbiome by examining relative percentage of ten specific phyla in three different placental tissues including the allantois (AL), cotyledon (COT), and the intercotyledonary membrane of the chorion (ICM). Phyla examined in this analysis include Euryarchaeota, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Elusimicrobia, Fibrobacteres, Firmicutes, Proteobacteria, and Spirochaetes. Tissue samples were obtained from multiparous beef cattle at parturition (n=10) with equal proportions of female and male progeny. Relative percentages made up by each phyla were obtained using QIIME2 and further statistical analysis of this data was performed using SAS two sample t-tests. T-tests were used to compare means across sexes and look for sex-influenced differences by examining p-values. The phyla examined in this analysis did not differ (P ≥ 0.18) among fetal sex. Based on this data we conclude that fetal sex does not influence the relative abundance of these specific phyla in the placental microbiome of cattle

21

12. The human endogenous retrovirus ERVW-1 promotes trophoblast stem cell proliferationand differentiation towards syncytiotrophoblast

First Author: Rachel West, RWest@FLColo.com. Colorado Center for Reproductive Medicine

Post-Doctoral Poster

Human endogenous retroviruses (HERV) are ancient remnants of retroviral infections that integrated into the germline millions of years ago. Of the 8% of retroviral DNA that comprises our genome, most is considered artifact and does not code for protein. The placenta, however, has co-opted HERV to drive fusion of the villous cytotrophoblast to become the multinucleated syncytiotrophoblast (STB) during the first trimester of pregnancy. Our laboratory previously found the presence of several HERV in the undifferentiated, progenitor cells of the peri-implantation stage primitive placenta. Interestingly, the HERV, ERVW-1, was found as early as embryonic day 8 in the primitive syncytium of peri-implantation stage embryos. ERVW-1 has been characterized in term placentas and primary trophoblasts. However, little is known about its role during the formation of the transient, primitive placenta that forms when implantation occurs. We hypothesized that, in addition to driving first trimester villous cytotrophoblast cell fusion, the endogenous retrovirus ERVW-1 may regulate placental stem cell proliferation and differentiation during implantation. We utilized trophoblast stem cells (TSCs) first described by Okae et al. in 2018. TSCs were treated with lentivirus containing shRNA designed to target ERVW-1 or a scramble nontarget control. Stable cell lines were generated by isolating and propagating monoclonal cell populations selected by using Puromycin. After selection, RNA was isolated from wild type (WT), scramble control (SC), and ERVW-1 knockdown (KD) cells. Knockdown TSCs had 86% lower ERVW-1 mRNA compared to SC cells. Using flow cytometry, we determined there were significantly fewer (p<0.01) ERVW-1 positive KD cells compared to WT and SC. To determine cell proliferation, WT, SC, and KD cells were collected 24, 36, 48, and 72 hours post-plating and cell doubling time was assessed. ERVW-1 knockdown cells had a significantly (p<0.005) longer cell doubling time compared to WT and SC cells (WT=26.5 hours, SC=25.4, KD= 43.7). Additionally, flow cytometry for the cell proliferation marker, Ki67, revealed significantly fewer Ki67 positive cells in KD compared to SC and WT control cells (p < 0.01). These data suggest that decreased expression of ERVW-1 causes altered cell proliferation in TSCs. Interestingly, when mRNA levels of additional HERV were analyzed, ERVFRD-1 was significantly increased in the KD cells. This suggests that, upon loss of ERVW-1, ERVFRD-1 can potentially compensate to continue to contribute to cell fusion. To test this hypothesis, we induced differentiation towards the syncytiotrophoblast (STB) lineage using Forskolin. KD cells grown in STB conditions had significantly lower mRNA levels of the STB markers CGB and PSG-1. Additionally, cells immunostained for CGB and SDC-1 exhibited lower levels of immunofluorescence. These data suggest that, even with increased ERVFRD-1, loss of ERVW-1 leads to impaired syncytialization. While the role of HERV is fairly well understood in the later stage placenta, the role of HERV is still not well understood during implantation. These data shed some light into how HERV might be contributing to the formation of the primitive syncytium leading to successful implantation and providing important insight into HERV function outside of their classically understood role of HERV in pregnancy.

22

13. Establishment of an immune-competent animal model of subclinical Zika virus infectionand its impact at the maternal-fetal interfaceFirst Author: Joseph Westrich, joseph.westrich@colostate.edu Colo State UnivPost-Doctoral Poster

Zika virus (ZIKV) infections, acquired during pregnancy, have been shown to cause fetal abnormalities. Although ZIKV is no longer in the global spotlight, it continues to spread and cause disease worldwide. ZIKV has been shown to cross the placental barrier and infect the fetus, even in mothers with subclinical infections. It is estimated that roughly 80% of ZIKV infections are asymptomatic, presenting significant risk to pregnancies in ZIKV endemic areas. Beyond infection of the fetus itself, the maternal anti-viral immune response may also negatively contribute to fetal health. Given the continued spread of ZIKV, greater understanding of how these mechanisms negatively impact fetal development is crucial. To evaluate these mechanisms, reliable animal models that recapitulate human disease are needed. The most common animal model for ZIKV is the interferon deficient mouse. While ZIKV pathogenesis studies have benefited greatly from these mice, they are not ideal to characterize the contribution of the immune response to ZIKV related pathologies. Previous studies have shown that immune competent mice and guinea pigs are susceptible to ZIKV infection, though due to transient and mild presentation of disease in depth characterization has not yet been performed. These subclinical infections may more closely mirror disease observed in the majority of human cases.To determine if subclinical ZIKV infections of immune competent animals could recapitulate human disease and exhibit deleterious effects on the fetus, we inoculated cohorts of mice and guinea pigs during the first trimester of pregnancy. This period is known to be a critical time for ZIKV infection. We utilized both subcutaneous and intra-vaginal routes of inoculation. Animals were monitored for symptomatic disease throughout the timecourse for evidence of ZIKV pathologies. To determine the extent of ZIKV infection, RT-qPCR and plaque forming assays were performed on a multitude of maternal and fetal tissues. Lastly, to understand how these infections impact the immunological and growth factor profile, we performed both protein and transcript analysis of tissues across a broad spectrum of factors commonly dysregulated during diseased or abnormal fetal development.We found that intra-vaginal, but not subcutaneous, inoculation of ZIKV resulted in a localized infection of the reproductive tract, which crossed the placental barrier and infected the fetus. These infections exhibited a highly transient viremia that did not establish in peripheral tissues outside the reproductive track. Futhermore, these localized infections displayed undetectable symptoms in the pregnant animals. Evaluation of the immunological and growth factor profile showed a significant increase of CXCL10 in all tissues infected with ZIKV. This finding was consistent across both animal models and is consistent with published literature finding increased CXCL10 in human sera of ZIKV infected mothers.These models demonstrate establishment of subclinical ZIKV infections that alters the immunological environment similarly to what is observed in human cases. Utilization of these models will assist to further explore and identify immunological factors involved in ZIKV related disease, as well as factors that are causative and preventative of ZIKV related fetal abnormalities. These findings will be of great importance as ZIKV continues to spread worldwide.

23

14. Mare diet supplementation alters oocyte lipid content and developmental competenceFirst Author: Giovana Di Donato Catandi giovanac@colostate.edu Colo State UnivPre-Doctoral Poster

The equine oocyte is dense in lipids, which may serve as an energy source for oocyte maturation and later embryonic development. However, the association between lipid content and fertility remains to be determined, as does the extent that diet can modify oocyte lipids. We hypothesized that diet supplementation can alter the oocyte lipid profile and subsequent developmental potential. In Study 1, we examined if oocyte triglyceride (TG) relative abundance was affected by dietary supplementation. Mares (16-22 yr, n=9) were fed grass/alfalfa hay and supplemented daily with a combination of commercially available feed additives designed to promote equine wellness and fertility [Equine GI™ (147 g daily), Potency® (28 g daily), Motility Plus® (23 g daily), Healthy Weight Oil (60 ml daily), Platinum Performance, Inc., Buellton, CA]. Oocytes were collected from the mares before (PRE) and after ≥ 8 weeks (POST) of supplementation during the natural breeding season. In Study 2, we compared oocyte developmental potential after injection of sperm into oocytes obtained from mares supplemented for ≥ 8 weeks with the same complex nutrient blend (CNB, 18-24 yr, n=5) or from a similar group of mares supplemented with a grain control diet (450 g of grain mix and 60 ml of corn oil daily, GRN, 19-23 yr, n=5). Oocytes were collected from dominant follicles (³ 35 mm) during estrus and at 20±2h after induction of follicular maturation. In Study 1, oocytes were denuded of cumulus cells after collection, snap frozen, and assessed for TG composition by non-targeted liquid chromatography-mass spectrometry using a Waters Acquity UPLC system. In Study 2, recovered oocytes were placed in culture medium for 22±2h before being injected with sperm from one stallion, and blastocyst formation was assessed in 7 or 8 days. A total of 131 annotated TG species were identified. Normalized peak areas for PRE and POST oocyte TG were compared using ANOVA with Kenward-Roger degrees of freedom and false discovery rate adjustments. Blastocyst development rates were compared by Fisher’s exact test. Relative abundance of 40 TG species differed (p £ 0.05) and 36 tended to differ (p £ 0.1) between PRE and POST; all TG species, as well as total relative abundance of TG were higher in oocytes from PRE when compared to POST. Blastocyst rates per sperm-injected oocyte were greater (p=0.03) for ADD (40%, 6/15) than GRN (5%, 1/19). Dietary supplementation of the complex nutrient blend to middle-age to older mares resulted in reduced relative abundance of TG in oocytes and improved developmental potential. We determined that oocyte lipid content can be modified through diet. The extent that diet supplementation improved oocyte competence by altering the lipid profile is still to be determined.

24

15. PEPCK Activity is Essential During Early Embryo DevelopmentFirst Author: Kyle Fresa, Kyle.Fresa@colostate.edu Colo State UniversityPre Doctoral Poster

Phosphoenolpyruvate carboxykinase (PEPCK), an enzyme known primarily for its role in gluconeogenesis, has recently been identified as major regulatory enzyme influencing nutrient uptake, biosynthesis, and cell proliferation in cancer cells. While PEPCK is present in early embryos, its influence during development is not known. We hypothesize that similar to cancer cells, PEPCK activity promotes the rapid cell division and biosynthesis that is required in the early embryo and may serve as a biomarker of embryo developmental potential. To investigate the importance of PEPCK activity in developing embryos, we used hydrazine sulfate (HS) to inhibit PEPCK during early embryo culture. Cumulus-oocyte complexes were aspirated from slaughterhouse-derived bovine ovaries, matured for 18 hours, and fertilized. After 20 hours post-fertilization, COCs were vortexed, and the resulting 1-cell structures were randomly added to culture media containing several concentrations of hydrazine sulfate for a total of 8 days. Groups included control (no HS), 0.01mM, 0.05mM, 0.1mM, 0.25mM, and 0.5mM HS. A total of 1196 embryos among 4 replicates were used to compare developmental endpoints including observation of cleavage on day 3, cell number on day 3, and blastocyst development by day 8. Statistical tests used to compare treatment groups to controls included logistic regression and Dunnett’s test. At a dose of 0.5mM HS, blastocyst development was completely inhibited and observation of cleavage and cell number on day 3 was significantly reduced (p<.0001). Although 0.25mM HS dose did not affect cleavage or cell count on day 3, it reduced blastocyst development by 88% compared to controls (p<.0001). At lower doses of HS, no developmental outcome was significantly different compared to controls. This differing response to PEPCK inhibition between cleavage stage embryos and blastocysts may reflect a greater reliance on proliferative mechanisms as cell division becomes more rapid. Our results suggest that PEPCK likely has a role in embryo development, which may be most important at the blastocyst stage when rapid cell proliferation occurs. To further confirm that this effect is a direct cause of reduced PEPCK activity, we are currently working to produce PEPCK knockdown embryos using siRNA vectors. Our studies will evaluate developmental potential, nutrient uptake, and regulation of metabolic enzymes associated with PEPCK activity.

25

16. Elucidating the Mechanism of Early Embryo Mortality in Holstein-Friesian CowsFirst Author: Carolina Gonzalez Berrios, clgonz@colostate.edu, Colorado State UniversityPre Doctoral Poster

Disruption of maternal-fetal crosstalk, at day (d) 16 of pregnancy, causes early embryo mortality (EM) but it is still poorly understood. It was hypothesized that EM pregnancies are associated with impaired action (paracrine and endocrine) of interferon-tau (IFNt) from the conceptus on the endometrium, corpus luteum (CL) and peripheral blood mononuclear cells (PBMC) and therefore, fails to prevent up-regulation of estradiol receptor 1 (ESR1) from activating the luteolysis cascade. For this study, twenty-two healthy multiparous lactating Holstein-Friesian cows were randomly selected and sorted into pregnant (P; exposed to semen; n=15) or non-pregnant (NP; not exposed to semen; n=7) group. All cows were enrolled into a synchronization program after a 60d post-partum voluntary wait period to induce ovulation. The program consisted of three intra-muscular (IM) injections: 0.1 mg/mL of GnRH (d0), 25 mg/mL of PGF2a (d7) and second dose of GnRH [56 hours (h) after d7]. Only cows in the P group were artificially inseminated 16h later from second GnRH injection, and this was considered d0 of the study. On d16 of pregnancy/estrous cycle, uterine flushings (UF) and tissues (endometrium, CL and PBMC) were collected for all cows but only conceptuses were collected for the P group. Cows in P group were re-sorted based on the conceptus morphology: Normal (N; n=9) pregnancies if conceptuses were elongated and translucent or EM pregnancies (n=6) if conceptuses were restricted in elongation and were pink, red and (or) opaque. Extracted RNA from conceptuses, endometrium, CL and PBMC were submitted for RNA-Seq analysis. Sequences were aligned to the ARS-UCD 1.2 genome reference assembly and were analyzed using the DESeq2 package in R. Submitted data into Ingenuity Pathway Analysis (IPA) had the following cutoffs: log2fold change ≤-1.5+≥ and P≤0.05. If IPA failed to identify key biological pathways (KBP), then a list of genes provided by IPA were evaluated for their interactions and roles. Previously, we reported EM were significantly shorter (P<0.006) and produced lower (P<0.0001) IFNt mRNA (from RNA-Seq) when compared to N conceptuses. Also, IFNt protein concentrations (ELISA) in UF were greater (P< 0.004) in N and EM tended (P < 0.07) to be greater than NP cows. IPA identified IFN signaling (P=7.54E-16) as the KBP for NvsNP endometrium, as expected. Likewise, both CL and PBMC had up-regulated the IFN signaling (ISGs) as the KBP for NPvsN. For EMvsN conceptuses, IPA identified the T-helper 1 cell activation to be up-regulated and as the KBP. While for endometrium, IPA failed to identify KBP and listed fifteen up-regulated genes. Only nine were found to be associated with ESR1 action for EMvsN. Furthermore, the primary KBP for CL were granulocyte adhesion and diapedesis, and hypercytokinemia in EMvsN. No differences were found in PBMC for EMvsN. In summary, EM pregnancies have compromised conceptuses with faulty IFNt release and upregulated adaptive immune responses, ESR1 luteotylic-mediated signals in the endometrium, localized overproduction of immune cells in the CL and thus, leading to pregnancy loss. USDA-NIFA-AFRI-2019-07133 & Zoetis, Inc.

26

17. Prolonged exposure of human blastocysts to hyaluronan-enriched transfer mediahas no effect on peri-implantation stage embryo development during in vitro cultureFirst Author: Deirdre Logsdon DLogsdone@ColoCRM.com Colorado Center for Reproductive Medicine

Pre-Doctoral Poster

Exposing human embryos to hyaluronan-enriched embryo transfer (ET) media (Embryoglue (EG), Vitrolife) before ET is a widely used protocol in IVF-ET cycles to aid in implantation of embryos and improve pregnancy success. However, the benefit of treating embryos with EG has been of much debate and various studies note no differences with EG treatments. More recently, a clinical study reported a surprising increase of pregnancy rate from 45% to 75% when blastocysts were exposed to EG for 3 h, but no effect was observed when exposure time is less than 1 h. Here, our objective was to determine whether the prolonged exposure of human blastocysts to EG was beneficial for human peri-implantation stage development in vitro. Additionally, we investigated whether the addition of a cocktail of estradiol (8nM), progesterone (200 ng/mL), pyruvate (1mM), and lactic acid (0.22% v/v) to EG would benefit human embryo development during the peri-implantation stage in vitro. Vitrified human blastocysts donated for research (WIRB study no. 1179872) were warmed and recovered in EG or EG with additives (EGA) for either 10 min or 3 h (EG10m, EGA10m, EG3h, and EGA3h). Embryos from each group were then fixed with 4% paraformaldehyde and stained for DAPI and antibodies against cleaved caspase-3 to examine apoptotic stress. Separate blastocysts were also treated (EG10m, EGA10m, EG3h, and EGA3h) and then introduced to an extended embryo culture (EEC) system (Deglincerti et al., Nature 2016) and cultured in vitro until EEC day 5. Embryo attachment, morphology, and trophectoderm outgrowth areas were assessed on each day during EEC. Finally, we performed surgical ET in mice to assess implantation and fetal developmental potential of in vitro produced CF1 embryonic day 3.5 mouse blastocysts exposed to EG or EGA for 3 h. Implantation and fetal development were assessed at day 17.5 post fertilization. No differences in total (EG10m: 5.71 ± 0.98 n=24; EGA10m: 6.62 ± 0.92 n=21; EG3h: 9.50 ± 2.59 n= 24; EGA3h: 8.14 ± 1.29 n= 21) or % apoptotic cells (EG10m: 7.99% ± 1.52%; EGA10m: 11.13% ± 1.53%; EG3h: 13.22% ± 4.04%; EGA3h: 12.63% ± 1.93%) were noted amongst treatments. There were also no differences in attachment, percent of normal development, or outgrowth areas during EEC. Finally, there were no differences in fetal development following surgical ET in mice (Fetus/Implantation: EG3h 21%, n=51; EGA3h 33% n=50). Our results suggest that prolonged exposure to EG up to 3 h has no effect on blastocyst cell apoptosis, peri-implantation development, or fetal development. Additives to the EG also do not seem to provide any benefit in promoting peri-implantation stage human embryo development in vitro, therefore, the likelihood of providing any benefit in a clinical IVF setting is slim.

27

18. Generation of Organoids Derived from Agricultural and Companion Animal Reproductive Tissue: Preliminary Findings.

First Author: Riley Thompson, riley.thompson2@colostate.edu Colo. State University Post-Doctoral Poster

Organoids are three-dimensional, spherical, cell clusters formed in vitro that are capable of regeneration and organization with similar function to their tissue of origin. Organoids overcome two major short-comings of the classic monolayer and explant cell culture approaches by 1) maintaining viability in vitro longer (months) and 2) retaining the ability to physiologically respond to various stimuli similar to its in vivo organ counterpart. Furthermore, organoids are superior to conventional 2D cell culture techniques in providing physiological structures that support intercellular interaction between multiple cell types and, therefore, offer a potentially superior model for exploring disease mechanisms, investigating potential therapeutics, and improving in vitro produced embryos using co-culture. Importantly, organoids reduce the need for whole animal studies, making research more efficient and cost effective while improving animal welfare. To date, equine endometrial organoids are the only reproductive organoids reported in domestic animals (non-rodent, non-human). Our first objective was to establish bovine oviductal organoids, canine endometrial organoids, and equine chorionic girdle organoids. Implementing similar culture conditions and methodology used to produce equine endometrial organoids enabled establishment of viable organoids from bovine oviducts (n=4), canine endometrium (n=5), and equine chorionic girdle (n=3; day 33-34 conceptuses). Our second objective was to assess the preliminary function of each organoid type. Firstly, bovine oviductal organoids were cultured for 77 days (passaged every 6-10 days), and conditioned media (CM) was collected every other day for isolation of extracellular vesicles (EVs) via ultracentrifugation and characterization by nanotracking technology. We found that bovine oviductal organoids produce EVs that are 50-150 nm diameter at a concentration of 2.7x106 particles/mL. As a potential model for endometritis and pyometra, canine endometrial organoids were cultured for 1-2 passages prior to stimulation with lipopolysaccharide (LPS). Three concentrations of LPS were tested (control, 1 mg/ml LPS, 3 mg/ml LPS, or 6 mg/ml LPS), and organoids were collected 6 and 24 hours after LPS exposure for assessment of inflammatory gene expression (IL8, CXCL10, and IFNb). Organoids demonstrated increased IL8 gene expression after 6 hours of LPS stimulation compared to control (p<0.001) but not at 24 hours (p=0.397). No significant differences were detected for CXCL10 at 6 hours (p=0.0537) or 24 hours (p=0.294) or IFNb at 6 hours (p=0.698) or 24 hours (p=0.487). Lastly, equine organoids derived from placental chorionic girdle cells were established and cultured for 16 days (1 passage). CM was collected every other day, and equine chorionic gonadotropin (eCG) concentration was measured via radioimmunoassay. Initial results were promising with CM from equine chorionic girdle organoids having concentrations as high as 242.3 ng/ml eCG during the first passage. In conclusion, these data are the first reproductive organoids generated in domestic animals beyond the equine endometrium. While further research is required, reproductive organoids may be an effective in vitro cell culture model for evaluating reproductive pathophysiology, trialing novel therapeutics, and potentially improving in vitro embryo production, which will facilitate reduced reliance on research animals and associated cost and welfare concerns.

28

19. Multiple IVF method using single frozen straw in bovine species. First Author:

Sung Woo Kim, sungwoo@korea.kr, Animal Genetic Resources Research Center,

National Institute of Animal Science, RDA, Korea

Research Scientist Poster

The frozen semen of selected animals is a valuable genetic resource that cannot be regenerated again without live animals. So, the number of remaining semen is decreased by using the semen, and the price of most wanted semen goes higher with time. The objective of present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with single frozen semen. To optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

29

20. The effect of Medroxyprogesterone Acetate on Neural Activity in the Ventral TegmentalArea of Reindeer BullsFirst Author: Katherine Agster, kagster@uvwy.edu, Univ of WyoUndergraduate Poster

Reindeer bulls are notably aggressive during the rutting period. Medroxyprogesterone acetatehas been used anecdotally to reduce aggression while maintaining sexual competency in rutting males during the breeding season; however, it is unknown if the reward pathway remains intact and functional in progesterone treated bulls. The Ventral Tegmental Area (VTA) is an integral part of the cortical network that composes the reward pathway and is responsible for various cognitive and emotional processes, including motivation and reward in relation to sexual activity. The objective of this study was to determine the effects of Depo-Provera (medroxyprogesterone acetate) on neural activity in the VTA of reindeer bulls (n=8) while in rut. Bulls (n=4) were treated with a single intramuscular injection of Depo-Provera (400 mg) two weeks prior to the start of rut with tissues collected in the early (n = 4) or mid (n = 4) rut. Brains were perfused and the VTA blocked using surface landmarks. Cryoprotected blocks were cut at (30µm) using a cryostat. Slices were incubated with rabbit anti-cfos antibody (primary antibody: 1:20,000 dilution, Calbiochem San Diego, California) using standard immunohistochemistry procedures, mounted on slides and coverslipped. Specificity was confirmed by lack of staining in the absence of cfos antibody. Positively stained neurons were quantified using Image J. Neural activity in the medial aspect of the VTA did not differ (P≥ 0.192) by treatment, collection period, or their interaction. This study suggest that Medroxyprogesterone acetate does not alter neural activity within the VTA. These findings may be translatable to males of other species when change in behavior and maintenance of reproductive competence is desired.

30

21. Stimulation of GnRHR-II Partially Rescues Steroidogenesis Despite Suppressed LuteinizingHormone Secretion in Neonatal BoarsFirst Author: Amy Desaulneirs, desaulniers@ucmo.edu, Univ of Central MissouriAssistant Professor Poster

Male pigs have three distinct populations of Leydig cells—fetal, neonatal, and adult. Testicular steroidogenesis is classically stimulated by luteinizing hormone (LH), but recent evidence suggests that the second mammalian form of GnRH (GnRH-II) and its receptor (GnRHR-II) are autocrine/paracrine regulators of Leydig cells in the mature boar. However, it remains unknown, if GnRH-II and its receptor also promote steroidogenesis within other populations of Leydig cells. The objective of this study was to evaluate steroidogenesis in neonatal boars treated with analogues targeting GnRHR-II and/or the classical GnRH receptor (GnRHR-I) on gonadotropes. Neonatal (d 14) littermate boars were randomly assigned to intramuscular treatment with either vehicle (n=3; 5% mannitol), the GnRHR-I antagonist cetrorelix (n=3; 10 µg/kg BW), or analogue 135-18 (n=4; 10 µg/kg BW), which simultaneously agonizes GnRHR-II and antagonizes GnRHR-I. Blood and testicular tissue were collected 4 h post-treatment. Serum was subjected to LH radioimmunoassay and testicular protein was extracted for StAR immunoblotting. Compared with vehicle-treated males (7.0 ± 0.2 ng/ml), both cetrorelix and 135-18 ablated LH secretion similarly (0.1 ± 0.2 ng/ml and 0.3 ± 0.2 ng/ml, respectively; P<0.0001). Abundance of StAR was reduced by 84% in males treated with either cetrorelix or 135-18 compared with controls (P=0.0001). In a subsequent experiment, neonatal (d 10) littermate boars were treated with either cetrorelix (n=3) or analogue 135-18 (n=3). Blood was collected prior to as well as 4 h post-treatment. Males were then treated every 24 h for 4 d. Blood was also collected 24 h after the last treatment (d 14). Serum was subjected to HPLC-MS/MS. An effect of time was detected for progestogens (17α-hydroxyprogesterone, progesterone) as well as androgens (androstenedione, androsterone, testosterone). Hormone concentrations were reduced 4 h post-treatment by approximately 77–91% (P<0.05). However, concentrations were similar to basal levels 24 h after the last treatment (P>0.05). Concentrations of dihydrotestosterone and estrone were similar prior to and 24 h after the last treatment (P>0.05); both were largely undetectable 4 h post-treatment. Cetrorelix more effectively inhibited steroidogenesis compared to 135-18 despite similar suppression of LH. Cetrorelix-treated males had a 55% reduction in DHEA concentrations (0.8 ± 0.8 nM) compared with 135-18 treated animals (1.8 ± 0.8 nM; P<0.0243). Estrone was reduced by 85% in animals treatedwith cetrorelix (0.05 ± 0.08 nM) versus 135-18 (0.34 ± 0.07 nM; P=0.0449). Androstenedioneconcentrations were reduced by 26% in males treated with cetrorelix compared with 135-18 (4.0 ± 0.7nM versus 5.4 ± 0.7 nM, respectively; P=0.0361). Testosterone levels tended to be reduced by 23% inmales treated with cetrorelix (1.0 ± 0.2 nM) compared with 135-18 (1.3 ± 0.2 nM; P<0.09). Theseresults demonstrate that both GnRH analogues potently inhibit steroidogenesis in a time-dependentmanner due to impaired LH secretion. Notably, GnRHR-II stimulation partially rescues gonadalsteroidogenesis despite potent suppression of LH. These data suggest that GnRH-II and its receptormay be autocrine/paracrine regulators of testicular steroidogenesis within neonatal pigs.

31

22. Estrus suppression in the mare through the use of altrenogest releasing intravaginal rings. First Author: Jess Lederman jess.leerman@colostate.edu Colo State UnivProfessor Poster

This study proposes an alternative method for delivering altrenogest, a synthetic progestin, to decrease adverse behavior in mares through hormonal manipulation. We hypothesize that an intravaginal ring designed for the mare will provide a novel and efficacious method for sustained release of altrenogest to suppress adverse behavior commonly associated with estrus. Importantly, it will allow the mare to resume normal cyclicity upon removal of the ring for pursuit of reproductive procedures. Mares often demonstrate undesirable behaviors such as biting, striking, kicking out, distraction, frequent urination and overt interest in other horses during the estrus phase (5-7 days) of their 21-22 day estrus cycle, due to elevated estrogen levels produced by the dominant ovarian follicle. Following ovulation, progesterone levels increase, overcoming the effects of estrogen for 11-14 days; often alleviating the undesirable behavior. The first aim of this study is to determine the ideal size of vaginal ring for prolonged placement in the vaginal vault of the mare. The second aim of this study is to evaluate the pharmacokinetic and pharmacodynamic values of altrenogest delivery through an intravaginal device in the mare. Altrenogest, currently delivered orally or by injection, provides estrus suppression through similar biologic activity as native progesterone. Mares often resist oral administration, posing public health concerns, and injections result in muscle soreness and inflammation, leading to noncompliance. The safety and ease for the application of intravaginal rings between the diameters of 13cm and 18cm was determined through pilot trials of control rings in the summer of 2020. Daily evaluation for ring size (14.2 cm) and shape was performed through inspection with a vaginal speculum and found to be effective for both mare retention and safety. The next phase will begin in the spring breeding season of 2021 to evaluate intravaginal altrenogest pharmacokinetic and pharmacodynamic values at the onset, steady-state and tail-end of vaginal altrenogest delivery. Treatment groups will receive varying dosages of altrenogest to determine the optimal level. Three groups will be evaluated for the purpose of this study: group one will receive an intravaginal ring integrated with altrenogest, group two will receive an intravaginal ring without altrenogest, and group three will be administered daily oral altrenogest and will not receive an intravaginal ring. Groups two and three will act as control cycles. Each mare will advance to the subsequent group after completing each 30 day cycle. Outward estrus behavior and physiological changes will be assessed through exposure to stallions, routine transrectal ultrasound examination and hormonal assessment. The overall goal is retention, steady-state levels of altrenogest at or above 0.5 ng/mL and behavioral estrus suppression for a 30 day duration. Comparative data will be analyzed using the Shapiro-Wilcoxon signed-rank test. Statistical significance will be set at P≤0.05.

32

23. Gonadotropin Response to Insulin and Lipid Infusion Reproduces the ReprometabolicSyndrome of Obesity in Eumenorrheic Lean Women: a Randomized Crossover Trial

First Author: Nanette Santoro, Nanette.santoro@cuanschutz.edu UC School of Med Professor Platform

Introduction: The reprometabolic syndrome of obesity is associated with reduced gonadotropins and impaired LH and FSH response to gonadotropin releasing hormone (GnRH). We sought to reproduce the reprometabolic syndrome in normal weight, eumenorrheic women by infusing a combination of insulin and lipid.Materials and Methods: 15 women, median age 32 [IQR 26, 36] and BMI 21.9 [20.2, 22.9] were recruited with intent to perform early follicular phase, 6-hour infusions of insulin (20-40mg/mU/m2/min) and lipid (Intralipid)—insulin/lipid infusion; or saline infusion (controls); 12 women completed both intended studies and an additional 3 women completed only one of the two studies. The first 4 hours of each study assessed endogenous gonadotropins; at 4hrs, a 75 ng/kg GnRH bolus was administered and sampling continued until 6hrs. Linear mixed model analysis was used to determine differences between insulin/lipid versus saline on endogenous LH pulse amplitude (primary outcome), mean FSH, and area under the curve (AUC) response to GnRH (secondary outcomes).Results: LH pulse amplitude, mean FSH, and both AUC responses to GnRH were reduced by insulin/lipid, mean FSH (P=0.03) and AUC for LH (P=0.05) were at or near statistical significance. LH response to GnRH was significantly reduced (P=0.02) when one participant with very high LH and AMH levels was excluded. Discussion: Acute infusion of insulin/lipid to eumenorrheic, normal weight women recapitulated the reprometabolic syndrome of obesity. These findings imply that specific circulating factors in obese women contribute to their subfertility and thus may be amenable to discovery and treatment.

33

24. Cell Type Specific Effects of Hyperlipidemia and Hyperinsulinemia, Characteristic ofReprometabolic Syndrome, on Pituitary Function.First Author: Rosemary McDonald, rosemary.mcdonald@cuanschutz.edu, CU Anschutz MedCampusPre - Doctoral Poster

Introduction: Obesity has a profound impact on reproductive function, reducing fertility and increasing the risk of pregnancy complications and birth defects. Obesity in women is associated with increased circulating free fatty acids (FFA) and insulin resistance and is characterized by decreased basal and GnRH-stimulated FSH and LH secretion from gonadotrophs in the pituitary. We have termed this phenotype ‘Reprometabolic Syndrome’. Our lab has previously shown that acute infusions of lipid/insulin into healthy, normal weight, cycling women recapitulates the reprometabolic phenotype observed in women with obesity.Aim: Our goals in this study were to investigate if lipid/insulin infusion impacted all cell types in the anterior pituitary similarly or whether effects were confined to gonadotrophs, in order to provide insights into the mechanism underlying the observed suppression of FSH and LH.Methods: This was a secondary analysis of samples from a study measuring FSH and LH in 8 normal weight women during two 6-hour visits with either a saline and heparin (control) infusion, or a hyperinsulinemic-euglycemic clamp with Liposyn (Abbott laboratories). Blood was drawn every 10 minutes during each visit, which occurred in a random order, between days 2-5, of 2 different menstrual cycles. Serum was pooled to measure creatinine and levels of TSH, prolactin (PRL), growth hormone (GH), thyroid hormones (fT4 and T3), cortisol, IGF-1, leptin and adiponectin in both saline and lipid/insulin infused participants.Results: In contrast to the previously observed decreases in FSH and LH, TSH levels increased in the lipid/insulin-treated women’s samples, with the most dramatic percent change after 160 minutes (28.19% increase), significantly different from TSH levels in the control (saline) infusions (p<.0005), which slightly decreased (-11.37%). Leptin also slightly increased at approximately 240 minutes in the lipid/insulin visit compared to the saline control (p<.02). There were no significant differences in Thyroid hormones (fT4 and T3), PRL, GH, IGF-1, cortisol, adiponectin or serum creatinine between saline and insulin/lipid infusion conditions.Conclusions: These results indicate that the observed decreases in FSH and LH were not due to potential hemodilution, as there was no change in serum creatinine between the saline and lipid infusions. Free T4 and T3 were unchanged, suggesting that the increase in TSH was a pituitary thyrotrope cell response to insulin/lipid and not due to alterations in thyroid function. Levels of the lactotroph hormone PRL and somatotroph GH were not impacted by insulin/lipid, confirming that effects on the pituitary are cell type specific. Consistent with the lack of effect on GH, IGF-1 was also not significantly different between visits. Cortisol, a known inhibitor of the hypothalamic-pituitary axes was unaltered in the lipid/insulin visits, thereby removing this as a potential mechanism for the differences seen in FSH, LH, and TSH. These observations imply that the impact of obesity on the hypothalamic-pituitary-gonadal axis is not simply suppression and extends beyond reproductive functions. Further research is needed to elucidate the mechanisms underlying the cell type specific selective modulation of pituitary trophic hormones in response to changes in diet and metabolism.

34

25. Can IV administration of rFSH correct the hypogonadotropic hypogonadism state seen in obese women?First Author: Than Ha Luu, thanh-ha.luu@cuanschutz.edu, Anschutz Medical CampusMD Poster

Maternal obesity is an independent risk factor for decreased reproductive fitness. Decreased gonadtrophin secretion in obesity is well documented but poorly understood. Furthermore, obese women secrete less protein and steroid hormones from their ovaries. We hypothesize that pituitary dysfunction, due to insufficient FSH pulsatility results in inadequate folliculogenesis and reduced ovarian steroid and protein production as seen in obesity. We will attempt to correct pulsatile FSH secretion in obese women by superimposing an exogenous FSH to reverse the suppressed circulating ovarian hormones in obese women. Our primary outcome is the change in maximum inhibin B between post and pre-treatment. Herein, we present results from our interim analysis. Reproductive aged, regularly menstruating, normal weight (NW) (BMI 18.5-24.9) and obese (OB) (BMI >30) women were recruited for an overnight study during the early follicular phase. Frequent blood sampling (q10min) for 10h was initiated to obtain baseline hormonal levels. At 10h cetrorelix acetate (3 mg) was given followed by a secondary dose (0.25mg) 6h later. At this time, hourly IV rFSH (30IU) was initiated and frequent blood sampling continued for 10h. LH, FSH, estradiol (E2) were measured by immunoassay (Centaur XP, Siemens). Inhibin B was measured using a commercial ELISA kit (Ansh labs). Differences between groups in change outcomes were modeled in linear regression models, where adjusted models include age and cycle day (continuous). The relationship between change in maximal inhibin B and change in maximal E2 was estimated in a linear regression. A total of 36 participants (19 NW and 17 OB) were included in our interim analysis. There were no differences in age, cycle day of study, race, and waist/hip ratio. There was a statistically significant change in maximum inhibin B and E2 levels within each group in response to pulsatile rFSH. The difference in maximal inhibin B change between the two groups were statistically significant when we adjusted for age and cycle day and removed outliers [-47.5 (95% CI -92.5, -2.6), p=0.039]. No difference was seen in maximal E2 response. Furthermore, there did not appear to be a relationship between inhibin B and E2 response [0.08 (95%CI -0.26, 0.42), p=0.634]. These early results show that pulsatile rFSH does not correct the hypogonadopic hypogonadism state seen in obese women and suggest that the ovary is the primary focus of dysfunction in obesity.

35

26. Hormonal Impact on Citrullination and Peptidyl Arginine Deiminase Activity in FemaleReproductive TractFirst Author: Amanda Christensen, achris36@uwyo.edu,Undergraduate Poster

Women are three times more likely to develop rheumatoid arthritis (RA), develop the disease earlier, and have poorer clinical outcomes compared to men. RA auto-immunity is hypothesized to develop when citrullinated (cit) proteins are generated at a mucosal surface by peptidylarginine deiminases (PADs) enzymes, which post-translational convert arginine to a non-coded residue citrulline. Citrullinated proteins can stimulate creation of anti-cit protein antibodies (ACPA) and systemic inflammation. Studies of non-reproductive mucosal sites have not explained the gender disparity of RA. To address this intriguing health disparity in RA, we investigated whether the female reproductive mucosa is a site of RA initiation. Specifically, we investigated if PAD activity and ACPA production are present in the cervico-vaginal fluid (CVF). Our results demonstrate that PAD activity and levels of citrullinated proteins change in women’s CVF over the menstrual cycle and in mouse vaginal fluid during estrous cycle. Given this, we aimed to identify the citrullinated proteins in female CVF and develop a mouse model to determine the effects of hormones on PAD activity and cit-proteins in CVF. To test this, CVF samples were collected across the estrous cycle and changes in PAD activity examined. Next, we tested if estrogen controls PAD expression and resulting fluctuations in cit-protein levels. CVF was collected from ovariectomized mice treated with vehicle or estrogen and PAD activity measured. Our results suggest that PAD activity and levels of citrullinated proteins are present in the CVF and change over the course of the estrous/menstrual cycle.

36

27. Targeted Loading of Heat Stress-Associated Mirnas Into Extracellular Vesicles: PotentialApplication to Modulate Response to Heat Stress in Bovine Granulosa CellsFirst Author: Samuel Gebermedhn, etay@colostate.edu, Colorado State University Post - Doctoral Poster

Elevated summer heat stress (HS) is reported to be the leading cause of stress in dairy and beef cows, which negatively influences ovarian function, steroidogenesis, and embryonic development. Extracellular vesicles (EVs), naturally occurring nano-sized, bilipid membrane-enclosed complexes, released by cells into the surrounding extracellular space mediate cell-to-cell communication. The impact of HS on the cellular and extracellular microRNAs (miRNAs) expression in bovine granulosa cells (GCs) is not fully studied. We previously showed that GCs subjected to HS release a significantly higher number of EVs into the extracellular space compared to the unstressed counterparts. Moreover, small RNA seq analysis revealed that the EVs released from the HS cells are significantly enriched with bta-miR-1246, bta-miR-374a, and bta-miR-2904. Moreover, the coincubation of the stressed EVs with GCs delivered protective signals against subsequent HS. Here we aimed to assess and optimize multiple approaches of loading selected miRNAs into the EVs. For this, we utilized two approaches to load the candidate miRNAs in EVs derived from the unstressed GCs, the high-voltage electroporation (500 V for 10 ms) to exogenously load the miRNAs (100 µM) into EVs obtained from unstressed GCs. Besides overexpression of the cellular miRNAs using miRNA mimics was used to endogenously load the candidate miRNAs into the EVs. Results showed that high voltage electroporation of EVs with the candidate miRNAs increased the diameter of the EVs compared to the non-electroporated EVs. Moreover, the expression of miR-1246 has significantly increased in both the electroporated EVs and non-electroporated EVs, which signifies lower efficiency of delivery due to the preexisting high copy number of the miR-1246 in the EVs. Contrary to this, the expression of miR-374a was significantly higher only in the electroporated EVs compared to the non-electroporated counterparts, signifying higher efficiency of delivery. For the endogenous loading of the candidate miRNA, it was shown that miR-1246, miR-374a, and miR-2904 significantly enriched in GCs transfected with the miRNAs mimics. Interestingly, the expression of these miRNAs in the corresponding EVs was also significantly higher compared to the EVs from mimic NC transfected GCs. In conclusion, the selective loading of heat-stress-associated miRNAs into EVs depends either on the preexisting reserve of that particular miRNA and the technique used for loading. It is worth noting that EVs have the potential to be used as natural molecule delivery tools since they can be engineered to shuttle selective bioactive molecules associated with pathophysiological conditions of interest.

37

28. The Role of Granulosa Cell Derived Extracellular Vesicles in Bovine Oocytes Exposed toThermal Stress During In Vitro MaturationFirst Author: Nico Menjivar, menjivar@rams.colostate.edu, Colorado State UniversityPre-Doctoral Poster

Pregnancy can be disrupted through a multitude of factors, among the most prevalent are elevated thermal environments, which are reported to increase stress and compromise reproductive functions. In early stages of oocyte maturation, crucial intercellular communication within the follicle microenvironment has proven essential for establishing gamete competency. Extracellular vesicles (EVs), which are naturally occurring complexes released by most cells into the surrounding extracellular space are emerging as important mediators of cell-to-cell communication, underpinning the maintenance of physiological functions. EVs assume a natural role in genetic information transfer acting as carriers of bioactive molecules. Furthermore, EVs from bovine granulosa cells subjected to heat stress are reported to shuttle protective messages within granulosa cells against subsequent heat stress. Accordingly, EVs derived from heat stress granulosa cells could reduce ROS accumulation, improve cell viability and reduce apoptotic levels in recipient cells exposed to thermal stress. Here, we aimed to assess the effects of supplementing granulosa cell derived EVs during bovine IVM on oocyte competence and response to heat stress. For this, we modeled a cell culture protocol to generate EVs from bovine granulosa cells, with the ability to subject the cells to differing ambient temperatures (38.5°C body temperature of the cow vs. 42°C heat stress), isolating the EVs released into the spent culture media for the stated experiment. Nano-tracking Analysis (NTA) was used to analyze the size and concentration of EVs. Both stressed and nonstressed EVs were supplemented to the maturation media at a concentration of 1.5 x 108 particles/mL, which constitute 20% of the total volume of the maturation media. Oocytes cultured in the presence of these two types of EVs were cultured for the first 8 hrs at 38.5°C and half of the treatment groups were transferred to an incubator adjusted to 42°C to complete maturation until 23h. All subsequent events including IVF and IVC were carried out under normal 38.5°C. Results showed that oocytes supplemented with stressed EVs showed an increase in cumulusexpansion, compared to those not supplemented with EVs following exposure to heat stress.Furthermore, cleavage rate and blastocysts rates at Day 7 and Day 8 were assessed. Interestingly,results also showed that irrespective of EV supplementation, cleavage rates were lower (p= 0.0008) forall treatments subjected to heat stress compared to those cultured under normal temperature (70.33±0.33 vs 94.67±2.67). However, elevated blastocyst rates were observed in oocytes supplemented withstressed EVs compared to those without EV supplementation after subsequent heat stress. Inconclusion, the supplementation of bovine granulosa cell derived EVs has the ability to reducedetrimental effects of heat shock on the function of the oocyte.

38

29. The Effects of Omega-3 Fatty Acids on Cytokine-Induced Regulated Cell Death in BovineLuteal Microvascular Endothelial Cells.First Author: Anika Shelrud, anika.shelrud@unco.edu, University of Northern ColoradoMasters Student Poster

Prostaglandin (PG) F2a is the endogenous luteolysin in domestic ruminants. We have reported that dietary supplementation of fish meal or oil, both containing omega-3 fatty acids, decreases luteal responsiveness to a low-dose intrauterine infusion of PGF2a. However, the cellular and molecular mechanisms by which fish meal or oil protects the corpus luteum (CL) from PGF2a are largely unknown. Prostaglandin F2a increases synthesis of tumor necrosis factor alpha (TNF-a) and interferon-gamma (INFg) in cells of the CL. Additionally, cytokines have been shown to induce cell death in bovine luteal endothelial microvascular cells (CLENDO cells). It is hypothesized that omega-3 fatty acids, specifically docosahexaenoic acid (DHA) and eicosapentaenoic (EPA), prevent cytokine-induced apoptosis in CLENDO cells. The objective of the current study was to determine the effects of DHA and EPA on CLENDO cells. CLENDO cells were seeded in 6-well dishes at a 1 × 105 seeding density. Cells were incubated in either control BSA medium or medium supplemented with BSA and omega-3 fatty acids (10 mM DHA+EPA) for 72 h to allow fatty acids to incorporate into biological membranes. Cells were then cultured in 1% fetal bovine serum containing BSA alone or BSA with DHA+EPA for 12 h and subsequently treated with TNF-a (2.3 nM) and INFg (2.5 nM) for 0, 12, 24, 36 or 48 h. Proteins in cell lysates were separated using SDS-PAGE and transferred to PVDF membranes. Membranes were probed for cleaved caspase-3. Protein loading and transfer was normalized using beta-actin as a control. Cleaved caspase-3 was not detected at the 0 or 12 h time points in both the control and DHA+EPA treated group. In control treated cells, cytokines induced abundance of cleaved caspase-3 at 24, 36, and 48 h time points, with the greatest abundance occurring at 36 h. The DHA+EPA treated cells, however, had a relative reduction in cleaved caspase-3 at the respective time points. These preliminary data show that omega-3 fatty acids, specifically DHA and EPA, attenuate cytokine-induced regulated cell death in CLENDO cells, potentially providing a mechanism to the previously observed luteal protective role of dietary fish meal or oil following PGF2a administration.

39

30. Fibroblasts of the Bovine Corpus Luteum Release Prostaglandin F2α Into theMicroenvironment in Response to Pro-Inflammatory Cytokines.First Author: Corrine Monaco, corrine.monaco@unmc.edu, University Nebraska MedicalCenterPre - Doctoral Poster

The ovarian corpus luteum (CL) is a transient endocrine gland that produces the progesterone required for embryo development, implantation, and maintenance of pregnancy. In the absence of an embryo, luteal progesterone secretion is disrupted and the CL regresses to form a fibrotic corpus albicans. Prostaglandin F2α (PGF2α) has been implicated as a major luteolytic hormone in many species, including domestic farm animals, primates, and humans. PGF2α-induced regression in cows rapidly elevates the expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF) and interleukin 1β (IL1B). In addition to steroidogenic cells, the CL contains vascular endothelial cells, pericytes, and fibroblasts. While the exact role of luteal fibroblasts is unknown, previous studies show that luteal fibroblasts produce components of a fibrotic matrix. We hypothesize that luteal fibroblasts respond to pro-inflammatory cytokines and alter the microenvironment by producing additional luteolytic factors that contribute to successful luteal regression. Fibroblasts were isolated from bovine CL tissue punches, passaged 2-3 times, and treated with 10 ng/mL of TNF or IL1B to examine early intracellular signaling responses and production of PGF2α. Western blot analysis showed significant increases in NFκB (p65) (8-fold, p < 0.05, n = 6), and p38 MAPK (5.6-fold, p < 0.001, n = 6) phosphorylation by 10 minutes of TNF treatment. TNF increased phospholipase A2 phosphorylation nearly 2-fold (p < 0.05, n = 4) after 4 hours of treatment and prostaglandin-endoperoxide synthase 2 (PTGS2) protein expression 2-fold after 12 and 24 hours (p < 0.05, n = 4). TNFα also induced a 42-fold increase in PGF2α production (n = 2). Responses to IL1B were not as robust as TNF, but p65 phosphorylation was increased 2-fold after 10 minutes (n = 6, p < 0.05) and p38 phosphorylation increased 2.4-fold after 30 minutes (p < 0.05, n = 6). IL1B also increased PTGS2 expression (3-fold, p < 0.05, n = 4) and PGF2α production 9-fold by 4 hours and 28-fold by 24 hours (n = 2) of treatment. These results indicate that cytokines implicated in CL regression activate signaling pathways in fibroblasts to stimulate PGF2α release into the luteal microenvironment, potentially exacerbating regression of the CL.

40

2021 Program CommitteeDr. Thomas (Tod) Hansen, Chair, Colorado State UniversityDr. Gerrit (Jerry) Bouma, Colorado State UniversityDr. Jason Bruemmer, USDA-APHIS and Colorado State University Dr. Andy Bradford, University of Colorado Anschutz Medical Campus Dr. Brenda Alexander, University of WyomingDr. Patrick Burns, University of Northern ColoradoDr. Ye Yuan, Colorado Center for Reproductive Medicine

41

RMRSS 2021ACKNOWLEDGEMENTS

RMRSS, a regional meeting focused on graduate student training, is supported by the Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, and Vice President for Research Office at Colorado State University.

The 2021 symposium was supported by:• The Colorado State University Animal Reproduction and

Biotechnology Laboratory• CSU Ventures• The Society for the Study of Reproduction

Special thanks to Ana Hilton from the Society for the Study of Reproduction forproviding expertise and assistance in the planning and launching of the 2021 RMRSsymposium. The online venue for the event was made possible by a grant and supportfrom the Society for the Study of Reproduction.