Molecular Mechanisms of Estrogen Signaling W. Lee Kraus, Ph.D. The Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Division of Basic Science, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, 75390.

Estrogens are a class of endogenous steroid hormones that are required for the normal development and function of reproductive organs, mammary glands, bone, heart, vasculature, adipose, and the central nervous system. The biological actions of estrogens are mediated by two estrogen receptor (ER) isoforms, ERα and ERβ, which direct the gene regulatory effects that underlie estrogen control of cell physiology and pathology. In spite of considerable research in this area, the underlying molecular mechanisms by which estrogens exert their effects have not been fully elucidated. ERα functions as a ligand-regulated, DNA-binding transcription factor in the nuclei of estrogen-responsive cells. It acts primarily as a nuclear transcription factor, binding to many thousands of ERα binding sites (ERBSs) across the genome, collectively called the ERα ‘cistrome’. The binding of ERα to genomic DNA promotes the coordinated recruitment of coregulator proteins that establish an active ‘enhancer,’ leading to chromatin looping and target gene transcription. In many respects, ligand- and DNA-bound ERα acts as a nucleation site and scaffold for the assembly of a multi-protein complex containing histone-modifying enzymes, ATP-dependent nucleosome remodeling enzymes, and Mediator, an RNA polymerase II (Pol II)-interacting coregulator. The transcriptional effects of estrogen signaling are rapid, on the order of minutes, resulting in transcription at both the target genes and their distal ERα enhancers. We have been exploring the molecular mechanisms of estrogen-dependent gene expression, in particular the molecular events leading to the establishment of active enhancers, using a variety of biochemical, molecular, cell-based, genetic, and genomic assays. Our studies are aimed at linking specific molecular mechanisms to physiological and pathological outcomes in the reproductive tract and mammary glands, related to pregnancy, parturition, and cancer.

The estrogen-related studies in the Kraus lab are funded by grants from the NIH/NIDDK and CPRIT, as well as the Cecil H. and Ida Green Center Endowments.

Transplantation of Immune Privileged Sertoli Cells: More Than Just Testicular Nurse Cells Jannette M. Dufour, Gurvinder Kaur, Lea Ann Thompson Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas Testis immune privilege was first described over two centuries ago. This immune privileged is important for protecting the developing auto-antigenic germ cells from immunological attack. Moreover, foreign tissue grafts transplanted into the testis enjoy prolonged survival. Sertoli cells (SC) are considered key players in creating this immune-privileged environment, which has led to their use in transplantation. For instance, SC survive and protect co-transplanted allografts or xenografts, such as pancreatic islets without the use of chronic immune suppressing drugs. However, the mechanism for this protection remains unresolved. Interestingly, we have found that successful co-transplantation of SC with islets is associated with the formation of tubule-like structures. Unexpectedly, the insulin-expressing islets were not located in the center of these tubules and yet enjoyed prolonged graft survival. This is similar to the testis where foreign tissue grafts and germ cells located outside of the blood-testis-barrier are still immune protected and suggests that, contrary to popular belief, testis immune privilege involves more than just sequestering auto-antigens behind the blood-testis barrier. In support of this, we have found that SC express several immunoregulatory proteins and survive transplantation by inducing regulatory cells (Tregs and macrophages) and creating a tolerogenic environment. In our model, the SC survival could be attributed to a significant decrease in inflammatory cytokines and M1 macrophages, while at the same time a significant increase in anti-inflammatory cytokines, M2 macrophages and regulatory T cells was observed. More recently, we have explored the possibility of using SC in cell based gene therapy by engineering them to express basal levels of insulin. When SC were engineered to transiently express insulin, using an adenoviral vector, they significantly decreased blood glucose levels after transplantation into diabetic SCID mice. To provide stable insulin expression, SC were engineered to deliver insulin using a lentiviral vector. These cells survived and expressed insulin for over 50 days when transplanted as allografts in diabetic BALB/c mice. Using a modified lentiviral construct, we have significantly increased the amount of insulin secreted by the SC and successfully lowered blood glucose levels in diabetic mice. Overall, our results demonstrate that SC immunoprotect co-grafted cells by creating an immune privileged environment. Additionally, SC stably produce basal insulin thereby, strengthening the use of SC in cell based gene therapy. Supported by NICHD HD067400, NIAID AI109398 and the Wilson Foundation.

Tspan8 Expression Distinguishes Spermatogonial Stem Cells from Progenitor Spermatogonia. Kazadi Mutoji1, Thu Nguyen1, Christopher B. Geyer2, Amy V. Kaucher3, Melissa J. Oatley3, Jon M. Oatley3, John R. McCarrey1 and Brian P. Hermann1 1Department of Biology, University of Texas at San Antonio 2Department of Anatomy and Cell Biology, East Carolina University 3Center for Reproductive Biology, Washington State University Spermatogenesis and male fertility are dependent upon formation of a pool of spermatogonial stem cells (SSCs) in the mammalian testis. SSCs arise from prospermatogonia, but only some prospermatogonia form the foundational SSC pool, suggesting there are subtypes of undifferentiated germ cells with discrete molecular signatures that predispose distinct fates. We recently used single-cell gene expression approaches to demonstrate considerable heterogeneity in mRNA and protein abundance of a panel of germ cell and stem cell genes in the P6 mouse testis. The results of that study supported the concept that multiple spermatogonial subtypes are present in the neonatal mouse testis which may represent functionally distinct spermatogenic cell types. Twenty-seven of the 172 genes examined exhibited bimodal mRNA levels among Id4-eGFP+ spermatogonia (i.e., present/absent), suggesting these genes mark two or more discrete cell subpopulations of Id4-eGFP+ undifferentiated spermatogonia. Flow cytometry and fluorescence-activated cell sorting (FACS) using antibodies against one cell surface marker encoded by a bimodal gene, TSPAN8, was used to sub-fractionate P6 Id4-eGFP+ spermatogonia. While no difference was observed in cell cycle state between Id4-eGFP+ spermatogonia that were TSPAN8-hi (top 1/3 brightest) and TSPAN8-lo (bottom 1/3 dimmest), RNA-seq analyses defined 132 and 159 genes which were differentially up-regulated ≥2-fold in TSPAN8-hi or TSPAN8-lo cells, respectively. Transplantation studies demonstrated that SSC activity was enriched among the TSPAN8-hi Id4-eGFP+ spermatogonia and depleted among TSPAN8-lo Id4-eGFP+ spermatogonia. These results suggest that bimodal Tspan8 expression among individual spermatogonia marks phenotypically and functionally-distinct subpopulations of P6 Id4-eGFP+ spermatogonia and defines stem and non-stem spermatogonia. This further supports our contention that the gene expression heterogeneity we recently reported reflects the existence of distinct subtypes of undifferentiated spermatogonia with differing functional capacities, including stem and progenitor spermatogonia fates.

This study was supported by NIH grants HD062687 (BPH), HD061665 (JMO), HD072552 (CBG), and GM092334 (JRM), NSF grant 1337513 (BPH), the Max and Minnie Tomerlin Voelcker Fund, the Kerr Foundation, the Kleberg Foundation, and The University of Texas at San Antonio.

Effect of Cabozantinib on HGF-MET Signaling in the Testis. Sean Kow, MD Anderson Cancer Center; Marie-Claude Hofmann, Ph.D., MD Anderson Cancer Center; Thomas Garcia, Ph.D., University of Houston-Clear Lake Objective: Testicular germline stem cells are crucial for male fertility. Crizotinib, a kinase inhibitor targeting MET activity, induces hypogonadism in men treated for metastatic non-small cell lung cancer. The mechanism causing hypogonadism by crizotinib—and whether inhibitors against MET can cause irreversible damage to the stem cell pool—is unknown. Hepatocyte growth factor (HGF), the ligand for the MET receptor, acts as a pleiotropic factor and cytokine in a number of tissues. Mounting evidence demonstrates MET expression in germ cells and HGF expression in somatic peritubular cells. However, the physiological function of HGF/MET in the testis has yet to be established. The objectives of this study were to further characterize the expression and activation of MET in the seminiferous epithelium using mice as a model, and to test the hypothesis that treatment of mice with cabozantinib, another kinase inhibitor against MET, can cause hypogonadism. Materials & Methods: qPCR analysis for Met and Hgf was performed with freshly isolated mouse germ cells and Sertoli cells. Expression of MET, pMET and HGF was tested by immunocytochemistry along with the germ cell markers GFRα1, POU5F1-GFP, PLZF, and KIT. Mice were administered cabozantinib via daily oral gavage, and testes were evaluated for germ cell numbers. Results: qPCR analysis of freshly-isolated germ cells and Sertoli cells revealed that MET is expressed by germ cells, while HGF is undetectable in mouse germ cells or Sertoli cells. Immunohistochemistry of mouse testes revealed stage-specific MET activation (staining for pMET) only in differentiating germ cells, but not in undifferentiated spermatogonia. Treatment with cabozantinib resulted in significant decreases in testes weights, along with decreases in tubule diameter and epithelial thickness. Further analysis revealed these decreases were due a loss of differentiating germ cells, but not stem cells or somatic cells. Conclusion: Taken together, these results demonstrate that the HGF/MET signaling pathway may be involved in germ cell differentiation, and that MET kinase inhibitors might induce hypogonadism without affecting the stem cell pool. Further study with other kinase inhibitors against MET and with mice treated and assessed longer-term is required to support the interpretation that there is impairment of fertility, yet likely to be reversible.

Karyotype and Y-Chromosome Microdeletion for Men with Testicular Failure: Subsequently or Simultaneously? A Cost-Effectiveness Analysis

Taylor P. Kohn1, Stephen M. Pickett2, Ranjith Ramasamy3, Larry I. Lipshultz4, Dolores J. Lamb4

Affiliations: 1Baylor College of Medicine, Houston, TX; 2Department of Economics, Rice University, Houston, TX; 3 Department of Urology, Miller School of Medicine, University of Miami, Miami, FL; 4 Scott Department of Urology, Baylor College of Medicine, Houston, TX Introduction and Objective: The American Urological Association guidelines recommend that both karyotype and Y-chromosome microdeletion testing (YCM) are offered to men with azoospermia and severe oligospermia (<5 million sperm/ml). We assessed the prevalence of karyotype abnormalities and YCM among men with testicular failure, and performed a cost-effectiveness analysis to determine if testing should be performed sequentially rather than simultaneously. Methods: We reviewed records of men with testicular failure (azoospermia or severe oligospermia) who underwent both YCM and karyotype between January 2000 and June 2015. A cost-effectiveness analysis examined if, and to what extent, a financial benefit exists when performing karyotype and YCM testing sequentially, rather than simultaneously. Three end outcomes were identified: genetic counseling, surgical sperm retrieval, and no surgical intervention. Results: This study represents one of the largest reported comparison of karyotype and YCM testing frequency at a single institution. 578 men were identified as having testicular failure and completed both karyotype and YCM testing. 57 men had karyotype abnormalities (9.8%) and 36 men had positive YCM (6.2%) (Figure 1). Of the 578 men, 11 men (2%) had both a karyotype abnormality (2 patients with 46 XX, 3 inverted duplications, 3 inversions, 2 translocations, and 1 deletion) as well as positive YCM (8 AZFab and 3 AZFabc). At our institution, the cost of a karyotype assay is $800 and a YCM assay is $500, with a cost of $1300 if run simultaneously. We examined which patients would not need a second test if they were run sequentially. YCM testing was unnecessary in 9.8% of the men who had either autosomal or X chromosomal abnormalities (4.3%) requiring genetic counseling, Klinefelter syndrome (3.6%) who underwent testicular sperm extraction (no men with Klinefelter syndrome had a positive YCM assay), and men with AZFa or b microdeletion (1.9%) who chose donor sperm or adoption (Figure 1). Sequential testing for karyotype first and then YCM, results in 9.8% of men savings of $500. This reduces the average cost of testing to $1,250.69 per patient, a savings of 3.8%. Conclusion: The probability of having both a karyotype abnormality and YCM in men with testicular failure is extremely rare. Sequential performance of karyotype, followed by YCM assay if there are no abnormal karyotype findings results in demonstrable financial savings.

Figure 1: Flowchart for YCM and karyotype testing.

IVF/ICSI

Title: A Defect At the Mitotic:Meiotic Switch Contributes to Testicular Germ Cell Tumorigenesis

Authors: Dawson, EP; Lanza, DG; Webster, NJ; Benton, SM; Heaney, JH.

Background: In the 129 inbred mouse model of human testicular germ cell tumors (TGCTs), tumors initiate between embryonic days (E)13.5 to 15.5, which coincides with the mitotic:meiotic switch: germ cells of both sexes lose pluripotent capacity, female germ cells (oogonia) commit to meiosis, and male germ cells (gonocytes) enter mitotic arrest. We previously discovered that genes expressed in pre-meiotic oogonia, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), are aberrantly expressed in tumor-susceptible gonocytes and appear to contribute to TGCT initiation by delaying gonocyte entry into mitotic arrest and suppression of pluripotency. Importantly, expression of these genes in oogonia and tumor-susceptible gonocytes at the same developmental time-points suggests that a signal normally active in the testis may be absent or diminished in both the ovary and tumor-susceptible testis. Therefore, we hypothesized that delayed activation of the male germ cell specification program contributes to TGCT initiation in mice.

Methods: To test whether components of the male germ cell specification program are repressed in tumor-susceptible strains, we compared a tumor-resistant strain, FVB/NJ (FVB), and two tumor-susceptible strains, the 129-Chr19MOLF/Ei chromosome substitution strain (M19; 80% affected) and the 129/SvImJ inbred strain (129; 8% affected). All strains harbor a germ cell-specific GFP transgene for immunostaining experiments or fluorescence activated cell sorting (FACS) followed by gene expression analyses.

Results: Although male differentiation signals from somatic cells, namely Fgf9 and Activin A/B, are not differentially expressed between tumor-resistant and susceptible strains, we determined there is a significant decrease in the expression of an indispensable male germ cell specification gene, Nanos2, in tumor-susceptible gonocytes. Importantly, expression of downstream effectors of Nanos2 involved in male germ cell specification (e.g. Dnmt3l, Tdrd1/9) was significantly decreased in tumor-susceptible gonocytes, indicating that there is a functional consequence for decreased Nanos2 expression. We are currently investigating whether a 129-specific SNP in Nanos2 is responsible for the differential expression of Nanos2 between tumor-resistant and susceptible strains.

Conclusion: We conclude that there is delayed expression of genes important to male germ cell specification in tumor-susceptible strains. Further experiments are necessary to determine whether this delay contributes to the induction of tumor-susceptible phenotypes.

Control of primordial oocyte reawakening by Kit

H. D. Saatcioglu, I. Cuevas, and D. H. Castrillon Department of Pathology and Cecil H. and Ida Green Center for Reproductive Biology Sciences, UT Southwestern Medical Center, Dallas, TX 75390-9148

Females are born with a finite number of oocytes stockpiled in the form of primordial follicles. Oocytes are “reawakened” via an irreversible, metered process whereby they are selected from this reserve pool to initiate follicle growth. Oocyte reawakening must be delicately balanced to ensure that some growing oocytes are available during each estrus cycle but at the same time, limit the number of growing follicles to avoid premature depletion of primordial follicles. Because the mechanisms controlling reawakening in effect balance fertility with reproductive senescence, their elucidation is of fundamental importance. The forkhead transcription factor Foxo3 is a critical regulator and suppressor of this process. Foxo3 serves as a molecular switch whose activity (localization in cytoplasm versus nucleus) is under the control of PI3K-AKT signalling within the oocyte.

However, the identity of

the presumptive upstream signalling factor(s) controlling the PI3K-AKT-Foxo3 axis has been controversial.

Kit is highly expressed in oocytes, but firm genetic evidence implicating Kit in oocyte reawakening has been lacking. To define the role of Kit in this process, we created two novel alleles in mice. In the first, we engineered a conditional knockin mutation (Kit

D818V), which results in constitutive Kit activity. In the second (Kit

-), the kinase

domain essential for Kit activity was floxed, permitting cell-specific conditional inactivation. These two alleles are complementary, as one is gain-of-function, while the other is loss-of-function. We employed the germ-cell specific Cre driver (Vasa-Cre) to explore the effects of Kit hyperactivity vs. inactivity within oocytes. Kit

D818V adult females

were infertile and showed elevations in both FSH and LH levels (P=0.0021 and 0.0114; n=3) consistent with ovarian failure. Histomorphological analysis revealed that oocyte diameters of the mutant females were larger on day 7, 14, and 28 (P<0.001; n>6), and their ovaries became depleted of all oocytes by 6 weeks of age, a classic global reawakening phenotype. Additionally, Foxo3 protein was exported from the nucleus in Kit

D818V oocytes,

demonstrating that reawakening was dependent on the action of Kit via PI3K-Akt-Foxo3. Kit- females were also

infertile with ovarian failure, but exhibited an opposite phenotype: they had smaller ovaries and viable oocytes with no significant oocyte loss up to 6 weeks of age. Histomorphological analysis revealed that oocytes failed to grow by 6 weeks (i.e. a complete failure of reawakening). Also, oocyte Foxo3 remained constitutively nuclear, demonstrating that loss of Kit activity caused the oocytes to remain in a state of “suspended animation” via constitutive Foxo3 localization. Taken together, our findings strongly implicate Kit as the key upstream signalling molecule and cell surface receptor, acting through the PI3K-AKT-Foxo3 axis, to control the reawakening of oocytes.

Protein Restriction and the Oocyte: Effects on Amino Acid Kinetics and Mitochondrial Structure and Function. A. Schutt,1 C. S. Blesson,1 J. W. Hsu,2 C. T. Valdes,1 W. E. Gibbons,1 F. Jahoor,2 C. Yallampalli1; 1Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, 2Children's Nutrition Research Center, USDA/ARS, Department of Pediatrics, Baylor College of Medicine, Houston, TX Introduction: Maternal dietary intake critically supports the oocyte through maturation, fertilization and until embryo genome activation. However, little is known about the effects of protein restriction on amino acid metabolism in the oocyte proper, and how these changes may impact antioxidant homeostasis and mitochondrial structure and function. Dietary protein provides substrates for the production of cysteine, which is the rate limiting amino acid for the synthesis of glutathione (GSH), the primary intracellular antioxidant. This series of experiments investigated the effects of an isocaloric low protein (LP) diet with and without folate supplementation on amino acid kinetics, glutathione production, and mitochondrial ultrastructure and function within the cumulus oocyte complex (COC). Methods: Rats (n=24) were assigned to three isocaloric dietary groups (n=8) for 40 days; control (C, 20% protein), low protein (LP, 6%) and low protein + folate (LPF, 6% + 5mg/kg folate). 15U of pregnant mare serum gonadotropin was given; 48h later animals underwent central catheterization. Isotopic tracers of 13C3-Serine and D2-Cysteine were infused as a 4h prime-constant rate. Blood sampling was performed at times 0, 3, 3.5, 3.75 and 4 hours. After sacrifice, germinal vesicle oocytes were denuded of cumulus cells, and liver specimens were obtained. The rate of appearance of each traced amino acid was calculated using the tracer dilution method. ANOVA followed by post-hoc comparisons were performed. For mitochondrial ultrastructure, ovarian sections were fixed. Transmission electron microscopy (TEM) was used to examine mitochondrial morphology in tertiary cumulus oocyte complexes (COCs); n=4 per group, oocyte and cumulus cell mitochondria were analyzed. Whole transcriptome amplification was utilized to enable the analysis of cohorts of 10-20 denuded oocytes per animal. mRNA expression levels of various genes involved in the regulation of mitochondrial structure and function via qPCR. Results: Preovulatory protein restriction increased the flux of serine and cysteine, and increased the concentration of GSH in RBCs and liver in LP and LPF groups compared to control (p<0.01 and p<0.001, respectively). Oocyte kinetics demonstrated a suppressed cysteine flux in the LP group (p<0.05) compared to control, suggesting an inability to compensate effectively in a state of protein starvation. Oocyte mitochondrial ultrastructure in LP and LPF groups demonstrated an increased prevalence of abnormal mitochondria (p<0.01 and p<0.05, respectively) compared to control (C = 14.6%, LP = 52.7%, LPF = 47.1%). Cumulus cell mitochondrial ultrastructure in LP and LPF groups also demonstrated an increased prevalence of abnormal mitochondria (p<0.001 and p<0.05, respectively) compared to control (C = 12.7%, LP = 52.4%, LPF = 38.9%). Additionally, protein restriction altered oocyte expression of genes related to mitochondrial biogenesis including Drp1, Opa-1, Mfn1/2, Parl and Ndufb6 (p<0.05) and Hk2 (p<0.01). Conclusions: Preovulatory protein restriction resulted in profound changes in amino acid metabolism and COC mitochondrial structure and function. Amino acid kinetics in the oocyte demonstrated a decrease in cysteine flux, suggesting an inability to compensate effectively for protein starvation. It may be inferred that the LP oocyte is vulnerable to oxidative stress as it cannot increase GSH production. Evidence for this is manifested via abnormal COC mitochondrial ultrastructure and altered oocyte expression of genes related to mitochondrial biogenesis. Ultimately, these metabolic and functional responses may affect the reproductive potential of oocytes.

Conditional depletion of the hedgehog pathway regulator Patched1 by S100a4 promoter-

driven Cre recombinase impairs ovarian and pituitary functions and fertility in female

mice.

Teresa Monkkonen1, 2, Michael T. Lewis1, 2, 3, JoAnne S. Richards1, and Yi Athena Ren1

1Department of Molecular and Cellular Biology and 2Lester and Sue Smith Breast Center, 3Department of Radiology, Baylor College of Medicine, Houston, TX 77030, USA

Abstract

The hedgehog (HH) signaling pathway is a key mediator of stromal and epithelial cell

interactions regulating development and homeostasis in many tissues. Previous studies of HH

signaling in the murine ovary indicate paracrine mechanisms of action, but the functional

consequences of HH in stromal cells remain to be clearly defined. In the canonical HH pathway,

Patched1 (PTCH1) functions as a suppressor of HH signaling activation; binding of HH ligands

releases this suppression and induces GLI-mediated transcriptional activity. To dissect the

functions of HH signaling specifically in stromal cells, we crossed the Ptch1f/f mice with mice

expressing S100a4 promoter-driven Cre recombinase. The female S100a4-Cre;Ptch1f/f mutant

mice are infertile. S100A4 is a small calcium-binding peptide expressed not only in fibroblasts

but also in myeloid cells (such as macrophages) and lymphoid cells. To determine the cell type-

specific expression of S100A4 in the ovary that may underlie infertility, we crossed S100a4-Cre

mice with the membrane-bound Tomato:EGFP Cre reporter mice. Green fluorescent protein-

positive (GFP+) cells were observed in the stroma but not in granulosa cells or oocytes. Co-

immunofluorescent (IF) staining of GFP and F4/80 demonstrated that many GFP+ cells were

also F4/80+, and thus are macrophages. Other GFP+ cells appeared to be fibroblasts according

to their morphological features but are not immuno-positive to antibodies against α-SMA+ or

vimentin. At 8 weeks of age, ovaries of the infertile S100a4-Cre;Ptch1f/f mice were devoid of

corpora lutea and had few large antral follicles; rather, abnormal, degenerating small follicles

with reduced FOXL2 immunostaining were observed, suggesting impaired follicular

development. Quantitative real-time PCR analyses documented that whereas expression of

some genes associated with growing follicles including Cyp19a1, Fshr, Cyp17a1, Insl3, and

Foxo1 was comparable between wild-type (WT), heterozygous and homozygous mutants,

expression of Lhcgr and Cyp11a1 was significantly lower in homozygous mutant ovaries

compared to WT. Importantly, mRNA levels of HH signaling components and activity read-out,

Gli1 and Gli2 appeared increased in homozygous mutant ovaries compared to WT. These

results indicate that HH signaling activity is up-regulated and that functional luteal tissue is

absent in the mutant ovaries. We have also observed that serum follicle-stimulating hormone

(FSH) and pituitary Fshb mRNA levels were comparable between mice of different genotypes,

whereas serum luteinizing hormone (LH) and pituitary Lhb levels were significantly lower in the

mutants at 8 weeks of age. Thus, pituitary function was also compromised in the S100a4-

Cre;Ptch1f/f mutant females. IF analyses confirmed that GFP+ cells were present in the pituitary

and, as in the ovary, mRNA levels of Gli1 and Gli2 appeared elevated in homozygous mutant

compared to WT, indicating that HH signaling activity is also up-regulated in pituitaries of the

S100a4-Cre;Ptch1f/f mutant mice. Collectively, these results provide provocative evidence that

disruption of PTCH1-dependent signaling in monocytes (such as macrophages) impairs

follicular growth, corpus luteum formation, as well as pituitary function, highlighting the impact of

altered monocyte function on the female hypothalamic-pituitary-gonadal axis and fertility. NIH-

HD-076980, T32 HD007165, NSF-1263742, NIH-CA-127857.

In vivo high-resolution imaging of reproductive events and ciliary behavior in the mouse oviduct

Shang Wang1, Jason C. Burton

1, Richard R. Behringer

2, and Irina V. Larina

1

1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030

2Department of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030

The understanding of the reproductive events and the molecular mechanisms regulating fertility and

infertility in humans relies heavily on the analysis of the corresponding phenotypes in mouse models.

While molecular genetic approaches provide significant insight into the molecular regulation of these

processes, the lack of live imaging methods that allow for detailed visualization of the mouse

reproductive organs limits our investigations of dynamic events taking place during the ovulation, the

fertilization and the pre-implantation stages of embryonic development. For its normal functions, the

mammalian oviduct relies heavily on the motility of cilia which line the lumen. The role of cilia is well

recognized, but due to their small size (~5-10 μm in length and ~300 nm in diameter), live visualization

of oviduct cilia and their activity in the lumen of the oviduct through tissue layers represents a major

challenge not yet overcome. For the first time, we developed an in vivo three-dimensional imaging

approach for visualizing the mouse reproductive events with micro-scale spatial resolution using optical

coherence tomography (OCT). Animal surgical procedures similar to the ones used for production of

transgenic mice are utilized to expose the reproductive organs for imaging in anesthetized females. OCT

imaging relies on the natural tissue optical contrast and does not require the application of any contrast

agents or vital reporters, which makes our approach easily applicable to analysis of mutant phenotypes.

We also developed functional imaging method based on spectral analysis of the OCT speckle variations

produced by the beat of cilia in the oviduct for in vivo micro-scale mapping of cilia and cilia beat

frequency (CBF) in the mouse oviduct. We will present in vivo structural imaging of the mouse oviduct

capturing the oocyte and the preimplantation embryo and then show the result of depth-resolved high-

resolution CBF mapping in the oviduct of live mice. We validated our approach with widely-used

microscopic imaging methods and demonstrated the ability of this approach to differentiate CBF in

different locations of the oviduct at different post-conception stages. Our data suggest that the described

structural and functional OCT imaging approach can be a useful tool for a variety of live investigations of

mammalian reproduction and infertility.

Figure: In vivo micro-scale structural and functional imaging of the

mouse oviduct.

Top: depth-resolved cross section from in vivo 3D OCT structural

imaging of the ampulla of the mouse oviduct shows the clear

visualization of the mucosa folds, oocytes and cumulus cells.

Bottom: depth-resolved micro-scale mapping of CBF in vivo in the

ampulla of the mouse oviduct at 0.5 days post conception.

Corresponding publications:

J. C. Burton, S. Wang, et al, Biomedical Optics Express, 6(7): 2713-2723, 2015.

S. Wang, J. C. Burton, et al, Scientific Reports, 5: 13216, 2015.

Uterine Activin-Like Kinase 4 Regulates Trophoblast Development During Mouse

Placentation

Fullerton PT1, Peng J1, Monsivais D1, Clementi C1, Su GH2, Matzuk MM1

1. Baylor College of Medicine, 2. New York University School of Medicine

After fertilization, the placenta is the first organ to develop. The placenta acts as the interface

between the developing fetus and the maternal uterus allowing the exchange of nutrients and

gases. In the past two decades, in vivo and in vitro approaches have been used to show that

members of the TGF-β superfamily regulate embryo implantation and placental development.

Nodal, a TGF-β superfamily ligand, is essential for mesendoderm formation and left-right axis

patterning during embryogenesis, and Nodal null mutants exhibit abnormal placental

organization with expansion of trophoblast giant cells and a decrease of spongiotrophoblast and

labyrinth. To better understand the importance of Nodal signaling in the uterus, we established a

mouse model to conditionally ablate activin-like kinase 4 (ALK4; the Nodal type 1 receptor)

using Cre recombinase driven by the progesterone receptor promoter sequences (Pgr-Cre).

Mating for six months with wild-type males showed that Alk4 conditional knockout (cKO)

females are subfertile with significantly fewer total pups, pups per month, litters per female, and

pups per litter. The subfertility is due to placental abnormalities and fetal loss in pregnancy. Alk4

cKO mothers have a placental disorganization phenotype similar to Nodal null mice including

expansion of trophoblast giant cells and a reduction of spongiotrophoblast and labyrinth at 10.5

days post-coitus. We hypothesize that Nodal functions as an indirect regulator of placental

development by binding to type 1 and type 2 receptors on maternal decidual cells to stimulate

expression of unknown regulators of placental development. Our study not only describes a

novel mouse model that enables study of Nodal signaling in placentation, but also highlights the

importance of signaling through ALK4 in the maternal decidua for placentation. A clearer

understanding of the mechanisms underlying placentation, particularly the maternal role, will

hopefully enable screening to identify women at risk for placental defects and the development

of treatments that can promote normal placentation.

Effects of Agmatine on Ovine Trophectoderm Cells for Production of Interferon Tau and Catecholamines, and Expression of Genes Related to Production of Polyamines

Yasser Y Lenisab, Xiaoqiu Wangab,1, Wanjin Tangab, Guoyao Wuab and Fuller W. Bazerab aDepartment of Animal Science, and bCenter for Animal Biotechnology and Genomics, Texas A&M University. 1Current address: National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 Embryonic mortality is an impediment to maximizing reproductive performance in animal production systems. Most embryonic losses occur during the peri-implantation period of pregnancy in sheep when conceptuses undergo a dramatic morphological transition from spherical to tubular and then filamentous forms. These changes and embryonic survival require histotrophic nutrition, which includes molecules secreted, or transported into the uterine lumen by uterine epithelia. L-Arginine (Arg) is a common substrate for synthesis of nitric oxide, ornithine, proline, glutamate, creatine, urea, polyamines, and agmatine. Agmatine (Agm), a product of arginine decarboxylation, is a substrate for agmatinase for synthesis of putrescine and other polyamines by the ovine conceptus. Polyamines are essential for conceptus development. The goal of this study was to compare effects of Arg and Agm in equimolar concentrations (200 µM) on secretion of interferon tau (IFNT) and catecholamines and expression of genes related to production of polyamines by ovine trophectoderm (oTr1) cells cultured in vitro. Arg increased (P< 0.05) proliferation of oTr1 cells by 2.2- and 3.6-fold at 48 and 96 h, respectively, while the combination of Arg and Agm increased (P< 0.05) proliferation of oTr1 cells by 2.0- and 3.5-fold at 48 and 96 h, respectively; however, Agm alone had no effect (P>0.05) on proliferation of oTr1 cells. The combination of Arg and Agm increased (P<0.05) migration of oTr1 cells by 1.8- and 1.5-fold, respectively, while Agm alone had no effect (P>0.05). Neither Arg nor Agm affected cell adhesion. After 96 h of culture1 of oTr1 cells, the total amount of IFNT in culture medium increased (P< 0.05) in response to Arg (1.6-fold) and Arg plus Agm (1.4-fold); however, Arg and Arg plus Agm decreased IFNT production per oTr1 cell. Arg alone and Agm plus Arg decreased secretion of dopamine and norepinephrine by oTr1 cells at 48 and 96 h of culture, but total DNA and RNA in oTr1 cells after 96 h of culture was not affected (P > 0.05) by treatment with Arg and Agm. Agm increased (P > 0.05) expression of mRNAs for SLC7A1, agmatinase and AZ2, while the combination of Arg and Agm decreased (P > 0.05) expression of mRNAs for ODC1, SLC7A1, AZ1 and AZ3 by oTr1 cells. Although Agm is considered an anti-tumor molecule and, in the present study, it did not stimulate proliferation, migration or adhesion of oTr1 cells or their secretion of catecholamines, Agm did increase transcription of SLC7A1 and agmatinase genes. This suggests that Agm has important roles regulating synthesis of polyamines in the ovine conceptus, particularly for enhancing transport of basic amino acids (particularly Arg) into trophectoderm cells for synthesis of polyamines and nitric oxide. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-20028 from the USDA National Institute of Food and Agriculture.

NELL1: A Genetic Factor Predisposing to Fibrosis Associated with Peyronie’s Disease Alexander W. Pastuszak1,2, Juan Bournat1, Yofre Cabeza-Arvelaiz1, Larry I. Lipshultz1,2, Dolores

J. Lamb1,2,3

1Center for Reproductive Medicine, 2Scott Department of Urology and 3Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, TX. Objectives: Peyronie’s disease (PD) is characterized by development of fibrotic plaques in the penile tunica albuginea (TA), triggered by an abnormal inflammatory process involving myofibroblasts and osteoblasts. The genetic factors predisposing to PD are unknown. We sought to identify genes involved in PD, and herein present data implicating NELL1, a gene involved in osteogenesis and inflammation. Materials and Methods: Genomic DNA from 16 men with both PD and Dupuytren’s Disease (DD) and 4 controls was used for microarray-based comparative genomic hybridization (aCGH). Copy number variations (CNVs) were identified and candidate PD genes were selected. Gene copy numbers were validated using gene-specific Taqman qPCR and DNA sequencing used to identify mutations in genes of interest. Fibroblasts from the TA of a patient with PD were treated with human recombinant NELL1 (hrNELL1) protein to determine effects on myofibroblast and osteoblast differentiation pathways using qPCR. Results: Using aCGH, 23.1kb and 16.0kb microdeletions at chromosome 11p15.1 encompassing the NELL1 gene were identified in 2/16 patients (12.5%) and validated. In contrast, in the DECIPHER database of ~50,000 patients not selected for specific conditions, the NELL1 CNV frequency is 0.02%. Sequencing of NELL1 in 16 men with PD and DD identified a missense mutation, R82Q, in exon 3 in 14 patients (87.5%), predicted to negatively affect protein function. The R82Q Variant is also associated with two other inflammatory disorders - Crohn’s disease and ankylosing spondylitis. NELL1 was highly expressed in TA fibroblasts derived from a PD patient, and overexpression of NELL1 in human TA fibroblasts resulted in downregulation of profibrotic genes including ACTA2, CTGF, and EZH2, and upregulation of the antifibrotic genes BMPR1B, GDNF, HGF, LIF, and STAT1. Conclusions: Microdeletions in NELL1, as well as a missense mutation linked to inflammatory conditions, were identified in men with PD. NELL1 is highly expressed in TA fibroblasts from men with PD, and overexpression of NELL1 induces expression of myofibroblast and osteoblast pathway markers. These results support a role for NELL1 in inflammatory pathway activation and pathogenesis in men with PD. Support: AWP is a National Institutes of Health (NIH) K12 Scholar supported by a Male Reproductive Health Research Career (MHRH) Development Physician-Scientist Award (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program (to DJL).

Osterix is a Novel Regulator of Male Reproductive Tract Differentiation

Rachel D. Mullen, and Richard R. Behringer

Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030

Early in development the reproductive tract of both sexes consists of the Wolffian duct

and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct and upper vagina. Previous studies in humans and mice have demonstrated that MD regression requires binding and signal transduction from the transforming growth factor-β family member anti-Müllerian hormone (AMH) secreted from fetal testis and its type 1 and 2 receptors found in MD mesenchyme. RNA-Seq analysis of YFP positive MD mesenchymal cells FACS sorted from E14.5 Amhr2Cre/+;R26Ryfp/+ reproductive tracts was used to globally identify transcriptome differences in the MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) during regression. This analysis identified Osterix (Osx)/ Sp7, a C2H2-type zinc finger transcription factor required for osteoblast differentiation, as a possible downstream effector of AMH-signaling during MD regression. Using in situ hybridization, Osx was shown to be expressed in a male-specific pattern in the MD mesenchyme during MD regression. Osx expression was lost in AMH signaling mutant males. Additonally, transgenic mice ectopically expressing AMH in females resulted in a male pattern of Osx expression. Together these results indicate that AMH signaling is necessary and sufficient for Osx expression in the MD mesenchyme. MD regression was delayed in functional null Osx LacZ/LacZ males. This suggests Osx contributes to MD regression. The BMP signaling effectors, Msx2, Dlx5 and Sp6 were also identified as potential mediators of AMH-signaling during regression. We are currently examining the in vivo role of these candidate factors during MD regression using available mouse models.

Is CD73’s Protection of the Epithelial Barrier Promoting Tumor Immunosuppression in Endometrial Carcinoma?

Jessica L. Bowser1,6, Yuexin Liu2, Michael R. Blackburn3, Gregory L. Shipley4, Jose G. Molina3, Kenneth Dunner Jr.5, Russell R. Broaddus2 Departments of Translational Molecular Pathology1,6 and Pathology2, The University of Texas MD Anderson Cancer Center, Houston, TX, USA, Department of Biochemistry & Molecular Biology3, The University of Texas-Houston McGovern Medical School, Houston, TX, USA, Shipley Consulting LLC4, Austin, TX, USA, Department of Cancer Biology5, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Interdisciplinary Translational Education and Research Training Program Fellow6, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Ecto-5’nucleotidase (CD73) is central to the generation of extracellular adenosine. Extracellular adenosine is a potent immunosuppressor and a major factor in CD73-mediated tumor progression. Unexpectedly, we found CD73 is down-regulated in carcinoma cells of poorly-differentiated and advanced stage endometrial carcinomas (EC). Loss of CD73 genetically in vivo and pharmacologically in vitro shows that 1) CD73-generated adenosine protects epithelial integrity (barrier function) in the normal endometrium; 2) this physiological reflex is intact in early stage EC, and 3) loss of CD73 is associated with tumor progression. CD73 induces epithelial integrity via adenosine A1 receptor-mediated actin polymerization. Contrary to other tumors, such as breast carcinomas, EC grow as inter-connected malignant glands with few intervening stromal cells and inflammatory cells. Considering the different tumor microenvironment, we hypothesize that CD73-mediated epithelial integrity may be promoting tumor immunosuppression by acting as a physiological barrier. We evaluated CD73 expression in The Cancer Genome Atlas (TCGA) EC data set [n=270 endometrial endometrioid carcinoma (EEC); n=53 uterine papillary serous carcinoma (UPSC)]. The TCGA data are in agreement with our work; CD73 expression is higher in EEC, especially grade 1 (G1) and grade 2 (G2), compared to UPSC. CD73 expression differs significantly in two TCGA molecular subtypes, hormonal and immunoresponsive. Both subtypes are composed almost entirely of low grade (G1 and G2) EEC. The immunoresponsive TCGA subtype of EC is characterized by higher expression of immune response-related genes; the hormonal subtype is characterized by higher expression of hormone receptors and genes induced by estrogen. CD73 expression was significantly lower in the immunoresponsive compared to the hormonal subset. Genes that characterize the immunoresponsive subset include CD74, CD14, TNFRSF1B, and CEBPD, which have been shown to activate inflammatory responses. We previously showed that G1 and G2 EEC expressing lower hormone receptors express higher levels of IL-10 and TGFβ2. CD73-mediated protection of epithelial integrity in EC is comparable to CD73 in maintaining the barrier in the gut. In the gut, breakdown of the intestinal epithelial cell barrier contributes to the development of inflammatory bowel disease. Important is that loss of the epithelial barrier results in the production of pro-inflammatory mediators by immune cells and epithelial cells. Together, our results provide a different perspective regarding CD73 in cancer. We provide the first basis of why CD73 loss promotes tumor progression and provide consideration of an indirect action of CD73-mediated tumor immunosuppression. (NIH 1P50CA098258)

C-Terminal Binding Protein 1 (CtBP1) and GATAD2B Serve As Novel Mediators of Progesterone/PR Suppression of Proinflammatory and Contractile Genes in the Pregnant Myometrium Imran Hussain, Chien-Cheng Chen, Alina P. Montalbano, Phuong Truong and Carole R. Mendelson

Progesterone (P4), acting through its receptor (PR), plays a central role in the maintenance of pregnancy by suppression of proinflammatory (e.g. COX-2, IL-8) and contractile (e.g. CX43, OXTR) genes in the myometrium. P4/PR exerts these effects, in part, by tethering to nuclear factor-κB (NF-κB) bound to the promoters of these genes, resulting in a decline in NF-κB transcriptional activity. Recently, we found that the PR DNA-binding domain (DBD) plays a crucial role in P4-mediated suppression of endogenous proinflammatory genes; however, this inhibitory effect of P4/PR was not mediated by direct DNA binding. We further observed that P4/PR transrepression activity occurred at the level of transcription initiation and was mediated by decreased recruitment of NF-κB p65 and RNA Pol II to the COX-2 and IL-8 promoter regions. Thus, we postulated that nuclear proteins interacting with the PR DBD may play an important role in P4/PR mediated transrepressive activity. Using immortalized human myometrial cells (hTERT-HM) stably expressing wild-type PR-B (PR-BWT) or PR-B containing a mutation in the DBD (PRmDBD), we identified two transcriptional repressors, CtBP1 and GATAD2B, that interacted strongly with PR-BWT, but poorly with PR-BmDBD. P4 treatment of PRWT hTERT-HM cells, caused enhanced recruitment of endogenous GATAD2B and CtBP1 to NF-κB-binding regions of the COX-2 and IL-8 promoters. Further, siRNA knockdown of endogenous GATAD2B or CtBP1 significantly reduced P4/PR-BWT transrepression of COX-2 and IL-8. Using RT-qPCR, we observed that GATAD2B and CtBP1 mRNA levels were significantly decreased in myometrial biopsies of pregnant women in-labor, as compared to those from women not-in-labor, at term. To gain further insight into expression and function of Gatad2b and Ctbp1 in the regulation of contractile gene expression during pregnancy and parturition, we analyzed myometrial tissues from timed pregnant mice. We observed that increased expression of Oxtr and Cx43 in mouse myometrium near term was associated with a marked decline in Gatad2b and Ctbp1 mRNA and protein. Using ChIP-qPCR, we found that increased expression of Oxtr and Cx43 in myometrium near term occurred in concert with a marked decline in binding of endogenous Gatad2b and Ctbp1 to the NF-κB-binding regions of the Oxtr and Cx43 promoters. This decline in corepressor recruitment was associated with a pronounced decrease in the repressive histone mark, H3K27me3. Together, these findings suggest that Gatad2b and Ctbp1 serve as novel mediators of P4/PR suppression of myometrial proinflammatory and contractile gene expression. The decreased expression and DNA-binding of these corepressors near term contribute to the decline in PR function and result in the induction of myometrial proinflammatory and contractile genes leading to labor.

Supported by: NIH 5-P01-HD011149 and March of Dimes Foundation Prematurity Initiative Grant #21-FY14-146

Novel Observations on the Relationship Between Fetal Synthesis and Placental Clearance of Cytokines During Parturition and Chorioamnionitis (Chorio) at Term Gestation.

I. Mir, L. Chalak, C.R. Rosenfeld. Neonatal-Perinatal Medicine, UT Southwestern Medical School, Dallas, TX USA

Background: Evidence suggests pro-inflammatory cytokines contribute to onset and progression of term parturition and are further elevated in the presence of Chorio. Anti-inflammatory IL-10 is also elevated and may contribute to pregnancy maintenance. However, their source and site of clearance in the fetal compartment are unclear.

Objective: To determine the fetal contribution to cytokine synthesis and if the placenta is a site for cytokine production or clearance during parturition and in the presence of Chorio.

Methods: 3 groups of term pregnant women were studied: Group 1 were uncomplicated elective cesarean delivery without labor (n=20); Group 2, uncomplicated vaginal delivery with labor (n=20); and Group 3, pregnancies complicated by maternal Chorio (fever >38.5°C, abnormal fetal heart rate and stat cesarean delivery; n=6). Umbilical arterial (UmA) and venous (UmV) blood was collected from double-clamped segments of umbilical cord for measurement of arterial blood gases and serum IL-1β, IL-2, IL-6, IL-8,TNFα, and IL-10 by ELISA. Data are means±SEM and were analyzed by rank-sum test, χ2 and ANOVA.

Results: UmA gases were similar in all groups. Group 1 UmA and UmV cytokine levels were below sensitivity of assays except for IL-10, which were similar (P≥0.1; 504±15 vs. 468±16 pg/ml, respectively). Group 2 UmA IL-6 and -8 increased 10-fold (P<0.001; 16.7±1.6 and 18.4±4.3 pg/ml, respectively) and were less in UmV (P<0.001; 0.29±0.2 and 0.74±0.3, respectively); thus, placental clearance was 98±0.6% and 97±1.3%, respectively. Group 2 IL-10 did not differ in UmA and UmV (P>0.05; 677±67 and 539±24 pg/ml). Group 3 UmA IL-6 was 2280±890, IL-8 690±277, and IL-10 1291±225 pg/ml; UmV IL-6 and IL-8 remained less than UmA (P<0.001;745±319 and 157±56pg/ml, respectively), placental clearance falling to 57±12% and 64±11%, respectively, and remaining absent for IL-10 (1227±215 pg/ml). IL-10/IL-6 and IL-10/IL-8 ratios were higher in Group 2 vs. 3, 53±8 and 54±8 vs. 2.2±1.1 and 5.0±2.0, respectively (P<0.001).

Conclusions: Fetal synthesis of inflammatory cytokines increase during labor and circulating levels are modulated by nonsaturable placental clearance. Chorio further increases inflammatory cytokines of fetal origin which are regulated by placental clearance. Anti-inflammatory IL-10 increases 34% during parturition and 156% with Chorio, but there is no placental clearance, providing a protective mechanism for maintaining fetal cytokine balance.

Blunted Ghrelin Signaling in Pancreatic Islets of Pregnant Rats Fed a Low Protein Diet

Haijun Gao, Eric Ho, Chandra Yallampalli. Department of Obstetrics & Gynecology, Baylor College of Medicine; Texas Children’s Hospital, Houston TX 77030

Enhanced insulin secretion by beta cells during pregnancy could compensate for insulin

resistance to ensure glucose supply for fetal growth. Our previous study demonstrated that in fed

status, plasma levels of insulin in pregnant rats fed a low protein (LP) diet remain lower in mid-

late pregnancy compared to controls, and in contrast, plasma levels of ghrelin, a hormone

inhibiting insulin secretion, were highly elevated in LP rats. In this study we hypothesized that

enhanced ghrelin signaling in beta cells inhibits insulin secretion in pregnant rat fed LP diet.

Pregnant SD rats were fed a diet with 20% (CT) or 6% casein (LP) from Day 1 of pregnancy

until killed on Day 21 (n=6-10 rats/diet) and plasma was collected for insulin measurement by

ELISA. Pancreatic islets were isolated and mRNA levels of Proinsulin 2, Ghrl (ghrelin), Mboat4

(membrane bound O-acyltransferase domain containing 4), Bche (butyrylcholinesterase) and

Ghsr (growth hormone secretagogue receptor) were measured by real-time PCR. Islets were also

cultured in presence of 2.8 or 16.7 mM glucose (n=3-4 rats/diet) together with ghrelin (10nM),

ghrelin antagonist (1μM) or their combination (COMB). Secreted insulin was measured by

ELISA. Results: 1) Plasma insulin levels were reduced (P < 0.05) by 2.3-fold in LP compared to

CT group; 2) mRNA levels of Proinsulin 2, Mboat4 and Bche was unchanged in LP rats; 3)

mRNA levels of Ghrl and Ghsr were increased by 1.8-(P < 0.01) and 1.6 (P < 0.05)-fold in LP

rats, respectively; 4) In presence of 2.8 and 16.7 mM glucose, insulin secretion was 1.6 (P <

0.01)- and 1.5 (P < 0.05)-fold higher in CT rats compared to LP rats, respectively. 5) In presence

of 2.8 mM glucose, insulin secretion was not changed by ghrelin and its antagonist in both CT

and LP rats, although insulin secretion was higher in CT rats for each treatment except COMB.

6) In presence of 16.7 mM glucose, in LP rats insulin secretion was unchanged by ghrelin and its

antagonist; in CT rats insulin secretion with the treatment of ghrelin was reduced to 69.8 % of

untreated group, but insulin secretion with the treatment of ghrelin antagonist and COMB was

increased (P < 0.001) by 2.8- and 2.6 -fold, respectively. These results indicate that despite

increase in both systemic and local production of ghrelin in pregnant rats fed LP diet, ghrelin

signaling in pancreatic islets was blunted and failed to inhibit insulin secretion in late pregnancy.

Testosterone Impairs Placental Vascularization and Oxygenation, Reduces Uterine Blood Flow, and Triggers Sex-Specific Placental Gene Expression in Pregnant Rats

K. Gopalakrishnan, PhD1, J. Mishra, PhD1, K. Vincent, MD1, G. Hankins, MD1, and Sathish

Kumar, PhD1

1Ob/Gyn, University of Texas Medical Branch, Galveston, TX, United States

Introduction: Placenta vascular formation is important for fetal growth and development. Among many factors, sex steroid hormones including estradiol and progesterone play an important role in placental vascularization. However, little is known about the role of androgens. Plasma testosterone levels are elevated in preeclampsia, PCOS mothers and pregnant African-American women, who often have fetal growth restriction. We previously showed that experimental increase in maternal testosterone levels cause fetal growth restriction, culminating eventually in adult onset hypertension that is more pronounced in males than females. Aim: We tested whether elevated testosterone affects placental vascularization and oxygenation, and if these effects vary in the male and female placentas. Methods: We injected pregnant Sprague-Dawley rats with vehicle or testosterone propionate (TP; 0.5 mg/Kg/day from gestation day 15-19) to increase plasma testosterone level twofold, similar to that observed in clinical conditions like preeclampsia. On gestation day 20, we quantified uterine artery blood flow using micro-ultrasound, visualized utero- and feto-placental arterial network using x-ray microcomputed tomography, determined fetoplacental hypoxia using pimonidazole and hypoxia-inducible factor 1α, and used Affymetrix array to determine changes in placental expression of genes involved in vascular development. Results: Plasma testosterone levels increased 2-fold in testosterone-injected rats. Placental and fetal weights were lower in rats with elevated testosterone. Uterine artery blood flow was lower and resistance index was higher in testosterone group. Radial and spiral artery diameter and length, number of fetoplacental arterial branches, and umbilical artery diameter were reduced in testosterone group. In addition, markers of hypoxia in the placenta and fetuses were elevated in testosterone group. The magnitude of disruption in placental vasculature and hypoxia were greater in male than female placentas. Intriguingly, different sets of angiogenic and blood vessel morphogenic genes were dysregulated in male and female placentas. Conclusion: Elevated maternal testosterone induces decrease in uterine arterial blood flow and fetal sex related uteroplacental vascular changes, which may set a stage for subsequent sex-differences in adult onset diseases.

Impact of Gestational Diabetes On Long-Term Maternal Health Using A Mouse Model Kathleen A. Pennington1, Nicola van der Walt1, and Laura C Schulz2

1Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston TX 77030 2Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia MO 62121

Pregnancy is a known stressor to maternal metabolism and physiology, the addition of gestational diabetes mellitus (GDM) further stresses the maternal environment. Women who have had a pregnancy complicated by GDM are more likely to develop type II diabetes and cardiovascular disease later in life. The mechanisms regulating the programming effect of GDM on maternal long term health and disease are poorly understood. Currently, no animal model has been developed to study the long-term effects of GDM on maternal health without preexisting complications of obesity. Previously we have shown that feeding mice a high fat diet (HFD, 45% kcal/fat, 17% kcal/sucrose) one week prior to mating and throughout pregnancy can induce glucose intolerance during pregnancy and that glucose intolerance resolves by weaning. The goal of the present study was to determine if dams exposed to glucose intolerance during pregnancy alone would develop long-term maternal health complications. Female C57BL/6J mice were placed on either a control diet (P-CD) or HFD (P-HFD) one week prior to mating and throughout pregnancy. On day of delivery dams were returned to normal chow until 7 weeks post-partum. To test the effects of diet exposure alone vs combine diet and pregnancy effects, a second set of female mice were exposed to the same diet regimes, either CD (NP-CD) or HFD (NP-HFD) for 4wks without pregnancy, followed by chow diet for 7wks. Following 7wks of chow diet, all groups (P-CD, P-HFD, NP-CD, NP-HFD) were given HFD to test whether previous GDM pregnancy would exacerbate or hasten the progression of glucose intolerance. Glucose tolerance tests were performed following 6wks of HFD. Following GTT, animals were sacrificed. Serum and tissue samples were collected for further analysis. GTT was not different between nulliparous females regardless of previous diet exposure. In contrast, females that had previously experienced GDM (P-HFD) had reduced glucose tolerance compared to females that had experienced a normal pregnancy (P-CD), as indicated by elevated blood glucose at 30 minutes post glucose injection (198.6±22.9 P-CD vs 293.1±43.1 P-HFD, p<0.05) . Weights did not differ with previous diet exposure, or pregnancy. Fasting serum insulin did not differ by previous diet exposure in the nulliparous or parous groups. Among the former CD groups, parous females had significantly decreased (p<0.05) serum leptin levels compared to nulliparous females (3.56±0.69 P-CD vs 8.28±1.02 NP-CD). This decrease was attenuated in 4HFD animals, in which pregnancy had been complicated by symptoms of GDM. Serum triglyceride levels were significantly decreased (p<0.05) in P-CD compared to NP-CD (0.79±0.12 vs 1.61±0.18). No differences in serum triglycerides between NP-HFD and P-HFD. Serum triglycerides were significantly elevated (p<0.05) in P-HFD compared to P-CD (1.31±0.05 vs 0.79±0.12). Overall, we present here an animal model of GDM that exhibits long term maternal health complications. Future studies will utilize this model to elucidate mechanisms that may regulate these adverse maternal health outcomes.

Pre and Perinatal Fluoxetine (FLX) Exposure of Shank3ex4-9 Mutant and WT Mice Affects Neurobehavior in Offspring and Indicates Gene-Environment (GXE) Interaction. Author Block: J. B. MURRY1, D. ENGLER 2, R. RAMAKRISHNAN 1, I. B. VAN DEN VEYVER 1; 1Baylor Coll. of Med., Houston, TX, 2Houston Methodist Res. Inst., Houston, TX. Abstract: Objective: We tested our hypothesis that perinatal exposures (FLX) in conjunction with ASD-associated risk alleles (Shank3) modulate neurodevelopment, increasing penetrance of offspring behavior disturbances. Study Design: Shank3ex4-9/+ (HET) dams were mated with HET males and given FLX (15mg/kg) in drinking water (+FLX n=36) or drinking water only (-FLX n=37) from day 0 of pregnancy (dpc0) until postnatal day 7 (pnd7). Adult male and female knockout (KO n=21, HET n=40, and wild-type WT n=38) +FLX and (KO n=42, HET n=73, WT n=33) -FLX offspring were tested for anxiety, repetitive, and social behaviors. Two-way ANOVA was used to test gene-environment (GXE) interactions, with Bonferroni posthoc test. Student’s t-test was used to test social preferences. Brains were harvested for histological and molecular characterization after testing. Results: Litter sizes and pregnancy outcomes were similar in HET(+FLX) and HET(-FLX) dams but KO female offspring of (+FLX) dams had significantly lower weights than those of (-FLX) dams at P49 and P56 (p<0.05). In all, four behavioral assays had significant differences in social and anxiety behaviors as summarized below:

�In the elevated plus maze, KO and HET(-FLX) males had more closed arm entries and traveled greater distance in the closed arm than WT(-FLX) males (p<0.05), with a significant GXE interaction (p<0.05) but FLX exposure resulted in less distance traveled in the closed arm (p<0.005). In the open field assay, vertical locomotion was lower in KO(+FLX) males vs. KO(-FLX) males (p<0.05), with a significant GXE interaction (p<0.05). There was no social preference in the 3-chamber test for both KO(+FLX) males (p=0.8) and females (p=0.4). In contrast, WT(+FLX) and HET(+FLX) males had significantly increased active social contact vs. WT(-FLX) (p<0.001) and HET(-FLX) males (p=0.05) in the direct social interaction test. No significant interactions were observed in tests for repetitive behaviors. No gross histological differences were observed. Conclusion: These data suggest chronic FLX exposure alters social behavior and reduces anxiety-like behaviors differently in Shank3-mutant and WT mice. Ongoing experiments will examine the molecular and structural correlates of developmental FLX exposure in these mice.

Epigenetic Changes Associated with In Utero Nicotine Exposure in the Rhesus Macaque

Stephen M. Saylor, M.S., Melissa Suter, Ph.D., Lyndsey Shorey-Kendrick Ph.D, Elliot Spindel Ph.D, Kjersti Aagaard, M.D., Ph.D.

Objective The use of electronic cigarettes and other smokeless tobacco products is rapidly increasing among women of reproductive age, making the effect of nicotine on pregnancy an important public health concern. Nicotine is associated with adverse effects on the fetus including reduced placental blood perfusion and decreased fetal pulmonary function. Utilizing a rat model of in utero nicotine exposure, we have revealed epigenetic alterations in the lung and brain of offspring at postnatal day 1. Previous studies in non-human primates show effects on fetal pulmonary function which correspond to the production of oxidants during nicotine metabolism. These effects are mitigated by treatment with the anti-oxidant Vitamin C (VC). Studies have also shown that nicotine is a potent histone deacetylase inhibitor. Due to nicotine’s impact on histone post-translation modifications (PTMs) and distinct phenotypes in both the fetal lung and placenta with in utero nicotine exposure, we hypothesized that differential histone PTMs would be detected in these tissues in response to nicotine that may be reversed upon VC treatment. Study Design In this study PTMs were investigated in histones isolated from fetal lung and placenta from near term Rhesus macaques. Mothers received VC or placebo, and nicotine or saline to yield six different treatment groups (n = 7-9); control (placebo + saline), saline + 50mg/kg VC or 250mg/kg VC, nicotine + placebo, and nicotine + 50mg/kg or 250 mg/kg VC daily. Western blots were performed for the following histone PTMs: H3K4me3, H3K9me3, H3K27me3, H3K9ac, H3K14ac, H3PanAc and H4PanAc. Total histone H3 was used for normalization. Results Analysis of histone PTMs demonstrate a significant difference between control and nicotine + 250mg/kg VC (NVC) groups by pairwise t-test in the placenta: H3K9me3, H3K9Ac, H3K27me3 (p = 0.016, 0.027, 0.027 respectively) and in the lung; H3K4me3 (p = 0.039). Conclusions Contrary to expectations neither nicotine nor VC treatments had a significant impact on global histone PTMs compared to controls. However, nicotine and VC demonstrated synergistic effect that caused a significant impact histone PTMs in both the placenta and the lung. Further study is warranted to investigate the effects of these modifications on lung and placental transcriptional networks in nicotine using patients treated with VC.

Selective Inhibition of Pge2 Receptors Ep2 and Ep4 Decreases Pelvic Pain in Endometriosis

Balasubbramanian, D 1, Lee, J.H. 1, Stanley, J.A. 1, Meagher, M.W. 2, Banu, S.K. 1, and Arosh, J.A. 1 1 Reproductive Endocrinology and Cell Signaling Laboratory, Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, USA 2 Department of Psychology, College of Liberal Arts, Texas A&M University, College Station, Texas, USA

dbalasubbramanian@cvm.tamu.edu; jarosh@cvm.tamu.edu

Endometriosis is an inflammatory gynecological disease of reproductive-age women. Two major clinical symptoms of endometriosis are chronic pelvic pain and infertility. Modern medical treatments aim at inhibiting the action of estrogen in endometriotic cells. However, these therapies are prescribed only for a short time because of their undesirable side effects. There is a fundamental need to identify nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis and this plays an important role in the growth of endometriosis lesions and in the clinical symptoms associated with the disease. The current study determines the pharmacological effects of the selective inhibition of EP2/EP4 on molecular pain pathways in endometriosis using human fluorescent endometriotic cell lines and chimeric mouse model of endometriosis. Human fluorescent endometriotic epithelial cell line 12Z and stromal cell line 22B were xenografted and peritoneal endometriosis was induced in Rag2g(c) mice. The experimental mice were treated with EP2 and EP4 inhibitors. The presence of endometriotic lesions was examined by gross visual examination and under fluorescence microscope. All lesions were dissected under the fluorescence dissection microscope. Mechanical hyperalgesia was assessed by stimulating the pelvic floor with von-Frey filaments. DRGs from L1 to S1 were isolated under dissection microscope. Immunocytochemistry was performed on endometriotic lesions and DRG sections, and digital images were captured. The results of the present study indicate that selective inhibition of EP2/EP4 decreases endometriosis-induced innervation, decreases the proinflammatory microenvironment of DRG neurons and suppresses pelvic pain associated with endometriosis.

Fetal Perfusion Pressure Responses to Labetalol Infused into Maternal and Fetal Compartments of Dually Perfused Placenta.

Andrey Bednov1, Jimmy Espinoza1, Ancizar Betancourt1, Michael Belfort1, Chandra Yallampalli1 Objectives: The use of antihypertensive medication is indicated for the treatment of severe hypertension during pregnancy to prevent adverse outcomes, including stroke and myocardial infarction. However, the use of antihypertensive drugs is not recommended in mild to moderate hypertension during pregnancy because of their association with fetal growth restriction. This study was designed to determine the effect of labetalol on the perfusion pressure in the chorionic plate vessels of dually perfused human placenta.

Methods: This study included placental tissues from women undergoing C-section at term with uncomplicated pregnancies (n=15). The dual-perfusion system was used and the maternal and fetal compartments were continuously perfused with Krebs solution equilibrated with 95% O2/5% CO2 and 95% N2/5% CO2 gas mixtures, respectively. Perfusion pressure in the fetal compartment was continuously monitored. After the stabilization of perfusion, labetalol was infused at varying doses (150 - 1,000 ng/ml) either to the fetal or maternal compartments. Changes in fetal perfusion pressure were assessed. Results: Labetalol infusion into the fetal compartment produced a dose-related decrease in the fetal perfusion pressure in intact vessels: with no effects at 0.3 mM, a 6% decrease at 2 mM, and 9% decrease at 3 mM. The 3-mM labetalol infused into the maternal compartment did not change a basal perfusion pressure in intact fetal arteries. In the vessels pre-contracted with the 16-nM U-46619 (a thromboxane analogue), the 1-mM, 2-mM, and 3-mM labetalol infused into the fetal circulation produced the 8%, 33%, and 60% drop in fetal perfusion pressure, respectively. 3-mM labetalol infused into the maternal system caused a 15% decrease in pressure in the fetal arteries pre-contracted with the 8-nM U-46619.

Conclusion: Administration of labetalol into fetal circulation caused a dose-dependent decrease in perfusion pressure both in basal and pre-contracted fetal arteries. Labetalol administration into the maternal system did not cause changes in the basal perfusion pressure, however, caused a decrease of pressure in pre-contracted vessels. It is possible that reduced fetal-placental perfusion pressure may partially account for the association between labetalol use during pregnancy and increased risk for fetal growth restriction.

Omental Artery Relaxations to CGRP Family Peptides are Greater in Women with Twins Compared to Singleton Pregnancy.

Ancizar Betancourt, Madhu Chauhan, Yuanlin Dong, Karin Fox, Jimmy Espinosa, Michael Belfort, Chandra Yallampalli

Calcitonin gene related peptide (CGRP), adrenomedullin (AM) and intermedin (IMD), acting through their receptor components, calcitonin receptor–like receptor (CRLR) and receptor activity modifying protein 1, 2, or 3 (RAMP), play important roles in vascular adaptions during pregnancy. All 3peptides relax of omental arteries (OA) in a dose-dependent manner, and responses are greater in pregnant compared to non-pregnant women. In this study we assessed if there are differences in relaxation responses to these peptides on OA from twin and singleton pregnant women.

Methods: Segments (2mm) of OA from 15 singleton pregnant and 3 twin pregnant women (normotensive) were mounted onto a wire myograph system. Cumulative dose-response curves were constructed for CGRP, AM and IMD (0.1nM-100nM), and bradykinin (BK, 0.1nM-1µM). The relaxation responses were calculated as a percentage of the U46619 induced contraction. Student t-test was used for statistical analysis.

Results: 1) CGRP, AM, IMD and bradykinin (BK) produced dose-dependent relaxation of human OA obtained from singleton and twin pregnant women; 2) vasodilatory responses to AM, IMD and BK were enhanced in twins compared with singleton (EMax AM: 52.5 ± 7.3 vs. 34.8 ± 2.4, P=0.007; IMD: 75.4+/- 4.4 vs. 48 +/- 2.6, P<0.0001; BK: 86.8 ± 4.8 vs. 52.2 ±2.1, P<0.0001; 3) CGRP induced vasodilatory responses are similar among twins and singleton pregnancies. (57.8 +/- 5.2 vs. 59.4 +/- 2.0)

Conclusion: Vasodilatory effects of AM and IMD but not CGRP are elevated in twin pregnancies and may involve differential expression of 3 RAMPs in OA, as co-expression of RAMP 1, 2 or 3 with CRLR dictates the specificity and sensitivity of CRLR to these peptides. Lack of enhanced effect of CGRP in twin pregnancies may relate to altered RAMP1 expression and/ or its post receptor signaling. Increased relaxation responses to BK, together with AM and IMD suggest these responses are endothelium related, and suggests a role for these peptides in supporting greater vascular compliance in twin pregnancies to accommodate increased blood volume.

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52.54 ± 7.37b 3

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Growth and Development of Rat Pups with Gestational Exposure to Sulfur Based Particulate Matter Jacob D. Brown1, Alyssa L. Miller1, Misti Levy2, Robert C. Burghardt3, Greg A. Johnson3, Fuller W. Bazer1, Guoyao Wu1, Cynthia J. Meininger4, Renyi Zhang2 and M. Carey Satterfield1, Departments of Animal Science1, Atmospheric Science2, Veterinary Integrative Biosciences3, and Medical Physiology4, Texas A&M University and Texas A&M Health Science Center, College Station, TX, USA Inhalation exposure to air pollutants has been associated with a higher incidence of preterm birth and reductions in fetal growth rates and head circumference. Most of these effects have been identified through epidemiological studies whereas few animal model studies investigated the effects of controlled air pollutant exposure on fetal development and future disease pathogenesis. Therefore, we conducted the following experiment. Pregnant Sprague Dawley rats were placed in individual cages within atmospherically controlled chambers from Day 0 to 18 of pregnancy. During this period, dams were exposed to either ammonium sulfate particulate matter (PM) at a concentration of 150-175 µg/m3 or to ambient clean air (n = 10/group). At Day 18 of pregnancy, gestating dams were removed and housed individually until parturition and weaning of pups. At birth (postnatal day 0, PND0), all pups were weighed and litters culled at random, to eight pups (four males and four females) per litter. The culled pups were immediately euthanized and organs were collected and processed for subsequent analyses. The remaining pups were reared on their dam until PND21 (weaning). At weaning, two males and two females from each litter, as well as the dams were weighed, euthanized, and processed as above. The remaining four (2 male and 2 female) pups were then fed either a high fat diet (HFD) or a low fat diet (LFD) for 12 weeks from PND21 until PND105. One male and one female from a litter were assigned to each of the two diets. On PND105, rats were weighed, euthanized and processed. Results indicated that PM exposure reduced gestation length (P<0.05), but did not alter litter size. Birth weights of offspring from exposed dams were lower than for control pups (P<0.001). Also on PND0, pups from exposed dams had lower weights of heart (P<0.001), spleen (P<0.01), brain (P<0.001), and total intestine (P<0.001), compared to control pups. At PND21, there was a reduction in weights of pups from exposed dams compared to control pups (P<0.05). Additionally, there was an increase in weight of the thymus in pups from exposed dams at PND21 (P<0.05). At PND105, offspring from exposed dams had increased brain (P<0.05) and kidney (P<0.01) weights, compared to control offspring, irrespective of dietary treatment. Interestingly, there was a sex by treatment interaction at PND105 in which the male offspring from exposed dams had increased thymus weights, compared to control males (P<0.05). At PND 105, offspring from exposed dams had higher concentrations of glucose in plasma, compared to controls (P<0.05). Pups from exposed dams exhibited reduced aortic ring relaxation in response to acetylcholine (P<0.05), an indication of cardiovascular dysfunction. Collectively, results indicate that maternal exposure to ammonium sulfate-based PM during gestation alters the developmental trajectory of offspring with perturbations remaining evident on PND105. Further work is required to elucidate the mechanisms regulating the growth and development of offspring exposed to air pollution during pregnancy. Keywords: pregnancy, pollution, fetal development, postnatal development

The Circadian Gene Npas2 Alters the Stereoidogenesis Pathway by Affecting the Expression of Star Cenk Cengiz, B.Sc.1,2,*; Amin Herati, M.D.1,2,*; Dolores J. Lamb, Ph.D.1,2,3 1Center for Reproductive Medicine, 2Scott Department of Urology, 3Department of Molecular and Cellular Biology, Baylor College of Medicine. Houston. Tx. *These authors contributed equally to this work. Background and Objective: Male hypogonadism is defined as the inability of the testis to produce testosterone and/or spermatozoa due to a dysfunction of the hypothalamic-pituitary-gonadal axis. Within the Leydig cells of the testis, cholesterol is converted in a series of reactions to form testosterone as part of the steroid biosynthetic pathway. This pathway is regulated by several mechanisms, many of which are extra-testicular. It is well established that circadian genes can modulate reproductive function; however, the exact mechanism by which this occurs is still unknown. Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm gene that exhibits both central and peripheral expression. We hypothesize that NPAS2 regulates testosterone production via modulation of the steroid acute regulatory protein (StAR) protein. The objective of our study, was to localize the expression of NPAS2 in the male reproductive tract and determine its effects on steroidogenesis. Material and Methods: Testis-specific expression NPAS2 was defined using Bouin’s fixed sections of testis obtained from 10-week old mice. Immunohistochemistry (IHC) was performed using commercial NPAS2 antibody (Santa Cruz Biotechnology, Tx, USA. Additionally, co-immunofluorescence (co-IF) staining with 3-Beta-Hydroxysteroid Dehydrogenase (3B-HSD) was performed on similar testis sections. To characterize the impact of NPAS2 on steroidogenesis, transfection studies were performed using NPAS2 siRNA (GE Dharmacon,USA) using lipofectamine (Thermo, USA) to transfect the immortalized mouse Leydig (TM3) cell line (ATCC, Virginia, USA). The expression of StAR was quantified using a Taq-Man (Thermo, USA) based qPCR assay. The efficiency of knockdown was analyzed using Western blot as well as qPCR. Results: Testis NPAS2 expression was visualized in the seminiferous tubules as well as in the interstitial cells of the testis. Co-IF studies confirmed staining of the Leydig cells in the interstitial compartment. Using qPCR, a (60%) reduction of StAR mRNA levels was observed in the siRNA-treated TM3 cells compared with scramble-treated control cells. Conclusions: NPAS2 is expressed within the mouse testis seminiferous tubules and Leydig cells. Silencing of NPAS2 reduced expression of StAR in the immortalized mouse Leydig cell line TM3. Confirmatory studies are ongoing to determine the effect on steroid biosynthesis by the TM3 transfected cells. Source of Funding: A.H. is a National Institutes of Health (NIH) K12 Scholar supported by a Male Reproductive Health Research Career (MHRH) Development Physician- Scientist Award (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program (to Dolores J. Lamb).

Blastocyst Formation in Single Cell Mouse Embryos Grown in Single versus Group Culture Utilizing Time Lapse Microscopy. Chappell N. Introduction: There exist several studies with data to support a benefit in growing embryos in communal versus single culture. Time lapse imaging (TLI) is a newer technology which allows for the direct study of an embryo in culture without disturbing the culture environment. Utilizing TLI, data have been collected that demonstrate morphokinetic parameters, specifically related to growth rate, can be positive prognostic markers for embryos and could aid in improved embryo selection, translating to higher pregnancy rates. The Embryoscope (Unisense Fertilitech, Aarhus, Denmark) offers use of TLI technology for embryos in vitro. Embryoslide wells compatible for culture with this device are only available for use with single cell culture. It is plausible, however, that if embryos could be cultured communally in these wells, there may be an additional benefit. We hypothesize that communal culture of single cell mouse embryos will have improved morphokinetic parameters when compared with single cell culture technique. Methods: To investigate, we performed a prospective, randomized study comparing single and communal culture of single cell mouse embryos on an Embryoslide. Each Embryoslide has 12 individual wells for culture arranged in a 4x3 fashion. Utilizing standardized protocols for quality control, single cell mouse embryos were thawed and cultured in groups of 1, 2, or 3 embryos per culture well in a randomized fashion (Table 1). They were then placed in the Embryoscope and imaged every 10 minutes in 5 focal planes for 120 continuous hours. The primary outcome of the study was time to blastocyst formation, with secondary outcomes being time to 2 cell, 4 cell, 8 cell, hatch, diameter at hatch, % 8 cell, and % survival (defined as % hatch / % 8 cell.) Results: We found no difference in the primary or secondary outcomes of the single cell mouse embryos grown in communal versus single culture. Time to blast was 62.57 hours for single culture and 64.1 hours for communal culture (p>0.05) (Figure 1). Total blastulation rate was 36.9%.

Conclusion: Growth of embryos in communal culture on the Embryoslide is not superior to standard single culture protocols.

Table 1. Example of Number and Distribution of Embryos on an Embryoslide (# embryos per well)

1 3 1 2

3 1 3 1

1 3 1 2

Figure 1. Overall Rate of Growth. The Blue line (Series 1) represents the embryos in Single culture while the Orange line (Series 2) represents the Group culture.

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Modulation of Rat Trophoblast Stem Cell Phenotype by Adrenomedullin2.

Madhu Chauhan; Meena Balakrishnan; Chandra Yallampalli

Introduction: Adrenomedullin2 (ADM2), secreted by human blastocysts and expressed in placenta, promotes trophoblast invasion in the 1st trimester of human pregnancy. Lower levels of ADM2 are associated with spontaneous abortion and inhibition of the endogenous ADM2 function in early rat pregnancy results in reduced implantation rate, restricted feto-placental growth and impaired placental vasculature. However, it is not known if ADM2 has a role in modulating the phenotypical changes in trophoblast stem cells. Current study was designed to assess the effect of ADM2 on the differentiation of trophoblast stem cells (Rcho-1) into giant trophoblasts.

Methods: RCHO-1 cells were seeded in 6-well plates in either stem cell medium (RPMI-1640 with 20% Fetal bovine serum, 50 μM 2-β-ME) for a 24 hrs treatment with ADM2 (10-8M-10-7M) at 370C, or in stem cell medium for 24 hrs followed by replacing the medium with differentiation medium (NCTC-135 medium with 1% horse serum, 50 μM 2-β-ME ) and culturing further at 370C for 3 days in presence or absence of ADM2 (10-8M-10-7M). Rcho-1 spheroids were prepared by hanging drop method using 700 cells per drop. Spheroids were transferred onto a monolayer of smooth muscle cells and cultured for 72 hrs in differentiation medium in presence or absence of ADM2 (10-7M). Morphological changes were assessed with Olympus U-TV1 microscope. Expression of mRNA levels was assessed by real time PCR.

Results: 1) ADM2 increases the formation of giant trophoblast cells in the stem cell population of Rcho-1 cells, 2) ADM2 increases the expression of both, stem cell marker Essrb1 and Id1, and differentiation markers prl3b1 and plf (P<0.05) in Rcho-1 cells, and these effects are inhibited by its antagonist, 3) ADM2 increases the expression of steroidogenic enzymes, STAR, sf1, 3bHSD1, 3bHSD2 and 17bHSD1 in differentiated Rcho-1 cells (P<0.05), and 4) ADM2 increases the differentiation of cells at the periphery of the Rcho-1 spheroids compared to the controls.

Conclusion: ADM2 induced increases in the expression of stem cell markers, differentiation markers and steroidogenic enzymes in Rcho-1 cells suggest an important role for ADM2 in regulating trophoblast stem cells phenotype to support placental formation.

Dysregulation of Hepatic Glucose Production in Gestational Low Protein Programmed Insulin Resistant Rat Offspring

Chellakkan S. Blesson1, Amy Schutt1, Daren T Tanchico1, Shaji Chacko2 Juan Marini2,3, Meena Balakrishnan1 and Chandra Yallampalli1 1Department of Obstetrics and Gynecology, 2Children’s Nutritional Research Center, 3Critical Care Medicine, Baylor College of Medicine, Houston, Texas.

Introduction: We have characterized a gestational low protein rat model that leads to insulin resistance and glucose intolerance during their adult life without obesity. We aimed to identify if the regulation of hepatic glucose production via gluconeogenesis (GNG) and glycogenolysis (GYG) is affected and if there are any sex differences.

Methods: Pregnant rats were fed control (20% protein) or isocaloric low protein (LP) (6%) diet from gestational day 4 until delivery. Standard diet was given during lactation and to pups after weaning. Fasting rats received a primed constant rate infusion of [6,6-2H2] glucose. Blood samples were drawn during steady states after 4h of fasting and following a euglycemic hyperinsulinemic clamp. Endogenous glucose production and a fraction of glucose derived from GNG pathway under fasting and euglycemic states were measured from steady state glucose enrichments after the infusion of [6,6-2H2] glucose and 2H2O tracers utilizing GCMS methodology. GYG was determined by calculating the difference between total and GNG. Hepatic gene expression of molecules involved in GNG and GYG were quantified using qPCR.

Results: Liver regulates glucose homeostasis by increasing glucose production during fasting and suppressing during the fed state. Glucose production was normal during fasting (Males: 7.3 mg/kg*min in controls & 7.5mg/kg*min in LP offspring, Females: 12.4mg/kg*min in controls & 10.3 mg/kg*min in LP). However under the simulated fed condition, glucose production was suppressed in controls (Males: 5mg/kg*min & Females: 6mg/kg*min) but not in LP rats (Males: 7.3mg/kg*min & Females: 10.2mg/kg*min). Thus LP rats showed inefficient suppression of glucose production (Males: 31% in controls vs. 2% in LP; Females: 47% in controls vs. 0% in LP) in a simulated fed state. Differential analysis shows that suppression of both GNG and GYG under fed state is affected in these LP programmed rats. These effects were greater in females when compared to males. Further, key genes involved in these processes such as G6Pase, Pepck, pyruvate carboxylase and glycogen phosphorylase in liver are dysregulated.

Conclusion: Our data shows impaired suppression of hepatic glucose production via GNG and GYG in gestational LP programmed rats with greater effects on females.

Keywords: Developmental Programming, Insulin Resistance, Glucose production

The Role of Wdr5 in Bovine Preimplantation Development

Cromwell, W1, Golding, M.1, Pryor, J.1, Snyder, M.1, Pinzon, C1, Westhusin, M.1

1. Texas A&M University, College Station, TX, United States of America wcromwell@cvm.tamu.edu; mwesthusin@cvm.tamu.edu

During preimplantation development in mammals, the epigenetic landscape in the embryo is established. Failure of this process can result in birth defects or abortion, and is thought to be the mechanism behind the increased association of these ailments with assisted reproductive technologies (ART) such as in vitro fertilization and cloning. Key to the generation and maintenance of epigenetic patterns is the group of enzymes known as the histone methyltransferases (HMTs). HMTs add methyl groups to the tails of histone H proteins resulting in an activating or repressing effect on the genes associated with that given histone. Lysine 4 of Histone H3 (H3K4) is a critical site which can be methylated, dimethylated or trimethylated by a group of HMTs known as the KMT2 family, resulting in higher expression. This family shares a common core, the WRAD complex, which is composed of WDR5, RBBP5, ASH2L, and DPY-30. Studies in human cells revealed that WRAD associates with a methyltransferase to methylate H3K4. WDR5 is the protein that holds the complex together and connects the SET HMT to H3K4. Understanding WDR5’s role during early development could provide information useful for the improvement of bovine ART. We quantified Wdr5 expression at different stages of preimplantation development in bovine embryos produced in vitro using qRT-PCR. Wdr5 transcripts are plentiful in the oocyte relative to reference genes until after the maternal to zygotic transition when they decrease. Currently we are using RNA interference to knock down Wdr5 expression to further elucidate its role in early embryonic development.

1

Targeting Adrenomedullin to Improve Insulin Sensitivity on Adipocytes in Diabetic Pregnancies Yuanlin Dong, Ancizar Betancourt, Michael Belfort, and Chandra Yallampalli. Department of Obstetrics and Gynecology, Baylor College of Medicine/Texas Children’s Hospital, Houston, TX

Introduction: Defective lipid metabolisms are features of insulin resistance in gestational diabetes mellitus (GDM), which adversely affects insulin sensitivity and increased incidence of perinatal mortality and morbidity. Recent studies have found that the circulating levels of adrenomedullin (AM), a multifunctional peptide, were elevated in patients with T2DM and GDM. Thus, present study was designed to determine the expressions of AM and its receptors components in the adipocytes in pregnancies with or without GDM, and assess the influence of AM on insulin signaling molecules and glucose transport in the adipocytes. Methods: Omental fat biopsies were collected from women during caesarian sections in term pregnancy with or without GDM (n=6 in each group). The existence of AM and its receptor components CRLR, RAMP2 and RAMP3 were determined by quantitative q-PCR and immunofluorescent staining. The effects of AM with or without its antagonists AM 22-25 on insulin signaling molecules and glucose transport were assessed by Western blotting. Results: Adipose tissues from normal pregnancies highly express the mRNA for AM and its receptor components CRLR, RAMP2 and RAMP3. Pregnancies with GDM display a significant increase in AM and their receptor components when compared to uncomplicated pregnancies (p<0.01). Immunofluorescent study confirmed significantly enhanced protein expressions for AM receptor components in adipocytes in GDM (p<0.01). Treatment with AM in adipose tissue explants (100 nM for 24 h) resulted in a significant reduction of insulin receptor substrate (IRS) -1 and GLUT4 proteins (p<0.05). In contrast, AM antagonists AM22-52 (1µM) counteracts AM’s actions and stimulates IRS-1 phosphorylation. Conclusions: Pregnancy complicated by GDM is associated with excessive AM and its receptor expressions by adipose tissues. AM inhibit insulin receptor substrate and GLUT4 expressions, thus may play a role in the pathogenesis of diabetic pregnancies via defective IRS-1-mediated insulin resistance. AM antagonist enhances the phosphorylation of IRS-1 in adipocytes, and thus may improve insulin sensitivity in hyperglycemic conditions of GDM.

Sustained Activation of Transforming Growth Factor Beta Signaling Using Growth

Differentiation Factor 9-Driven iCre Disrupts Postnatal Folliculogenesis and Promotes

Malignant Transformation of Ovarian Somatic Cells

Yang Gao and Qinglei Li

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and

Biomedical Sciences, Texas A&M University, College Station, TX, 77843

During folliculogenesis, the oocyte plays a leading role, and, via secretion of paracrine factors,

regulates granulosa cell development and function including, but not limited to, apoptosis,

differentiation, steroidogenesis, and metabolism. Transforming growth factor beta (TGFB)

signaling regulates multiple cellular functions and key reproductive events via transmembrane

receptors (TGFBR1/TGFBR2) and intracellular mediators of SMAD proteins. Until now, the

function of TGFβ signaling in mouse oocytes remains elusive. Available genetic evidence

suggests that the canonical TGFβ signaling may not be required for oocyte development.

However, gain-of-function studies are not available and the functional consequence imposed by

enhanced TGFβ signaling in the oocyte is unknown. To explore this question, we generated a

mouse model containing a constitutively active TGFBR1 (TGFBR1CA) in the oocyte using Cre

recombinase driven by growth differentiation factor 9 (GDF9)-iCre that is expressed in the

oocytes from the primordial follicle stage. Upon Cre-mediated recombination, the “stop”

sequence preceding the TGFBR1CA gene was removed, leading to sustained activation of

TGFBR1 (hereafter termed TGFBR1-CA/G9). During a 3-month fertility test period, the

TGFBR1-CA/G9 females were sterile in contrast to wild-type controls (8.25 ± 0.26 pups/litter

and 1.22 ± 0.07 litter/month). We showed increased phosphorylated SMAD2 levels in the

TGFBR1-CA/G9 ovary versus wild type controls at 2 month of age by western blot. Quantitative

real-time PCR was performed to determine potential effect of TGFBR1 activation on TGFB

target genes and genes crucial for ovarian development and function. The results showed

increased expression of TGFB target genes including plasminogen activator inhibitor-1 (Pai-

1)/Serpine1 and Smad7 in the ovaries of TGFBR1-CA/G9 mice as early as postnatal day 3 (PD3),

when the expression of inhibin alpha (Inha) and zona pellucida glycoprotein 3 (Zp3), genes

important for granulosa cell and oocyte function, was not significantly altered. However,

elevated mRNA levels of Inha and reduced expression of Zp3 transcripts were found at PD7.

Immunofluorescence staining of alpha smooth muscle actin (ACTA2; a theca cell marker),

DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4; an oocyte marker), and INHA (a

granulosa cell marker) demonstrated defects in postnatal folliculogenesis, evidenced by the

presence of disrupted follicle structures in the TGFBR1-CA/G9 ovaries. Remarkably, these mice

developed ovarian neoplasms reminiscent of sex cord-stromal tumors based on both

morphological and molecular criteria (i.e., expression of INHA, FOXO1, and SOX9), suggesting

that sustained activation of TGFBR1 using Gdf9-iCre is sufficient to induce malignant

transformation of ovarian somatic cells. The neoplasms were highly proliferative and contained

mitotic figures and abundant expression of MKi67. Further studies are to determine the

mechanism of ovarian tumor development and the potential stage-specific role for TGFBR1

activation during follicular development [Research was supported by the office of the Assistant

Secretary of Defense for Health Affairs, through the Ovarian Cancer Research Program under

Award no. W81XWH-15-1-0082 and in part by Texas A&M University New Faculty Start-up

funds].

Deregulation of the 16p11.2 Transcription Factor MAZ Results in Genitourinary Hypoplasias

Meade Haller Baylor College of Medicine

Molecular & Cellular Biology Ph.D. Program Advisor: Dolores J. Lamb, Ph.D.

Genitourinary (GU) birth defects comprise some of the most common yet least studied

congenital malformations and range in severity from conditions such as undescended testes (cryptorchidism), ventrally misplaced urethral meatus (hypospadias), and renal agenesis to highly complex malformations such as bladder exstrophy epispadias complex (BEEC) and ambiguous genitalia. Genomic copy number gains and losses (CNVs) frequently result in congenital malformations of the GU tract, and can be identified using array comparative genomic hybridization (aCGH). CNV overlap mapping revealed over 30 patients with GU defects harboring CNVs in the syndromic genomic region, 16p11.2 – the most common pathogenic CNV hotspot in humans. The only gene covered collectively by all the mapped 16p11.2 CNVs in patients with GU defects is MYC-associated zinc finger (MAZ), which encodes a transcription factor. In situ hybridization and immunohistochemistry staining on mouse embryos showed robust expression of Maz in the developing GU tract. Prospective CNV qPCR screening of genomic DNA from 258 patients with GU defects revealed an incidence of MAZ copy variation at 6% in GU-abnormal patients, an enrichment compared to the general population at <1% (Tucker et al, 2013).

As predicted based on the similarity of its consensus sequence to that of WT1, knockdown of MAZ in human embryonic kidney (HEK293) cells results in differential expression of several WNT pathway transcripts including morphogens WNT3A/4/5B/7B/8A/11. Knockdown of MAZ in HEK293 and RWPE1 normal prostate cells results in suppressed proliferation favoring stasis in G0/G1 over entry into S-phase, and deregulation of cell cycle genes including CCND1. In contrast to knockdown, overexpression of MAZ does result in differential expression of WNT pathway transcripts, but does not affect proliferation, possibly because MAZ is already robustly expressed in most cell lines. A murine CRISPR deletion model for Maz reveals that heterozygote animals exhibit bladder hypoplasia, and null embryos exhibit a 60% penetrance of congenital anomalies of the kidney and urinary tract (CAKUT) including but not limited to hypoplasias and ageneses consistent with suppressed proliferation. Once thought to be a simple housekeeping gene, MAZ encodes a transcription factor that influences WNT signaling and cell cycle progression, and contributes to the high frequency of congenital urogenital malformations associated with 16p11.2 dosage variation. Research supported by NIH grants T32DK007763 and R01DK078121

IFT140 is a Novel Candidate Gene for Impaired Spermatogenesis: Identification by Whole Exome Sequencing and Validation with Sanger Sequencing Amin Herati*, Peter Butler, Cenk Cengiz, Matthew Bainbridge, James Lupski, Richard Gibbs, Larry Lipshultz, Dolores J. Lamb, HOUSTON, TX INTRODUCTION AND OBJECTIVES: One third of male factor infertility cases are idiopathic and are likely caused by genetic factors. Non-obstructive azoospermia (NOA) is characterized by the absence of sperm in the ejaculate secondary to the lack of sperm production in the testicle. The aim of our study was to identify novel variants implicated in the pathogenesis of NOA through whole-exome sequencing (WES). METHODS: Homozygosity mapping with WES was performed using the NimbleGen SeqCap EZ Exome V3 sample preparation kit and the Illumina HiSeq 2000 next-generation sequencing platform to sequence the exomes of two siblings with NOA from a consanguineous family. Genetic mutations that passed filter criteria were validated using Sanger sequencing. We identified one candidate gene, Intraflagellar Transport Protein 140 (IFT140), based on its expression pattern in testes according to the Human Protein Atlas, as well the mutation’s damage prediction based on two online bioinformatics resources. We subsequently performed Sanger sequencing on 114 NOA and 43 fertile men. RESULTS: WES identified 442 variants in the index patients. Four (0.9%) of these variants passed filter criteria, one of which was a six-nucleotide deletion (HG38, chr16:1,519,961 TCTTGGC>T) in exon 22 of IFT140. Family segregation showed that this mutation was homozygous in the two brothers who underwent WES, as well as in a third brother with NOA and their sister. This mutation was not identified in the 1000 Genome Project and was predicted to be deleterious by the bioinformatics tools, MutationTaster and PROVEAN. The Exome Aggregation Consortium database of 60,706 genomes was also queried which identified a minor allele frequency of 0.1%. Sanger sequencing validated the mutation in this family and identified it as a heterozygous mutation in 1 (0.9%) of the 114 NOA men and none of the 43 fertile controls in our cohort. Pathologic correlation of this unrelated patient showed hypospermatogenesis with a clinical history significant for three failed cycles of in vitro fertilization (IVF). CONCLUSIONS: We identified a small indel mutation in IFT140 as a novel candidate variant for infertility. This mutation was detected in our cohort of NOA men in addition to the index family, with a prevalence of 0.9%, which is higher than the minor allele frequency in an unselected general population. Further studies are required to determine the role of IFT140 in spermatogenesis. Source of Funding: A.H. is a National Institutes of Health (NIH) K12 Scholar supported by a Male Reproductive Health Research Career (MHRH) Development Physician- Scientist Award (HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program (to Dolores J.Lamb).

Treatment with Low-Dose Aspirin During Gestation Does Not Prevent Maternal Hypertension and Vascular Dysfunction in Response to TLR9 Activation Paresh A. Jaini, An Nguyen, Melissa Valdes, Oluwatobiloba Osikoya, Styliani Goulopoulou University of North Texas Health Science Center, Institute of Cardiovascular and Metabolic Diseases, 3500 Camp Bowie Boulevard, Fort Worth, Texas Background: Daily low-dose aspirin after 12 weeks of gestation is recommended as a preventive intervention for women at high risk for preeclampsia, a hypertensive disorder of pregnancy with high rates of maternal and fetal mortality and morbidity. Activation of the innate immune system during pregnancy is implicated in the development of preeclampsia. Maternal exposure to synthetic CpG oligonucleotides (CpG ODN, specific ligand of the innate immune receptor Toll-like receptor 9) induces maternal hypertension, vascular dysfunction, and upregulation of cyclooxygenase enzymes in pregnant rats. Hypothesis: We hypothesized that maternal treatment with low-dose aspirin during gestation would ameliorate TLR9-induced hypertension and vascular dysfunction in pregnant rats. Methods: Pregnant Sprague-Dawley rats were treated with a synthetic CpG ODN (ODN2395) or vehicle on gestational day (GD) 14, 16, and 18. Aspirin treatment (or control) started on GD10 and continued throughout gestation for all groups [control (no treatment), ODN2395 (300 μg), aspirin (1.5 mg/kgBW), aspirin+ODN2395]. Blood pressure was measured on GD19 using the tail cuff method and mesenteric resistance artery (MES) function was assessed on GD21 using wire myography. Results: ODN2395-treated rats had higher blood pressure on GD19 compared to vehicle-treated dams and aspirin did not ameliorate ODN2395-induced hypertension (control: 97 ± 0.4 mmHg, ODN2395: 121 ± 7 mmHg, aspirin: 101 ± 5 mmHg, aspirin+ODN2395: 121 ± 7 mmHg, p<0.05). Aspirin treatment increased MES sensitivity to PE (pEC50, ODN2395: 5.6 ± 0.1 vs. aspirin+ODN2395: 5.9 ± 0.1, p<0.05) and reduced sensitivity to acetylcholine (ACh) in ODN2395-treated rats (pEC50, ODN2395: 7.6 ± 0.1 vs. aspirin+ODN2395: 7.0 ± 0.1, p<0.05). Fetal and placental weights did not change in response to any treatment. Conclusion: Treatment with low-dose aspirin throughout gestation did not prevent the development of TLR9-induced maternal hypertension, augmented vascular sensitivity to α1-adrenergic receptor activation, and attenuated endothelium-dependent dilation in rats exposed to innate immune system activation. Low-dose aspirin treatment alone may not be adequate to prevent increases in maternal blood pressure induced by bacterial infections. Funding: American Heart Association, University of North Texas Health Science Center, Texas College of Osteopathic Medicine Summer Research Fellowship

E2F1 is a Master Regulator Influencing Testicular Descent, Function and Health

Authors: Carolina J. Jorgez1,2, Ching H. Chen1, Nathan Wilken1,2, Juan C. Bournat Ph1,3,

and Dolores J. Lamb1,2,3 1Center for Reproductive Medicine, 2Scott Department of Urology, 3Department of

Molecular and Cell Biology, Baylor College of Medicine, Houston, TX, 77030.

Abstract

Currently, recognized causes of infertility include endocrine disorders, cryptorchidism,

obstruction of the genital track, ejaculatory defects, karyotype abnormalities, Y-

chromosome microdeletions, and gene mutations. However, the etiologies of infertility

and cryptorchidism remain unknown and studies suggest a genetic cause may underlie the

defect. We reported that gene dosage changes resulting from microdeletions and

microduplications encompassing E2F1 were present in a subset of infertile men. More

importantly, targeted deletion or overexpression of E2f1 in transgenic mice caused

spermatogenic failure, recapitulating the human phenotype observed in the clinic. To

define the functional consequences of E2f1 gene-dosage changes, E2f1-null mice were

carefully phenotyped. E2f1-null mice were cryptorchid with severe gubernaculum defects

and displayed a progressive depletion of germ cells beginning as early as 3 weeks of age,

resulting in a Sertoli cell only phenotype. Testicular failure was associated with

significantly decreased testicular weight, as well as impaired sperm production and poor

motility. Abnormalities in the blood testis barrier and in the expression of cell cycle and

Wnt signaling pathways genes were identified. Inactivation of E2f1 resulted in the loss of

germ cell polarity and abnormal tight junction assembly, which in turn contributed to

germ cell loss. Interestingly, germ cell depletion in the adults did not occur by apoptosis

but probably by altered Wnt4 signaling. In development, Wnt4 promotes female sex

differentiation and represses male external sexual differentiation. However, the function

of Wnt4 in the adult testis is not well established. Testicular Wnt4 gene and protein

expression significantly increased as E2f1-null mice matured and aged. Increased Wnt4

expression correlated with germ cell loss, suggesting a novel role of Wnt4 in

spermatogonial germ cell survival. E2F1 gene-dosage changes cause infertility and

cryptorchidism associated with cell cycle and Wnt4 signaling expression dysregulation.

Extended Spectrum Beta-Lactamase Urinary Tract Infections During Pregnancy Kelley B, Beninati B, Davidson C, Clark S, Eppes C. OBJECTIVE: Extended spectrum beta-lactamases (ESBLs) are enzymes produced by gram-negative bacilli that result in resistance against most antibiotics. We sought to evaluate the epidemiology and impact of ESBL urinary tract infections (UTIs) during pregnancy. METHODS: We performed a case control study comparing outcomes in pregnant women with optimally treated ESBL UTIs, sub-optimally treated ESBL UTIs, and non-ESBL UTIs from 2012-2014. Sub-optimal treatment was defined as treatment without carbapenems or post-treatment negative urine culture. For those with ESBL UTIs, we identified antecedent infections, prior antibiotic use, and the treatment course. RESULTS: 206 reproductive age female patients with ESBL UTIs were identified, of whom 19% (n=39) were pregnant. Of these, 28 had pregnancy outcome data. Suboptimal treatment (use of non carbapenem antibiotics or lack of post-treatment culture) was noted in the majority of cases. 6 of 6 patients with ESBL pyelonephritis had been treated previously for cystitis compared with 6 of 21 women with non-ESBL pyelonephritis. (p=0.0031). Only 1 of 21 women with non-ESBL pyelonephritis received suboptimal treatment, as defined above, whereas all 6 of 6 women with ESBL pyelonephritis had received suboptimal treatment (p=0.0001). No cases of pyelonephritis, chorioamnionitis or preterm birth were seen in the optimally treated ESBL group, while 10 such outcomes occurred in the group without optimal treatment. CONCLUSIONS: Sub-optimal treatment of ESBL infections in pregnancy is common. Our data support the importance of more aggressive treatment and follow up of pregnant women with ESBL UTIs to prevent secondary clinical pyelonephritis.

Shock Index: A Potential Criterion for a Maternal Early Warning System Jaden R. Kohn1, Catherine Eppes MD MPH1, Gary Dildy MD1 EDUCATIONAL OBJECTIVE: At the conclusion of this presentation, participants should be able to explain the relationship between shock index (SI) and postpartum hemorrhage (PPH) & need for transfusion. ABSTRACT: OBJECTIVE: Postpartum hemorrhage (PPH) is one of the most common causes of maternal mortality and severe morbidity in the United States and worldwide. Shock index (SI), heart rate divided by systolic blood pressure, has been proposed as a metric to predict early hypovolemia and need for transfusion or operative management in trauma settings. We explored how SI relates to PPH and need for transfusion. STUDY DESIGN: SI and delta-SI (peak SI - baseline SI) were calculated for all peripartum vital signs in 50 controls and 25 cases. ROC curves were plotted. Data were reported as mean +/- SD. RESULTS: Descriptive statistics are displayed in the table. Baseline pre-labor SI was the same for each group. Peak SI differed significantly between controls and cases (p=0.03). Delta-SI was significantly higher in cases than controls (p=0.01). SI was not a sensitive predictor of transfusion, however delta-SI was highly predictive; (threshold 0.34, 91% sensitivity, 90% specificity). Delta-SI was more predictive of need for transfusion than estimated blood loss (ROC area 0.9512 versus 0.9075, p=0.4). For the 6 patients who required hysterectomy following PPH, delta-SI threshold of 0.373 predicted need for surgical intervention with 100% sensitivity and 89% specificity. CONCLUSION: SI or a variant such as delta-SI may be a valuable predictor of maternal deterioration, the need for transfusion, or operative intervention in the setting of PPH. Shock index

Control N=50

All PPH N=25

PPH with transfusion N=8

PPH with hysterectomy N=6

Baseline 0.74 +/- 0.13 0.74 +/- 0.13 0.71 +/- 0.21 0.77 +/- 0.27 Labor 0.79 +/- 0.20 Peak 0.88 +/- 0.15 1.28 +/- 0.34 1.20 +/- 0.32 1.3 +/- 0.33 Delta-SI 0.19 +/- 0.23 0.39 +/- 0.24 0.50 +/- 0.20 0.53 +/- 0.20

Identifying a Novel Role for X-Prolyl Aminopeptidase (Xpnpep)2 in Crvi-Induced Adverse Effects on Germ Cell Nest Breakdown and Follicle Development Sivakumar, K. K 1, Stanley, J. A 1, Arosh, J. A 1, Burghardt, R. C 1, Banu, S. K 1 1 Texas A&M University, College Station, TX, USA ksivakumar@cvm.tamu.edu ABSTRACT Environmental exposure to endocrine disruptors (EDCs) is one of the causes for premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC, widely used in more than 50 industries including chrome plating, welding, wood processing and tanneries. Recent data from USEPA indicate increased levels of Cr in drinking water from several cities in the U.S., which potentially predisposes Americans to various health problems. Our recent finding demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm potassium dichromate (CrVI) from gestational day (GD) 9.5 – 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on embryonic day (E) 15.5, 17.5 or postnatal day (PND)-1, -4 and 25, and various analyses were performed. Results showed that gestational exposure to CrVI: (i) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; (ii) increased X-prolyl aminopeptidase (Xpnpep)2, a POF marker in human, during GCN breakdown; (iii) decreased Xpnpep2 during postnatal follicle development; (iv) Xpnpep2 inversely regulate the expression of Col1, -3 and -4 in all the developmental stages studied; (v) increased co-localization of Xpnpep2 with Col3 and -4. Thus CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Differentiation of Human Cytotrophoblast to Syncytiotrophoblast is Associated with

Profound Genetic and Epigenetic Changes

Youn-Tae Kwak, Sribalasubashini Muralimanoharan, and Carole R. Mendelson

Departments of Biochemistry and Obstetrics and Gynecology, The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038

Proper placental development is critical for successful pregnancy outcome. Defective placental implantation and vascularization with associated hypoxia are implicated in preeclampsia (PE), a leading cause of maternal and neonatal morbidity and mortality. Expression levels of a number of genes are highly regulated during differentiation of proliferative cytotrophoblasts (CytT) to multinucleated syncytiotrophoblast (SynT). The SynT performs essential functions to ensure growth and survival of the fetus, including transport of O2 and nutrients and synthesis of protein and steroid hormones. However, the genetic and epigenetic mechanisms underlying placental development remain incompletely defined. Mid-gestation human trophoblasts in primary culture provide a robust and biologically relevant system for defining chromatin structural changes associated with altered expression of a number of genes that are highly regulated during differentiation of CytT to SynT. Several of these genes are also markedly impacted by O2 tension. Using this system, we observed that expression levels of steroid receptor coactivator 3 (SRC-3/NCOA3), hCYP19A1/aromatase P450 and the critical transcription factors, estrogen-related receptor γ (ESRRG), estrogen receptor α (ESR1) and glial cells missing 1 (GCM1), are dramatically upregulated during differentiation of CytT to SynT, while others (e.g. achaete-scute homologue 2 [ASCL2] and upstream stimulatory factors [USF] 1/2) are downregulated, implying differences in transcription factor binding and in the underlying epigenetic mechanisms. In the present study, mid-gestation human trophoblasts in primary culture were used to define chromatin structural changes associated with altered gene expression during SynT differentiation. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) was used to analyze changes in binding of key transcription factors and histone modifications at the promoters of these differentially-expressed genes. We observed that Pol II binding was enhanced at the transcription start sites (TSS) of genes, NCOA3, CYP19A1, ESR1, and ERRG differentially upregulated in SynT, compared to CytT. The active histone mark, H3K9Ac, was increased at the promoter regions of these genes in association with increased RNA polymerase (Pol) II binding. Studies are in progress using ChIP-qPCR and ChIP-seq to analyze changes in binding of crucial transcription factors and modified histones to these highly regulated genes, as well as genome-wide. Epigenetic modifications of key regulatory genes in cells maintained under hypoxic conditions and in placentas of PE vs. normotensive women will provide important insight into the mechanisms underlying this devastating disorder.

Supported by: NIH-5-R01-DK031206

Development of a Surgical Procedure For Removal of a Placentome During Gestation in the Ewe Colleen Lambo, DVM; Ashley B. Keith, PhD; Fuller W. Bazer, PhD; M. Carey Satterfield, PhD Abstract:

In recent decades, there has been a growing interest in the impact of insults during pregnancy on postnatal health and disease. It is known that changes in placental development can impact fetal growth and subsequent susceptibility to adult onset diseases, however, the ability to collect sufficient placental tissues during gestation without compromising the pregnancy have not yet been described. The sheep has long been an established animal model for the study of fetal development. Due to its cotyledonary placental type, the sheep model has potential for surgical removal of materno-fetal exchange tissues, i.e., placentomes. Our objective was to establish a technique for the study of placental phenotype and gene expression throughout pregnancy with minimal risk to maintenance of the pregnancy. Placentomectomies were performed on Day 70 of pregnancy in control-fed (fed 100% NRC; n=10) and nutrient-restricted ewes (fed 50% NRC; n=41). A group of control-fed ewes (fed 100% NRC; n=4) were subjected to sham surgical procedures. For the procedure, the gravid uterine horn was partially exteriorized through a ventral midline abdominal incision. A placentome on the antimesometrial side of the uterus, just distal to the amnion was identified through palpation, and a 2 - 3 cm incision was made proximal to the edge of the placentome. Care was taken to avoid rupture of the chorioallantoic membrane. The placentome was exteriorized, and connections to the chorioallantois were ligated and transected. Finally, the stalk of the placentome was ligated and transected using a modified transfixational ligature. Uterine and abdominal tissues were closed normally. As of Day 112 of pregnancy, four nutrient restricted ewes died on Days 74, 89, 99, and 103 of pregnancy. Respiratory signs had been noted in the ewe which expired 4 days post-operatively, and she had been treated with antibiotics 2 days prior. Body condition scores in the 4 ewes averaged 1.6 at the time of death. The remaining 51 ewes were evaluated by ultrasound to confirm the presence of fetal heartbeats and maintenance of pregnancy post-operatively. Fetal weight comparisons will be made to confirm the viability of the technique, and adding to the value of our model. Placentomes acquired at surgery and necropsy will be subject to extraction of RNA for gene expression analyses. Differences in gene expression can be assessed between control and nutrient restricted ewes, as well as amongst nutrient restricted ewes, when comparing dams with low-weight fetuses and normal-weight fetuses to better understand early adaptive changes within the placenta in response to nutritional insult. With this technique, gestational studies in the sheep model could provide insight into the onset and complexity of changes in gene expression in placentomes resulting from undernutrition (as described in our study), overnutrition, alcohol or substance abuse, and environmental or disease factors of relevance and concern in humans. Research is supported by NIH 1R01HD080658-01A1.

Duration of Testosterone Therapy and Male Age Predict Time to Return of Normal Total Motile Sperm Count after Human Chorionic Gonadotropin Therapy

Matthew R. Louis1, Taylor P. Kohn1, Stephen M. Pickett2, Mark C. Lindgren3, Alexander W.

Pastuszak3, Larry I. Lipshultz3

Affiliations: 1 Baylor College of Medicine, Houston, TX; 2 Department of Economics, Rice University, Houston, TX; 3 Scott Department of Urology, Baylor College of Medicine, Houston, TX.

Keywords: Infertility, Azoospermia, Testosterone Introduction and Objective: Testosterone therapy (TTh) reduces sperm counts in men. For men with testosterone-associated azoospermia and severe oligospermia, treatment includes TTh cessation and human chorionic gonadotropin (hCG) therapy. However, optimal duration of hCG therapy needed for sperm recovery or the expected extent of sperm recovery remains unknown. Here we identify factors affecting the length of time needed for sperm recovery and for sperm counts to reach >5 million/mL after TTh and subsequent hCG therapy. Methods: Retrospective chart review of men seen at a single academic andrology clinic between 2004-2015 was performed. All included patients presented with infertility and were on TTh at the time of the visit, and had an initial semen analysis with azoospermia or severe oligospermia. Men ceased TTh and were started on hCG, and had a second semen analysis within 6 months of starting hCG. The number of men with total motile sperm count (TMC) >5 million was determined. Logistic regression analysis evaluated the relationship between the number of men with TCM >5 million and time on TTh, age, T formulation, combination therapy with or without clomiphene citrate, initial T level, and initial sperm count. Results: A total of 55 men were identified with a mean age of 40.0 (range 27 – 69) years and a mean duration of TTh of 42.0 (range 0.5-240) months. Of the 55 men, 36 (65.5%) achieved a TMC of >5 million within 6 months of stopping TTh and starting hCG. Two variables were identified to have strong associations with the likelihood of achieving TMC >5 million: age (p = 0.04) and time on TTh (p = 0.01). T formulation, initial T level, initial sperm count, and the use of clomiphene citrate were not significantly associated. Multivariate logistic regression demonstrated that both time on TTh (OR=0.781, p=0.016) and patient age (OR=0.941, p=0.050) decrease the probability of achieving a TMC >5 million within 6 months (Figure 1). Conclusion: Men with infertility associated with TTh can resume sperm production adequate for initiating pregnancy by IUI using hCG combined therapy and even obtain natural pregnancy. The likelihood of achieving sufficient sperm counts within 6 months of TTh cessation and initiation of hCG is related to patient age and duration of TTh. Clinicians should counsel their older

patients or patients on long-term TTh desiring future fertility that recovery of spermatogenesis in these men may be slower than in other men.

Figure 1: Predictive Probability of Achieving TMC > 5 million within 6 months for age and duration of TTh. Predictions are calculated using the margins command in Stata 12.1. The multivariate logistic regression used: Pr(𝑇𝑇𝑇 ≥ 5 𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑤𝑚𝑤ℎ𝑚𝑚 6 𝑚𝑚𝑚𝑤ℎ𝑠) = 𝛽0 + 𝛽1 ×𝐴𝐴𝐴 + 𝛽2 × 𝑇𝑚𝑚𝐴𝑇𝑚𝑇𝑇𝑇 + 𝛽3 × 𝐼𝑚𝐼𝐴𝐼𝑤𝑚𝑚𝑚 + 𝛽4 × 𝑇𝑚𝑚𝑚𝑚𝐶 + 𝛽5 × 𝐼𝑚𝑚𝑤𝑚𝐼𝑚𝑇𝐴𝑠𝑤𝑚𝑠𝑤𝐴𝐼𝑚𝑚𝐴 + 𝛽6 × 𝐼𝑚𝑚𝑤𝑚𝐼𝑚𝐼𝐼𝐴𝐼𝑚 + 𝜀.

Gestational Exposure to Sulfur Based Particulate Matter During Gestation Alters the Fetal and Postnatal Development of the Spleen and Thymus

Alyssa L. Miller1, Jacob D. Brown1, Misti Levy2, Robert C. Burghardt3, Greg A.

Johnson3, Fuller W. Bazer1, Guoyao Wu1, Renyi Zhang2, and M. Carey Satterfield1 Departments of Animal Science1, Atmospheric Science2, and Veterinary Integrative

Biosciences3, Texas A&M University, College Station, TX, USA Gestational exposure to air pollution can have adverse effects on the developing fetus, including the fetal immune system. Air pollution is commonly broken down into two classifications: gaseous (e.g. ozone) and particulate matter (PM) of various sizes and compositions. To investigate the effect of PM exposure during pregnancy on fetal and postnatal development, we exposed gestating Sprague Dawley rats (n=10) to 150-175 ug/m3 of ammonium sulfate PM in airtight chambers from gestational Day 0 to 18, while control dams (n=10) were exposed to ambient air. At birth (postnatal day 0, PND0), litters were culled at random to four male and four female offspring. All additional offspring were harvested, weighed, and tissues preserved for further analyses. At weaning, four of the eight remaining rats (two males and two females), were necropsied as above. The final four (2 male and 2 female) pups were then treated with either a high fat diet (HFD) or low fat diet (LFD) for a 12-week period from PND21 until PND105. At PND105, rats were necropsied as above. Results indicate that on PND0 spleen weight was lower in the offspring from polluted dams compared to the controls (P<0.05). However, spleen weight did not differ between treatment groups at PND21 or PND105 (P>0.1). In contrast, weight of the thymus increased in offspring from polluted dams at PND21. On PND105 there was an interaction between sex and treatment such that the thymus was heavier in male offspring from polluted dams compared to male offspring from control dams (P<0.05). Weight of the thymus did not differ between female offspring from polluted and control dams (P>0.1). H&E staining of the spleen at PND21 and PND105 showed no morphological differences. Immunohistochemical analyses of the proliferation marker Ki67 identified greater staining in spleens from rats fed a HFD compared to a LFD, although there was no effect of pollution exposure on staining. Evaluation of steady state mRNA levels within the spleen on PND105 found that CD8 mRNA expression was increased (P<0.05) in offspring from polluted dams compared to offspring from control dams. In contrast, CD19 mRNA expression was reduced (P<0.05) in offspring from polluted dams compared to offspring from control dams. Data highlights dynamic changes in patterns of postnatal organ development within the immune system in response to maternal PM exposure. Results suggest that maternal PM exposure induces a shift in the ratio of B and T cells within the spleen at PND105, with the result being a more cytotoxic environment. Further studies are needed to determine the effects of gestational exposure to PM air pollution on immune system function.

Enalapril Normalizes Endothelium-Derived Hyperpolarizing Factor-Mediated Relaxation in Mesenteric Artery of Adult Hypertensive Rats Prenatally Exposed to Testosterone Jay S. Mishra,1 Amar S. More,1 Gary D.V. Hankins,1 Chandra Yallampalli2 and Sathish Kumar1 1Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas, 2Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas.

Elevated maternal testosterone, a common factor observed in preeclampsia or gestational protein restriction, leads to endothelium-derived hyperpolarizing factor (EDHF)-mediated vascular dysfunction and hypertension in rat offspring. We tested whether the angiotensin-converting enzyme inhibitor enalapril restores blood pressure and EDHF function through regulating the activities of small (SK3) and intermediate (IK1) conductance calcium-activated potassium channels in the mesenteric arteries of adult rats exposed to elevated testosterone during fetal life. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg/day from gestation day 15–19, subcutaneously), and their 6-month-old male offspring were examined. A subset of rats in these 2 groups was administered enalapril (40 mg/kg/day, an angiotensin converting enzyme blocker) for 2 weeks through drinking water. Blood pressures were significantly higher in testosterone offspring compared to controls, and treatment with enalapril significantly attenuated blood pressure in testosterone offspring. EDHF relaxation in mesenteric arteries of testosterone offspring was reduced compared to controls, and it was significantly restored by enalapril treatment. IK1 channel expression and function were similar between control and testosterone rats, and it was not affected by enalapril treatment. The relaxations mediated by the SK3 were impaired in testosterone offspring, and it was normalized by enalapril treatment. Furthermore, enalapril treatment restored expression levels of SK3 channels. These results indicate that renin angiotensin signaling may be the underlying mechanism that links prenatal androgen exposure and adult life endothelial dysfunction.

Uterine BMP7 Guides the Process of Embryo Implantation Diana Monsivaisa,e,f, Caterina Clementia, Jia Penga, Martin M. Matzuka,b,c,d,e,f Departments of aPathology and Immunology, bMolecular and Human Genetics, cMolecular and Cellular Biology, dPharmacology, and Centers for eDrug Discovery, and fReproductive Medicine, Baylor College of Medicine, Houston, TX 77030 Achieving embryo implantation remains as a major roadblock to the success of assisted reproductive technologies (ART). The process of embryo implantation involves dynamic communication between the embryo and uterus; thus, determining the secreted growth factors that guide embryo apposition, implantation and decidualization will improve the success of ART. Previous studies showed that the bone morphogenetic proteins (BMPs) and their receptors are necessary for fertility in female mice. For example, uterine-specific deletion of BMP2 and the BMP type 1 receptor, ALK2, result in defective endometrial stromal cell decidualization and in female infertility. We recently showed that the BMP type 1 receptor, ALK3 is required for embryo attachment and implantation, whereas the BMP type 2 receptor, BMPR2 is important during late gestation and its absence results in female sterility. However, the BMP ligands that signal throughout the process of implantation and in late gestation have not been identified. To determine the contribution of BMP7 to female fertility, we mated Bmp7flox/flox females to Pgr-cre+/-

males to generate Bmp7flox/flox-Pgr-cre+/- females (Bmp7 cKO). Fertility analyses demonstrated that compared to control mice, the absence of uterine BMP7 resulted in subfertility. We determined that ovarian function in the Bmp7 cKO mice was normal, suggesting that the subfertility in Bmp7 cKO mice was the result of uterine defects. We also demonstrated that Bmp7 was highly expressed in the early decidua, suggesting that BMP7 is important during early pregnancy and necessary to establish a healthy pregnancy during late gestation. Indeed, analysis of 4.5 and 5.5 dpc implantation sites demonstrated defective uterine crypt formation and defective embryo implantation in the Bmp7 cKOs. We also detected elevated expression of estradiol-responsive genes (Lcn2, Clca3, Muc1) and decreased expression of implantation-associated genes (Bmp2, Wnt4, Ptgs2, Ereg) at 4.5 and 5.5 dpc in the Bmp7 cKOs. These early implantation defects had adverse effects in late gestation, causing 88% of all implantation sites to become resorbed by 10.5 dpc, as well as defective expansion of the trophoblast layer and decreased vascularization in the Bmp7 cKO implantation sites. Our results indicate that BMP7 coordinates maternal-embryonic communication during implantation that determines healthy fetal development during late gestation. These studies were supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development grant R01-HD32067 and the Institutional Research and Academic Career Development Award (IRACDA) K12-GM084897.

Angiotensin, Aldosterone and Vasopressin Levels are Dysregulated in Prenatal Testosterone Exposed Adult Hypertensive Female Rats

Amar More, PhD1, J. Mishra, PhD1, G. Hankins, MD1, and Sathish Kumar, PhD1. 1Ob/Gyn, University of Texas Medical Branch, Galveston, TX, United States, 77555-1062

Introduction: Plasma testosterone (T) levels are elevated in preeclampsia, PCOS mothers and pregnant African-American women. We have shown that prenatal T exposure leads to cardiovascular dysfunction in adult females. However, the underlying mechanisms that contributes for blood pressure increase is not known.

Aim: To evaluate whether prenatal T exposure leads to alterations in angiotensin-aldosterone and vasopressin systems in adult females.

Materials and Methods: Pregnant Sprague Dawley rats were injected with vehicle or T propionate (TP) (0.5 mg/kg/day from gestation day 15-19) to increase plasma T levels twofold, similar to that observed in clinical conditions like preeclampsia. In the offspring, at 6 months of age, mean arterial pressure was recorded using telemetry system; plasma levels of aldosterone, angiotensin-II and vasopressin levels were measured by ELISA; serum and urine Na+, K+ levels were measured; small resistance arteries were isolated to determine vascular response to angiotensin II (in a wire myograph) and vasopressin and adrenals were used to determine expression of mRNA transcripts of steroidogenic genes.

Results: T exposure during prenatal life significantly increased arterial pressure in female adult offspring. Aldosterone levels were lower (70.27 ± 10.22 vs 172.07±23.87 pg/mL in controls) whereas plasma angiotensin II levels were higher in T females (1.05±0.05 vs 0.65±0.07 ng/mL in controls). Plasma arg8-vasopressin levels were increased in T females (28.2±0.7 vs 21.3±0.5 pg/mL in controls). The mRNA levels of CYP11B2 (aldosterone synthase) was decreased in the adrenals of T females. Despite decrease in plasma aldosterone levels in T females, serum and urine Na+ and K+ levels were similar to that in controls. Vascular contractile response to vasopressin was unaffected but responses to angiotensin II in mesenteric arteries were enhanced in T offspring (logEC50 = -9.04±0.10; Emax = 60.9±6.93) compared with control (logEC50 = -8.56±0.08; Emax = 33.3±7.95).

Conclusion: These results suggest that prenatal T exposure down regulates adrenal CYP11B2 expression leading to decreased plasma aldosterone levels. Elevated angiotensin II and vasopressin levels along with enhanced responsiveness to angiotensin II may serve as an underlying mechanism to maintain plasma Na+ and K+ levels and mediate hypertension in adult T females.

Gene Expression In Developing Goat Testes: Sequencing, Assembly And Identification Of Caprine Spermatogenesis Transcriptome.

Authors: Brenda Barcelos, Sara Fuentes-Soriano, Jessica Watts, Floyd Williams, Flavio Ribeiro, Bill Foxworth, Louis Nuti, Gary Newton, and Shaye Lewis

Understanding gene networks necessary to establish and maintain spermatogenesis will provide insight into the development of molecular markers for potential fertility of individual animals. The objective of the current study was to identify candidate genes and gene networks important for the establishment and maintenance of spermatogenesis in the goat testes. We performed a high-throughput RNA sequencing project of caprine testis using Alpine goats as a model system. Sampling included pre-meiotic and meiotic testis parenchyma from 2-month and 4-month-old Alpine bucks (n=3 animals/time-point). Total RNA isolations were used to generate six single-stranded cDNA libraries, each treated for poly-A selection. In total, the raw reads for this project represented 436,408,082 sequences reads of an average length of 801 bp. To compare available transcriptomic information of Capris hircus (Yunnan goat) with our high-throughput sequencing project data we mapped raw reads against publically available goat transcriptome data (ftp://ftp.ncbi.nih.gov/genomes/Capra_hircus). A total of 343,704,188 reads mapped against the reference and 92,703,894 reads were unmapped. Unmapped reads were used to generate a de novo testes transcriptome assembly of Alpine goat. Mapping information provided insights into the top 1,000 highly differentially expressed genes. Results showed an age-specific enrichment for the apoptosis pathway (19 genes; KEGG). Apoptosis is important to establish and maintain spermatogenesis. The expression of 19 apoptosis genes decreased between 2-month and 4-month testes. To determine the presence of the apoptosis gene network in the de novo testes transcriptome, we generated a custom BLAST database and searched 19 genes and 25 variants involved in the apoptosis pathway. BLAST searches and alignment examinations showed evidence for 17 proteins and 20 variants members of the apoptosis pathway. The protein kinase, cAMP-dependent catalytic beta protein (PRKACB) showed the highest number of variants. Interestingly, this protein signals cell survival and integrity and is not involved in the execution of cell apoptosis. We have identified gene networks important during pre-meiotic and meiotic phases of spermatogenesis. Future studies will focus on implementing additional bioinformatics approaches to exhaustively and carefully compare completeness of current de novo transcriptome assembly by analysis of additional de novo transcriptome assemblies generated from each individual sample studied in the project (e.g., tissue, age, individual). In addition research efforts will focus on validating the expression of apoptosis genes and other candidate genes by qPCR and in situ hybridization and characterizing the biological significance of the expression of these pathways during caprine testes development.

Key Words:

RNA-sequencing, de novo assembly, apoptosis

Steroid Hormones Reorganize Cervical ECM Structure in Pregnancy Shanmugasundaram Nallasamy1, Kyoko Yoshida2, Meredith Akins1, Kristin Myers2, Mala

Mahendroo1

1Department of Obstetrics and Gynecology and Green Center for Reproductive Biological Sciences, University of Texas Southwestern Medical Center, Dallas, TX; 2Department of

Mechanical Engineering, Columbia University, New York, NY;

Cervical remodeling, the process by which the cervix transforms from a closed rigid to a

compliant structure that can open to allow safe passage of the fetus, is an essential feature for

normal parturition. Perturbations in the remodeling process can lead to premature remodeling

and a risk of preterm birth. Inappropriate reorganization of the cervical extracellular matrix

(ECM) can result from defects in processing and assembly of fibrillar collagen or from changes

in other ECM molecules that modulate collagen structure and strength. The proteoglycan

decorin has key functions in collagen fibrillogenesis. Loss of decorin perturbs collagen fibril

shape and mechanical integrity in connective tissues such as the tendon and skin. Decorin null

females have normal parturition but their cervical collagen fibril ultrastructure exhibits large and

irregular shaped aberrant fibrils interspersed among normal fibrils in the non-pregnant and early

pregnant cervix. Unexpectedly, we also identify abnormal elastic fibers. The transient

disappearance of aberrant collagen fibrils and elastic fibers in later pregnancy and reappearance

postpartum suggested a potential regulation by steroid hormones during pregnancy. Progesterone

treatment of ovariectomized nonpregnant decorin null mice resolves abnormal collagen fibrils in

the cervix. Estrogen treatment of ovariectomized nonpregnant decorin null mice resolves

abnormal elastic fiber structures. This study uncovers a direct role of steroid hormones to

regulate ECM architecture and provides a mechanism by which progesterone supplementation

could potentially be beneficial in preventing preterm birth in women.

The Circadian Machinery of the Neonatal Liver (Npas2) Regulates Metabolic Homeostasis in Response to Metabolic Stress in a Murine Liver Specific Conditional Knock Out (cKO)

O’Neil D, Goodspeed D, Shope C, Belfort B, Aagaard K.

Objective: Despite being in an environment absent of light-dark signals, the fetus establishes and then maintains the circadian machinery. Previously, we have demonstrated that a maternal high fat diet (MHFD) is associated with the disruption of a key peripheral circadian gene (Npas2) in the fetal liver. We developed a novel Npas2 cKO mouse model of the peripheral clock (liver) to elucidate the role of Npas2 in contributing to life-long metabolic disease.

Study Design: Npas2 cKO mice were generated by targeting the deletion of the loxp flanked region of exon 3 in the neonatal liver (postnatal day 7) by cre expressed under regulation of the Albumin gene promoter (Albcre). Due to the postnatal expression of cre, Npas2 is normally expressed during fetal development. Npas2 cKO (Albcre;Fl/Fl, n=62), heterozygous (Albcre;Fl/+, n=48), and control mice (Fl/Fl, n=49) were generated for this study. At weaning mice were placed onto control (CD) or high fat diet (HFD, 42% fat) and weighed biweekly. After 25 weeks, an oral glucose tolerance test (OGTT, 2g/kg glucose) was performed after a 6-hour fast. On a separate cohort of mice, we restricted food availability to 4 hours each day for 16 days; mice were weighed every 2 days.

Results: Our findings from over 150 mice are shown in the Figures. In sum, male cKO mice on a HFD had significantly increased mass when compared to control mice on a HFD starting at 16 weeks post-weaning (p<0.01). Moreover, under a restricted feeding regimen (hunger) cKO females have a significantly greater loss of weight (abnormal satiety signal) compared to controls (p<0.05).

Conclusion: Comprehensive initial phenotyping of our novel mouse model indicates that neonatal (prior to eye opening) expression of hepatic Npas2 plays a necessary role in regulating weight gain, lipid accumulation, and glucose regulation. Additionally, hepatic Npas2 regulates weight loss when food is restricted (females), demonstrating the importance of the peripheral liver clock independent of the central (light-dark) clock. These data underscore the importance of the neonatal hepatic Npas2 circadian responses in orchestrating both hunger and satiety signals throughout life.

Adenylyl Cyclase 2 Copy Number Variants Are Associated With Congenital Genitourinary Anomalies

O’Neill, Marisol1,2; Lamb, Dolores J.1,2,3 1 Center for Reproductive Medicine, 2Department of Molecular and Cellular Biology, 3Scott Department of Urology, Baylor College of Medicine. Houston, Texas

Birth defects are the leading cause of infant death in the United States, accounting for 20% of infant mortality. Genitourinary (GU) anomalies are among the most common categories of birth defects and consist of externally recognizable anomalies and congenital anomalies of kidney and urinary tract (CAKUT). Externally recognizable anomalies include hypospadias (1 in 125 male births), cryptorchidism (3-6% of full term male births) and ambiguous genitalia (1:2000-1:4000 births). Though the prevalence of GU anomalies is high, their causes are poorly understood. A genetic change which may contribute to disease phenotypes is a copy number variant (CNV; a micro-deletion or micro-duplication of a small chromosomal region). When CNVs occur in dosage sensitive genes, a disease phenotype can arise. In our laboratory, CNVs harboring candidate genes were identified in patients with GU phenotypes through the use of array comparative genomic hybridization (aCGH). Through the use of aCGH, we identified adenylyl cyclase 2 (ADCY2) CNVs in two patients. ADCY2 is a member of the adenylyl cyclase class III family of genes that are responsible for converting ATP into cAMP. cAMP, is involved in several essential processes required for GU development and function including testicular descent and androgen production. We have thus far identified an incidence of ADCY2 duplications in 1.9% of our cohort patients with GU anomalies whereas CNVs of ADCY2 occur in only 0.19% of the general population. Adcy2 expression was localized in the kidneys, testis, bladder, and urethra of developing murine GU tract through immunohistochemistry (IHC). IHC performed on both murine and human testis revealed high level of expression in Leydig cells; the site of androgen production within testis. Expression of ADCY2 in testis and urethra correlates with the cryptorchidism and hypospadias phenotypes observed in patients with ADCY2 CNVs. The presence of ADCY2 in Leydig cells leads us to believe that ADCY2 may play a role in the production of testosterone. Testicular descent and urethral growth, during genital development, both depend on proper androgen signaling. To verify the involvement of ADCY2 CNVs in disruption of steroidogenesis, we overexpressed Adcy2 in the murine Leydig cell line TM3. Overexpression of ADCY2 led to decreased steroidogenic acute regulatory protein (StAR) production. StAR, a protein which mediates the transport of cholesterol into mitochondria, is essential for steroidogenesis. Results suggest that ADCY2 copy number variants contribute to the development of GU anomalies through the partial inhibition of steroidogenesis.

This study was supported in part by NIH grant R01DK078121 from NIDDK (to DJL).

Title: Uterine Perivascular Adipose Tissue Potentiates Contractile Responses in Uterine Arteries From Pregnant Rats

Authors: Oluwatobiloba Osikoya, Styliani Goulopoulou

Affiliation: University of North Texas Health Science Center, Institute for Cardiovascular and Metabolic Diseases, Fort Worth, TX

Background: Perivascular adipose tissue (PVAT) has vasoactive effects, mostly via paracrine actions. The effects of PVAT vary with anatomic location; PVAT has anti-contractile effects in peripheral vascular beds in animals and in humans but it potentiates contractions in coronary vascular smooth muscle. Pregnancy is characterized by adipose tissue expansion as well as structural and functional changes in the uterine vasculature. However, the effects of PVAT on uterine artery reactivity during pregnancy are not understood.

Hypothesis: We hypothesized that uterine PVAT has a functional role in uterine artery contractile and dilatory responses and this role is modified by pregnancy.

Methods: Pregnant Sprague-Dawley rats were sacrificed on gestational day 16 (term=21-22 days). Uterine arteries and their surrounding PVAT were harvested and cleaned for study. Concentration response curves (CRCs) to potassium chloride (KCl, 4.7 – 80 mM) and phenylephrine (PE, 10-9 - 3x10-5 M) were performed using wire myography. CRCs were performed in the presence and absence of the surrounding PVAT (0.1 g) or PVAT-conditioned media. Arteries were incubated with PVAT or PVAT-conditioned media for 30 minutes. To make the media, we incubated 0.4 g of PVAT in 15 ml physiological salt solution for 60 min (37oC - 5% CO2, 95% O2).

Results: Uterine arteries incubated with PVAT (+PVAT) had greater contractile responses to KCl compared to control vessels [KCl (30 mM), control: 4.0 ± 0.81 mN vs. +PVAT: 14.7 ± 1.68 mN; KCl (40 mM), control: 14.4 ± 0.68 mN vs. +PVAT: 19.6 ± 0.88 mN, p<0.05]. PE-induced contractions were greater in uterine arteries exposed to PVAT compared to control vessels (pEC50, control: 6.0 ± 0.08 vs. +PVAT: 6.3 ± 0.10, p<0.05). Incubation with PVAT-conditioned media also increased uterine artery contractile responses [KCl (30 mM), control: 5.3 ± 0.52 mN vs. +PVAT-media: 12.6 ± 1.05 mN, p<0.05; PE (pEC50), control: 6.0 ± 0.09 vs. +PVAT-media: 6.3 ± 0.09, p<0.05].

Conclusions: Our data show that uterine PVAT has a pro-contractile effect on uterine arteries from pregnant rats. We propose that uterine PVAT provides signaling from the outer layer to the inner layers of the vascular wall that determines uteroplacental vascular adaptations to pregnancy.

Repression of GDNF Transcription by Jag1/Notch Signalling in Sertoli Cells

1Parag Parekh, 1,2Thomas Garcia, 1,3Krishna Sinha, 1Marie-Claude Hofmann 1University of Texas MD Anderson Cancer Center, Houston TX; 2University of Houston Clear Lake, Houston, TX; 1,3University of Texas Health Sciences Center, Houston, TX Self-renewal and differentiation of spermatogonial stem cells (SSC) are fundamental processes ensuring normal spermatogenesis throughout life. Within the spermatogonial stem cell niche, GDNF produced by Sertoli cells and peritubular cells plays a major role in the maintenance of a pool of undifferentiated spermatogonia. While GDNF production by Sertoli cells is positively driven by FSH and FGF2, we postulate that negative feedback regulation may also be involved in homeostasis. We previously demonstrated using mouse models and in vitro culture experiments that activation of NOTCH signaling in Sertoli cells reduces their GDNF expression. To investigate which cells in the seminiferous epithelium are driving NOTCH in Sertoli cells, we analyzed levels of expression of several NOTCH ligands by different types of germ cells isolated by FACS (GFRA1+, KIT+/DAZL+ and KIT-/DAZL+). Our data demonstrate high expression of JAG1 by GFRA1-positive germ cells, which decreased as germ cells differentiate. We next investigated whether downstream effectors of NOTCH signaling such as HES/HEY transcriptional repressors directly bind to the GDNF promoter. Our analysis revealed several N- and E-binding sites in the proximal promoter. ChIP-PCR using HEK293 cells and primary Sertoli cells demonstrated specific binding of HES1 and HEY1 to the GDNF promoter, indicative of direct regulation of GDNF transcription by Notch signaling. Further, to verify the functional activity of HES1 and HEY1, we performed Luciferase assays using HEK293 cells co-transfected with a GDNF promoter-luciferase reporter construct and HES1 or HEY1 expression vectors. Our results demonstrate significant down-regulation of luciferase activity by both HES1 and HEY1 proteins. Altogether, our data conclusively establish that the JAG1/NOTCH/HES/HEY axis down-regulates GDNF expression in Sertoli cells and that GFRA1 positive cells may ensure homeostasis through negative feedback regulation.

Increased Progesterone Receptor A is Associated with Increased Contractility in the Pregnant Mouse Myometrium. Mary Peavey1, Margeaux Wetendorf1, William Gibbons1, Corey Reynolds1, Francesco DeMayo2, John Lydon1. 1Baylor College of Medicine, Houston, TX; 2National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC. Objective: Preterm birth complicates 10-12% of pregnancies with substantial healthcare and economic burdens. Progesterone is necessary for establishment and maintenance of pregnancy. Humans undergo a functional progesterone withdrawal for initiation of labor, possibly due to an increase in the PR-A isoform at the time of labor. Our goal is to determine the role of PR-A in the maintenance of pregnancy by increasing the expression of PR-A in the mouse myometrium. Design: A mouse model was generated in which the PRA isoform is overexpressed specifically in the myometrium. The effect of increasing PRA within the myometrium was investigated by assessing fertility, number of implantation sites, pregnancy viability, length of gestation, uterine contractility, and myometrial gene expression patterns. Materials and Methods: To achieve overexpression of PR-A isoform, the cDNA for the coding region of PR-A was inserted into the CAAG-LSL-NR vector and targeted to the ROSA26 locus. These mice were then crossed to the myosin 11 heavy chain (Myh11) cre mice for tissue-specific smooth muscle/myometrial transgene expression. Embryo implantation number and location, fetal growth and viability were assessed via ultrasound throughout the gestation. After achieving pseudopregnancy, myometrial contractility was assessed ex vivo by exposing uterine strips to uterotonic agents and recording force with an isometric transducer. Results: Overexpression of PR-A is associated with a 2-fold increase in uterine contractility when held at equivalent tension as well as in response to uterotonics. Additionally, overexpression of PR-A was associated with increased expression of GJA and decreased ZEB1. Mice with overexpression of PR-A had an average of 4.6 pups/litter vs 8.3 pups/litter in control mice due to an approximately 50% early pregnancy failure rate, with pregnancy loss occurring at around day 7.5-8.5 of gestation. The gestational length was unchanged. Conclusions: Mice with overexpression of PR-A in the myometrium have increased uterine contractility, reduced litter sizes due to early pregnancy failure, and increased expression of contraction-associated proteins. This research was supported by NIH RO1: HD-042311(NICDH) and the Burroughs Wellcome Fund.

Comparison of in Vitro vs. Interspecies Oviductal Culture of In Vitro Produced Bovine Embryos on Embryonic Development and Cryotolerance.

C. Pinzón1; M. Snyder1; J. Pryor1; A. Castro2; V. Vishwanath2; M. Westhusin1;

C. Long1

1. Reproductive Science Complex, Department of Veterinary Physiology and Pharmacology, College of

Veterinary Medicine and Biomedical Sciences, Texas A&M University. 2. Sexing Technologies, Navasota, Texas.

In vitro production (IVP) of bovine embryos has transformed the beef and dairy cattle industries worldwide. However, despite extensive efforts in the improvement of vitro embryo production and cryopreservation in vitro produced embryos demonstrate lower quality scores and higher lipid content, which is associated with decreased cryotolerance and decreased pregnancy rates when compared to their in vivo counterparts. The aim of this study was to examine the effect of in vitro culture (IVC) versus ovine oviductal culture (OVC) of bovine embryos produced using sexed semen on embryonic development and cryotolerance.

To assess the embryo development and cryotolerance, abattoir-derived oocytes were matured and fertilized using X chromosome sorted Gyr semen (Sexing Technologies®). Eighteen hours post fertilization presumptive zygotes were stripped of remaining cumulus cells and abnormal or lysed zygotes were discarded. Remaining zygotes (n= 3423) were divided randomly in two groups IVC and OVC. The IVC medium consisted of Bovine Evolve® (Zenith Biotech) + 4 mg/ml Probumin® BSA + 1 µg/ml gentamicin (Invitrogen), in a 38.5 C, 5% CO2, 5% O2, 90% N2 humidified incubator (n=1355). For OVC (n=2068), an average of 50 zygotes were surgically transferred into each ligated sheep oviduct of 20 sheep carrying a 0.3gr of progesterone insert (EAZI-Breed™ CIDR®, Pfizer).

At day 6 or 7 post IVF, embryos were surgically recovered from the oviducts (average recovery rate= 67.3% ± 4.1). All embryos were graded and staged according to the IETS guidelines. Quality grade 1 and 2 embryos (OVC n=329, IVC n=271) were cryopreserved by either direct transfer (DT) (1.5M ethylene glycol + 0.4% BSA + 0.1M sucrose, ViGRO™) (IVC-DT n=163, OVC-DT n=173) or vitrified (VT) using pellet method (Sexing Technologies®) (IVC-VT n= 161, OVC-VT n= 156). Furthermore, 40 quality grade 1 and 2 embryos from each treatment were lysed in RLT lysis buffer ( Quiagen®) for future qPCR analysis. Unpaired student’s t-test was performed using GraphPad® Prism software.

A higher proportion of cleaved embryos was observed in the IVC compared to the OVC (IVC= 88.37% ± 1.327, OVC=79.29 % ± 2.792, P < 0.05), although this was influenced by the lower recovery rate in the OVC. The proportion of grade 1 and 2 embryos was similar among groups (IVC= 83.5 ± 3.5, OVC= 84.0 ± 2.7, P = 0.9). OVC tended to have a greater proportion of viable embryos (grade 1, 2 and 3; IVC=25.2 ± 3.1, OVC=36.6 ± 4.3, P=0.07). The cryopreserved embryos are awaiting transfer in order to assess pregnancy rates as an indicator of cryotolerance and developmental potential. This study demonstrates that IVC and culture in ovine oviducts are similar in developmental potential. Completion of this experiment will determine whether culture treatment affected cryotolerance as determined by changes in pregnancy rates.

Prenatal maternal stress and Shank3ex4-9 mutation alter hippocampal stratum radiatum white matter (WM) in male murine offspring Rajesh Ramakrishnan, Jaclyn. B. Murry1, Fang He, Alfred Balasa, Gary Stinnett, Steen Pedersen2, Robia Pautler2, and, Ignatia B. Van den Veyver Department of Obstetrics & Gynecology, 1Department of Human & Molecular Genetics, 2Department of Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, TX 77030 Prenatal maternal stress has been found to affect offspring development and health. We wanted to investigate the effects of prenatal maternal stress in presence of an autism predisposition allele on offspring behavior and neurodevelopment. Using diffusion tensor imaging (DTI), we examined WM fractional anisotropy (FA) in Shank3ex4-9 mutant (KO) and WT male offspring from dams exposed to Chronic Unpredictable Mild Stress (CUMS). Stress-exposed dams (ST) underwent a CUMS battery from prior to mating until E17.5 of gestation. Shank3ex4-9/+ HET dams were mated with HET males and single-housed upon presence of plug. Non-stressed (NST) HET dams were mated in a similar manner. From both treatment groups, 8 week-old WT (NST: n=5; ST: n=2) or KO (NST: n=4; ST: n=3) male offspring were randomly selected for DTI to assess WM tractography. The mice were anaesthetized and transcardially perfused with paraformaldehyde (PFA). The skull was exposed, fixed in PFA and stored in 5mM gadopentate dimeglumine at 4°C until imaging. Brains were imaged for DTI using 20 gradient directions and data processed using DTI Studio to yield FA volume maps. All brains were masked, upsampled and aligned to template mouse brain FA map. Brains were then segmented into left and right components for 29 regions and the mean FA values for the voxels computed. Two-way ANOVA was applied to a whole brain voxel-voxel and to the average FA values in regions of interest (ROI). We analyzed WM tractography for genotype (G) (WT vs. KO) and environment (E) effect (NST vs. ST) and gene-environment interaction (GxE). In all, 15 ROIs had significant differences in FA between the tested groups. The stratum radiatum showed significant change in FA in WT vs. KO NST offspring and in WT NST vs. WT ST offspring. WT ST and KO NST offspring had similar changes, but stress did not have an additive effect on stratum radiatum FA in KO offspring. We have identified a contiguous region (stratum radiatum) within the hippocampus whose FA is significantly altered by genotype and environmental stress interaction. This supports the hypothesis that prenatal maternal stress can influence the phenotype of autism-predisposing mutations in offspring.

Discovery of Microfractures in Fetal Membranes: Site of Epithelial-Mesenchymal Transition

L. Richardson1, G. Vargas, PhD2,3, G. Saade, MD1, T. Brown2, S. Sheller1, J. Trivedi, MS1 and R. Menon, PhD1.

1Dept. of OB/GYN, UTMB, Galveston, TX, United States, 2Dept. Of Neuroscience and Cell Biology , UTMB, Galveston, TX, United States, 3Center for Biomedical Engineering, UTMB, Galveston, TX, United States

Objective: Remodeling of fetal amniochorionic membranes and its extracellular matrix (ECM) involves balanced activities mediated by oxidative stress (OS) and inflammation. Any disturbance to these processes leads to membrane structural alterations which may be detrimental to pregnancy status. We obtained visual, biochemical and histologic evidence of structural alterations in term amnion layer and determined their association with Epithelial-Mesenchymal Transition (EMT).

Methods: Amnion membranes were collected from not in labor cesarean deliveries (TNIL, n=5) or laboring vaginal deliveries (TL, n=5). To document the effect of OS, TNIL samples in an organ explant culture were exposed to cigarette smoke extract (CSE)(n=4). Tissue sections were imaged using a combination of multiphoton autofluorescence and second harmonic generation microscopy. Subcellular imaging was performed at a high resolution using a wavelength of 840 nm. Images were analyzed for presence of microfractures using ImageJ software. Immunohistochemistry for vimentin and cytokeratin18 [CK18] was done to show apical-basal polarity rearrangement leading to EMT.

Results: 3D microscopic analysis of amnion membranes revealed the presence of pits or microfractures on the surface, which resulted from the migration of amnion epithelial cells through the basement membrane into the stroma. These appeared to associate with ECM fibroblast cells. Microfractures were characterized by membrane indentation, altered amnion morphology and ECM degradation, primarily basement membranes. Histologic and biochemical analysis revealed positive expression of markers of EMT at this site, characterized by CK18- Vimentin+ in TL & CSE treated TNIL membranes compared to CK18+Vimentin- cells in TNIL controls.

Conclusions: Amnion membranes microfractures are associated with EMT. Higher microfractures during labor and its increase by OS showed its physiologic and mechanical relevance in parturition. We posit that microfractures and its resealing by collagen remodeling is a physiologic process during pregnancy. Extension of microfractures and failure to reseal may predispose them to rupture and adverse pregnancy outcomes.

SMAD Signaling is Required for Structural Integrity of the Female Reproductive Tract and Uterine Function During Early Pregnancy in Mice

Amanda Rodrigueza,b,, Swamy K. Tripurania,, Jason C. Burtonc, Caterina Clementia,d, Irina

Larinae and Stephanie A. Pangasa,f,g*

aDepartment of Pathology & Immunology, bGraduate Program in Molecular & Cell Biology, cGraduate Program in Integrative Molecular and Biomedical Sciences,

dGraduate Program in Developmental Biology, eDepartment of Molecular Physiology & Biophysics, fDepartment of Molecular & Cellular Biology, gCenter for Reproductive Medicine,

Baylor College of Medicine, Houston, TX 77030 USA

Pregnancy is a highly regulated process involving transfer of the morula from the oviduct to the uterus, followed by blastocyst attachment and implantation that initiates decidualization, a proliferation and differentiation of the uterine stroma that supports the developing embryo. In mice, dysregulation of TGFβ superfamily ligands and receptors results in infertility or reproductive diseases. Previously, our laboratory demonstrated that double conditional deletion of the bone morphogenetic protein (BMP) receptor signaling SMAD transcription factors, Smad1 and Smad5 using Amhr2 promoter-driven Cre recombinase (Amhr2-cre), results in subfertility and development of metastatic granulosa cell tumors. SMAD1 and SMAD5 form trimeric transcription complexes with the common SMAD4, and Smad4 conditional knockout (cKO) females are subfertile with folliculogenesis and ovulation defects but do not develop tumors. In addition, neither Smad4 cKO nor Smad1/5 cKO have known defects in oviduct or uterus function even though Amhr2-cre is known to be expressed in the smooth muscle layer of the oviduct and uterus as well as the uterine stroma. In contrast to these two mouse models, when we generated triple Smad1 Smad5 Smad4 cKO using Amhr2cre, we found that the female mice were sterile. To determine why this mouse model was sterile, we analyzed the ovary, oviduct, and uterus, and found extensive defects in the reproductive tract. Utilizing the emerging imaging technique of optical coherence tomography (OCT), we obtained extensive high-resolution 3D images of the Smad1/5/4-Amhr2cre female reproductive tract. We observed striking abnormalities in the oviduct that include wall thickening and disorganized luminal folds. Quantitative PCR analysis determined that loss of Smad1/5/4 resulted in altered expression of genes essential for oviduct development and smooth muscle differentiation, including the smooth muscle and cardiac specific transcriptional co-activator, myocardin (Myocd), which is a master regulator of smooth muscle contractile gene expression. Furthermore, we found that smooth muscle development of the uterus was also defective, and during pregnancy, the uterine lumen fails to close, and there was complete pregnancy loss by mid-gestation (E9.5). During both artificial and natural decidualization, we found that these defects are associated with loss of expression in key signaling factors, Bmp2 and Wnt4 in the maternal decidual, as well as in expression of Ptgs2 in decidualizing cells at the mesometrial pole of implantation sites during early pregnancy (E5.5). These data provide the first genetic evidence that both BMP and TGF-β SMAD pathway are critical for oviduct and uterine function during pregnancy, and that loss of Smad1/5/4 results in structural and functional abnormalities of the reproductive tract that cause sterility and early pregnancy loss. This study was supported by NIH grants R01 CA138628, R01 HD076980, T32 HD007165, R01 HL120140, T32 GM008231, P30 CA125123, and a Burroughs Wellcome Career Award in the Biomedical Sciences.

Syncytial Trophoblast Fusion and Breaching of Uterine Luminal Epithelium during Implantation in Sheep Heewon Seo1, Fuller W. Bazer2, Robert C. Burghardt1, Greg A. Johnson1 1Department of Veterinary Integrative Biosciences, and 2Department of Animal Science, Texas A&M University, College Station, TX 77843 During the peri-implantation period, multinucleated syncytia are formed in the sheep placenta. For over 20 years the scientific consensus has been that, during trophoblast syncytialization in sheep, binucleate trophoblast giant cells (BNCs) differentiate from the mononuclear trophoblast cells (MTCs), fuse with LE cells to form trinucleate cells, and BNCs continue to migrate to the uterine luminal epithelial (LE) layer and fuse with these growing trophoblast-LE syncytial plaques. There is no loss of LE cells, but rather they are incorporated into syncytial plaques, and there is no lateral fusion between syncytial plaques, rather all fusion events involve newly formed BNCs. However, this idea was based on electron microscopy studies, without the benefit of molecular markers of BNC and LE to support the conclusion. Therefore, we performed double immunofluorescence staining to observe interactions between BNCs and uterine LE cells using molecular markers for BNC and uterine LE including pregnancy-associated glycoprotein (PAG), E-cadherin, and cytokeratin. Our results provide new insights into syncytia formation in the sheep placenta because we observed: 1) syncytial cells containing more than two nuclei within the MTC layer suggesting fusion between MTCs is not limited to the formation of BNCs, and the term ‘trophoblast giant cell (TGC)’ may be more appropriate than BNC; 2) empty spaces within the LE layer at sites of implantation suggesting that loss of LE occurs during TGC migration and fusion to form syncytial plaques; 3) rapid enlargement of syncytial plaques that replace uterine LE suggesting fusion is not limited to the incorporation of new BNCs, but involves the lateral fusion between growing syncytial plaques; 4) expression of activated caspase 3 (CASP3), an executor in the apoptosis cascade, as trophoblast cells syncytialize and invade the uterine LE layer, suggesting apoptosis of uterine LE during syncytial fusion; and 5) engulfment of LE cells by TGCs, suggesting elimination of apoptotic or live uterine LE cells through phagocytosis and/or entosis, respectively. In light of these results, we hypothesize that MTCs fuse with each other to become multinucleated TGCs. Large numbers of TGCs simultaneously migrate to insert themselves between the uterine LE cells and induce apoptosis of neighboring LE cells through direct physical interactions and/or the release of paracrine factors. TGCs then eliminate the apoptotic uterine LE cells by phagocytosis and/or engulf remaining live uterine LE cells by entosis. TGCs then fuse with each other to form an extensive syncytial layer that fills the spaces left by removal of uterine LE and provides the direct interface between uterine and placental tissues within the placentomes of sheep. This new hypothesis for syncytialization at the uterine-placental interface of sheep is very similar to what is proposed to occur during the two trophoblast fusion events during placental development in humans, making the sheep an increasingly compelling in vivo model to study human placentation.

Contractile Gene Activation of Myometrial Cells Treated with Amnion Epithelial Cell – Derived Exosomes S Sheller, R Urrabaz-Garza, T Kechichian, G Saade, R Menon OBJECTIVE: Exosomes are cellular vesicles that carry various signals and function as communication vehicles between tissues and organs. Exosome contents represent the character and physiologic state of the cell of origin, which can alter the phenotype of target cells. We hypothesize that increased oxidative stress (OS) at term causes fetal cells to release exosomes, which carry signals to the myometrium that initiate parturition. The objectives of this study were to isolate and characterize the contents of primary amnion epithelial cells (AECs) derived exosomes, under standard and OS states, and to document their functional impact on human myometrial cell function. METHODS: AECs from normal term not in labor placenta were grown in media under standard conditions or in response to a validated model of OS (exposure to cigarette smoke extract [CSE]) for 48 hours. Exosomes isolated by differential ultra-centrifugation were characterized by transmission electron microscopy (particle size and morphology), western blot (exosome markers and contents, specifically damage associated molecular pattern markers after OS exposure (DAMPs – HMGB1 and cell free fetal telomere fragments), and confocal microscopy (contents within exosomes). Myometrial cells were treated with exosomes from AEC grown under standard and OS conditions were stained with 7.5 uM carboxyfluorescein succinimidyl ester to monitor myocyte exosome uptake. COX-2, Connexin-43 expressions and NF-kB activation/RelA phosphorylation were determined in myocytes treated with exosomes from AECs cultured under standard and OS induced conditions. Student’s t-test evaluated significant changes (p<0.05). RESULTS: Primary AECs, under standard and OS conditions secrete exosomes with an average size of 50 nm that express established exosome markers (CD9, CD63, CD81 HSP70, and Alix) along with the amnion stem cell marker, Nanog. Evidence of OS in AECs was manifested by colocalization of DAMPs (HMGB1 and cell free telomere fragments) in exosomes. Exosomes from OS induced AECs increased myocyte COX-2 and Connexin 43 expression and RelA phosphorylation (all p<0.05) in myocytes compared to exosomes from standard AEC cultures. CONCLUSION: AEC derived exosomes contain bioactive contents with characteristics of DAMPs that can activate myocytes and potentially induce uterine contractility. We conclude that OS-mediated AEC damage can initiate labor through exosomal cargo transport of labor signals.

A Role for Expanded CGG Repeats In Fragile X-Associated Ovarian Insufficiency Shelly K, Nelson D. Fragile X-associated Primary Ovarian insufficiency (FXPOI) is the most common known genetic cause of premature ovarian failure, affecting roughly 20% of women heterozygous for an FMR1 premutation allele. Characteristics of this disorder in humans include alterations in hormone levels, increased follicular atresia, and premature cessation of menses. A subset of these features was reported in previous investigations of Fmr1 premutation mouse models, coupled with aberrations in granulosa cell populations and the presence of intranuclear inclusion within ovarian stroma. These available premutation models cannot distinguish the effects of alterations of Fmr1 transcript levels from the effects of expanded CGG RNA. We hypothesize that ovarian dysfunction in these models and in FXPOI patients results predominantly from the expression of premutation-sized CGG repeat tract RNA (rCGGs) that sequester rCGG-binding proteins within the ovary. To determine whether ectopically expressed premutation-sized rCGG repeats are sufficient to confer phenotypes associated with FXPOI, we designed a construct to enable tissue-specific expression of 87 CGG repeats fused to either FMR1 or fluorescent ZsGreen cDNA. To assess the effect of expanded repeats in vitro these constructs have been transfected into HEK293T and KGN (granulosa) cell lines. The constructs will also be used to generate mouse models expressing FMR1 or ZsGreen from the Rosa26 locus. We predict that expression of expanded CGG repeats in vivo will recapitulate phenotypes previously associated with FXPOI. Ultimately, these mouse models will enable us to determine whether ectopically expressed CGG repeat tracts are sufficient to confer features of FXPOI, and will potentially allow us to assess the contributions of specific tissues to these phenotypes.

Effects of obesity induced by a diet high in plant-derived saturated fats on reproductive and immune parameters in female Ossabaw mini-pigs

Cassandra Skenandore1, Katherine Lenz2, Romana Nowak3, Bryan White3,4, Andrea

Braundmeier-Fleming2, Annie E. Newell-Fugate1

1Dept. of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, TX 2Dept. of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois School of Medicine, Springfield, IL 3 Dept. of Animal Sciences, University of Illinois, Urbana-Champaign, IL 4Institute of Genomic Biology, University of Illinois, Urbana-Champaign, IL

Diets high in saturated fats lead to obesity and have been linked with compromised fertility in

females. This study examined the effects of obesity induced by a diet high in plant derived

saturated fats on reproductive and immune parameters in female Ossabaw mini-pigs. Five

nulliparous, sexually mature female Ossabaw mini-pigs were fed a diet of 17.4% hydrogenated

soybean oil, 4.7% coconut oil, and 22.6% fructose (obese; n = 3) or a control diet consisting of

57.5% cornmeal and 40% soybean meal (lean; n = 2) for nine estrous cycles. The diet-induction

period was six estrous cycles in duration (~ 3 months). During the diet-induction period, pigs

had ovarian ultrasound, blood, and vaginal swabs collected on d 1 and d 8 of each cycle. After

diet induction, pigs remained on the same diets and went through intensive sampling for cycles

7 and 9. During intensive sampling, ultrasound, blood, and vaginal samples were collected on d

1 (day of estrus), 4, 8, 12, 16, 18, 20, and every other day until the pig entered estrus again.

Heat detection was conducted in the presence of a boar and was used to measure estrous

cycle length. Weights and morphometric measurements were taken weekly. Transrectal

ultrasound was used to identify follicles and corpus lutea on the ovaries. Blood was collected to

measure circulating concentrations of glucose, estradiol, and cytokines. All estrous cycle and

cytokine data was assessed for normality with PROC UNIVARIATE (SAS, Inc, Cary, NC) and

logarithmically (continuous variables) or square root (follicle counts) transformed as necessary

prior to being analyzed using repeated measures ANOVA. Microbiome data was assessed by

principal component analysis. Compared to lean pigs, obese pigs had increased body weight

and morphometric measurements (p<0.02). There was no difference between the lean and

obese pigs for any reproductive parameter. There was also no difference in glucose

concentrations between lean and obese pigs (lean: 68.07 ± 4.45 mg/d; obese: 69.85 ±

3.73mg/dL; p=0.96). However, obese pigs had decreased concentrations of cytokines known to

drive inflammation (IL-1β (p<0.05) and TNF-α (p=0.2)) and, subsequently, decreased anti-

inflammatory cytokines (IL-4, IL-10 (p<0.05)). Obese pigs also had an increased diversity of

vaginal bacteria when compared to lean pigs. The fact that obese pigs fed the above diet have

decreased inflammation is intriguing, since obesity alone is known to drive inflammation. One

key difference between the high fat and control diet was the inclusion of coconut oil in the high

fat diet. We postulate that there is a potential role for coconut oil in the modulation of the

immune system, possibly through regulation of microbial community dynamics. Obesity has

been implicated as a major cause of type 2 diabetes and infertility. However, our results indicate

that obesity alone may not be responsible for these pathologies. Instead, the fat source itself

may modulate glucose homeostasis and reproductive function, even in the context of obesity.

SUPPRESSION OF ASH2L ALTERS DNA METHYLATION AND HISTONE PATTERNS

DURING BOVINE EMBRYONIC DEVELOPMENT

M. D. Snyder*, J. H. Pryor, M. D. Peoples, G. L. Williamson, W. M. Skiles M. C. Golding, M.

E. Westhusin, C. R. Long

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and

Biomedical Sciences, Texas A&M University, College Station, TX, USA

Epigenetic patterns established during early bovine embryogenesis via DNA methylation and histone

modification patterns are essential for proper gene expression and embryonic development. We have

previously discovered that suppression of absent, small, or homeotic-like (ASH2L) with small interfering

RNA (siRNA) had no significant effect during in vitro embryo development when compared with its

respective control (31.3 ± 2.0% standard error of the mean, n = 466 v. 34.8 ± 1.9%, n = 418). Analyzing

DNA methylation and histone modifications via immunocytochemistry will further explain the role

of ASH2L during embryonic development, specifically at the blastocyst stage. In this experiment, we

obtained mature bovine oocytes from a commercial supplier (De Soto Biosciences, Seymour, TN) and

preformed IVF following standard laboratory protocol. Eighteen hours after IVF, presumptive zygotes

were divided into 3 treatments: noninjected controls, nontargeting siRNA injected controls (siNULL), and

injection with siRNA targetingASH2L (siASH2L). Each embryo was injected with ~100 pL of 20 nM

siRNA previously verified to suppress expression of ASH2L by ~79%. Embryos were cultured in Bovine

Evolve (Zenith Biotech, Guilford, CT) supplemented with 4 mg mL–1

of BSA (Probumin, Millipore) for 7

days. Blastocysts from each treatment (N = 601) were fixed and prepared for immunocytochemistry

following standard laboratory protocol. The following primary antibodies were used to target specific

DNA and histone methylation marks: 5mc mAb (Epigentek, Farmingdale, NY), 5hmc pAb, H3K4me3

pAb (Active Motif, Carlsbad, CA), H3K4me2 pAb, H3K9me2–3 mAb, and H3K27me3 mAb (Abcam,

Cambridge, MA). Embryos were fluorescently labelled with the following secondary antibodies: Alexa

Flour 488 Goat Anti-Rabbit, Alexa 488 Donkey Anti-Goat, and Alexa Flour 594 Goat Anti-Mouse

(Invitrogen, Carlsbad, CA). The DNA was stained with Hoechst 33342 (Invitrogen). Fluorescent images

were captured using the Zeiss Stallion digital imaging work station. Ratio averages (targeting mark/DNA)

were calculated and statistical analysis performed using one-way ANOVA and Tukey’s honestly

significant difference to assess treatment effects. The ratio of DNA methylation to total DNA increased in

siASH2L as compared with control and siNULL embryos (0.35 ± 0.01, 0.26 ± 0.02, and 0.30 ± 0.01,

respectively; P < 0.01). The 5hmC was inversely related to 5mC levels and decreased in siASH2L

embryos (0.75 ± 0.01, 0.93 ± 0.02, 0.87 ± 0.02, respectively; P < 0.0001). The H3K4me3 and H3K27me3

are also inversely related with decreased H3K4me3 in siASH2L versus control and siNULL embryos

(0.48 ± 0.02, 0.57 ± 0.02, 0.58 ± 0.02, respectively; P< 0.001) and increased H3K27me3 (0.62 ± 0.02,

0.053 ± 0.01, 0.54 ± 0.02, respectively; P < 0.001). No differences were observed in H3K9me2–3 or

H3K4me2 labelling across treatments. These results indicate that ASH2L may play a role in DNA

methylation by decreasing 5mc and 5hmc conversion, which is a key event during early embryonic

development. Suppression of ASH2L also alters global levels of H3H4me3 and H3K27me3, which may

lead to transcription aberrations. Further analysis of siASH2L embryos via RNA-seq will help define its

role during early embryonic development.

Resveratrol Protects the Ovary Against Chromium-Toxicity by Enhancing Endogenous Antioxidant Enzymes and Inhibiting Metabolic Clearance of Estradiol. Stanley, JA1, Sivakumar, KK1, Arosh, JA1, Burghardt, RC1 and Banu, SK1. 1 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA. jstanley@cvm.tamu.edu Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) has not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in more than 50 industries. Environmental contamination with CrVI in the U.S. is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. The present study focused to determine the protective effects of RVT on CrVI-induced ovarian toxicity. Lactating mother rats were exposed to CrVI (50 ppm) from postpartum day 1-21 through drinking water with or without RVT (10 mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother’s milk. On postnatal day (PND) 25, blood and organs were collected from the F1 females for analyses. CrVI increased follicle atresia by increasing cytochrome C and caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E2) biosynthesis and enhancing metabolic clearance of E2, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by up regulating cell survival proteins and AOXs; and restored E2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E2. This is the first study to report the protective effects of RVT against any toxicant in the ovary.

Maternal BMI is Significantly Correlated with Endotoxin Core Igm Antibodies in Both Maternal and Neonatal Serum

Maike Kahr, Min Hu, Kjersti Aagaard, Melissa Suter

Department of Obstetrics & Gynecology, Baylor College of Medicine, Houston, TX

Obesity, diabetes and inflammation have long been associated, but the cause of the inflammation is unknown. One hypothesis is that bacterial products, such as lipopolysaccharides (LPS) are translocated across the gut in association with a high-fat diet which drives inflammation, a condition termed metabolic endotoxemia (ME). Data have shown that an increase in serum LPS and reduced levels of endotoxin-core IgM (endoCAb IgM) antibodies are associated with both obesity and diabetes. In this study we sought to determine if changes in serum LPS and endoCAb IgM are associated with maternal obesity and if the fetus is exposed to these markers of ME. We also tested whether markers of inflammation (TNF and IL-6) were increased with maternal obesity and if these markers were observed in the neonate. Commercially available ELISA assays were utilized to measure LPS and endoCAb IgM levels in matched maternal and neonatal (cord blood) serum samples (N=146). The Milliplex system was used to measure TNF and IL-6 through the DDC core. While we did not find any significant change in LPS levels in either maternal or neonatal samples, we found that maternal endoCAb IgM is significantly decreased in obese women compared to normal weight. When stratifying the data based on prepregnancy BMI into the categories of normal weight, overweight and obese we find that both maternal and neonatal endoCAb levels negatively correlate with BMI category. There is also a significant increase in TNF in maternal serum in obese women. We also found that maternal and neonatal TNF and IL-6 are positively correlated. While we do not find significant changes in LPS levels in the mother or neonate, the finding of altered endotoxin IgM levels is of interest. Because IgM antibodies do not cross the placenta, the endoCAb IgM detected in neonatal serum is likely due to the fetal immune response to circulating LPS levels. Correlation of maternal and neonatal TNF and IL-6 may similarly point to fetal exposure to markers of maternal ME during gestation.

STEROID RECEPTOR COACTIVATOR-2 IS A CRITICAL REGULATOR OF ENDOMETRIAL CANCER CELL METABOLISM Maria M Szwarc1, Ramakrishna Kommagani1, Nagireddy Putluri1, Cristian Coarfa1, Rainer B Lanz1, Francesco J DeMayo2, John P Lydon1, and Bert W O’Malley1 1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston TX 2Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC Background: Steroid receptor coactivator-2 (SRC-2) is a member of the p160/SRC family of coregulators, which also includes SRC-1 and SRC-3. SRCs exert a wide-spectrum of physiological processes ranging from mammary morphogenesis, metabolic homeostasis, to endometrial function. Importantly, deregulation of SRC expression levels is a causal factor for numerous pathologies. In the case of the endometrium, clinical studies reveal that SRC-2 and -3 levels are elevated in the endometrium of patients diagnosed with polycystic ovary syndrome. The endometrium of these patients displays severe defects in functionality, including increased endometrial cancer risk. Elevated expression of these two coregulators has also been found in hyperplastic and neoplastic endometrium. These clinical findings suggest a causal link between SRC function and the emergence of endometrial disorders. This is further supported by our previous findings showing that increased expression of SRC-2 in the endometrium of a transgenic mouse model leads endometrial hyperproliferation. This study focuses on determining the molecular mechanism by which SRC-2 promotes endometrial hyperplasia and cancer progression. Materials and Methods: In vitro proliferation, colony formation, and soft-agar assays and measurements of glycolytic and mitochondrial capacity were performed following siRNA-mediated SRC-2 knockdown in endometrial cancer cells. Under the same conditions, cellular metabolite levels were measured with mass-spectrometric, luminometric, and colorimetric methods. A microarray followed by quantitative PCR has been utilized to investigate regulation of genes by SRC-2 in endometrial cancer cells. These studies were complemented with in vivo studies in a xenograft model in which SRC-2 expression was modulated by shRNA delivered by lentivirus. Results: Knockdown of SRC-2, but not SRC-1 and -3, lead to a downregulation of endometrial cancer cell proliferation. Decreased levels of SRC-2 also caused reduced anchorage independence of cells. Importantly, SRC-2 is required to maintain glycolytic capacity and oxygen consumption of these cells indicating SRC-2’s role in regulation of endometrial cancer cell metabolism. Measurements of levels of key glucose metabolites revealed that SRC-2 is critical for the maintenance of phosphopentose and nucleotide levels generated by the pentose phosphate pathway. These metabolites are crucial for cellular proliferation as they are part of the biomass needed for dividing cells. This is accompanied by a reduction of one of key enzymes for phosphopentose production, ribose-5-P isomerase (RPIA), which may be a direct target of SRC-2 at the transcriptional level. Importantly, siRNA mediated downregulation of RPIA, similarly as in the case of SRC-2, leads to significant reduction of cell growth and both the glycolytic flux and oxygen consumption. Additionally, decrease of SRC-2 levels also attenuates tumor expansion in vivo. Together these findings indicate that SRC-2 is regulator of cell metabolism critical for endometrial cancer cell growth.

Cross-talk Between the Estrogen and Relaxin Signaling Pathways in the Myometrium Sudeshna Tripathy, Mala Mahendroo, and W. Lee Kraus Cecil H. and Ida Green Center for Reproductive Biology Sciences and Department of Obstetrics and Gynecology, UT Southwestern Medical Center, Dallas, TX 75390. Parturition requires coordinated remodeling of the cervix and contractions of the myometrium. These processes are controlled by hormones, such as relaxin (Rln) and estradiol (E2), which initiate gene regulation programs that prepare the tissues and initiate the physiological processes that lead to delivery. Rln, a polypeptide hormone secreted by the corpora lutea during the latter half of pregnancy, stimulates the growth and extensibility of the lower reproductive tract and facilitates parturition. E2, a steroid hormone produced by the ovaries in increasing amounts near term, promotes myometrial contractility and other parturition-related processes. How E2 and Rln work together to promote a common set of physiological outcomes is unknown. Previous studies have shown that the nuclear actions of estrogen receptors (ERs), such as ERα, can be modulated by cross-talk with polypeptide hormone signaling pathways (e.g., TNFα, IGF-1, EGF, TGFβ, etc.). This cross-talk can occur at the genome by creating an altered ERα cistrome (i.e., repertoire of ERα binding sites), which leads to a new transcriptome (i.e., regulated gene set) and altered physiological outcomes. To explore the potential cross-talk between the E2 and Rln signaling pathways in relevant tissues under physiological conditions, we performed time course and dose-response experiments on myometrial and cervical tissue from ovariectomized mice treated with vehicle, E2, Rln, and E2+Rln. Gene expression analyses using RT-qPCR demonstrated an antagonistic effect of Rln on the E2-dependent regulation of a set of classical estrogen target genes (e.g., Pgr, Esr1, Nrip1). To get a broader view of the transcriptome changes, we are subjecting total RNA from the samples to RNA-seq. In addition, we are using molecular approaches to examine how Rln affects E2-dependent ERα enhancer function. Ultimately, our goal is to use this information to understand the biological outcomes of such cross-talk during parturition.

Pancreatic Vasculature is Reduced in a Non-Obese Mouse Dietary Model of Gestational Diabetes

Nicola van der Walt and Kathleen A Pennington

Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston TX 77030

Gestational diabetes mellitus (GDM) is one of the most common obstetrical complications, affecting 7-18% of all pregnancies. GDM is defined as diabetes that is diagnosed during pregnancy and usually resolves after parturition. GDM has immediate and long term effects on the health of both mother and fetus. In a normal pregnancy beta-cell mass expands to cope with the increased demand for insulin but this process in deficient in GDM. Vascular remodeling of the pancreas is required prior to beta-cell expansion and insufficient remodeling contributes to the development of type II diabetes. However, this process has not been studied in the context of GDM. Despite the seriousness of GDM and its high prevalence, there is no widely accepted animal model. We therefore aimed to develop a mouse dietary model of GDM and determine its effect on pancreatic vasculature. C57Bl/6 mice were placed on either a 10% kcal/fat, 0% kcal/sucrose control diet (CD) or a 45% kcal/fat, 17% kcal/sucrose high fat diet (HFD) 1 week prior to and throughout pregnancy. A glucose tolerance test was performed on mice after 1 week on the diet, prior to mating (d0) and at d13.5 and d17.5 of pregnancy. Mice were either sacrificed at d13.5 or d17.5 and the pancreas was collected for immunohistochemical analysis. Antibodies against glucagon, insulin and CD31 were used to analyze alpha- and beta-cell number and islet vascularization respectively. Mice on the HFD did not differ in weight from those on the CD at d0 (18.6 ± 0.052 g vs. 18.2 ± 0.23 g), d13.5 (27.3 ± 0.44 g vs. 27.1 ± 0.83 g) or d17.5 (31.13 ± 0.78 g vs. 31.26 ± 0.93 g). Glucose tolerance was not different in mice on the HFD or CD prior to conception (AUC d0 HFD 23052 ± 700 vs. AUC d0 CD 20671 ± 1134). However, during pregnancy glucose tolerance was significantly impaired in mice on the HFD (AUC d13.5 HFD 26331 ± 963 vs. AUC d13.5 CD 16089 ± 1190; AUC d17.5 HFD 23921 ± 2902 vs. AUC d17.5 CD 12474 ± 721). Litter sizes, fetal weights and placental weights were not different at d13.5 or d17.5 of pregnancy. At d17.5, there was a reduction in the number of alpha cells in mice on the HFD, although it was not significant (p = 0.054). The number of beta-cells was reduced significantly (p < 0.05) in HFD-fed mice. A significant reduction in the number of endothelial cells and the ratio of endothelial/beta-cells was also significantly reduced at d17.5 (p < 0.02) in HFD-fed mice. Pancreatic vasculature at d13.5 is currently being evaluated along with pancreatic gene expression for key vascularization markers. In conclusion, pancreatic vasculature was reduced in this non-obese mouse dietary model of GDM. Future studies will use this model to further elucidate the remodeling of pancreatic vasculature in a pregnancy complicated by GDM and its effect on long term maternal health.

Molecular Mechanisms of Estrogen Receptor alpha (ERα) Enhancer Formation: The Roles of DNA sequence and ERα Acetylation by p300 in Uterine Biology Yasmin M. Vasquez, Ying J. Long, Kirk Tran, Shino Murakami, Rachel Ramirez, Tulip Nandu, Anusha Nagari, Rosemary N. Plagens, Christina K. Matulis, and W. Lee Kraus The Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA Estrogen receptor alpha (ERα) is a ligand-regulated, DNA-binding transcription factor that plays a critical role in the physiology of reproductive and non-reproductive organs. Liganded ERα binds to many sites across the genome, many of which contain estrogen response elements (EREs) and promotes enhancer formation by recruiting transcriptional coregulators, including the protein acetyltransferase p300, a key regulator of enhancer activity. In this study we are examining molecular determinants that may regulate ERα enhancer formation. We are focusing on the sequences of the ERE and post translational modifications of ERα, such as p300-mediated acetylation. We utilize inbred mouse strains, C57BL6/J and 129S1/SvmlJ, with well-defined genealogy and single nucleotide polymorphisms (SNPs) to explore the role of DNA sequence in controlling ERα enhancer function. We have identified approximately 200 strain-specific ERα binding sites in the uterus that contain a SNP in the ERE with quantifiable differences in ERα binding between strains by ChIP-seq. We are analyzing how the DNA sequence in the ERE, as well as the flanking regions, affect 1) ERα binding to the chromatin and 2) assembly and functions of the enhancer. In parallel to this line of investigation, we previously showed that p300 acetylates hERα at lysines 266 and 268, resulting in enhanced ERα binding to DNA in vitro. We have now generated a gain-of-function mouse line in which a gene coding for an acetylation mimic (lysine to glutamine substitution) mERα K270/272Q (homologous to K266/K268Q in hERα) is knocked-in at the endogenous ESR1 gene locus (generating the ERαAcKI mice). The female KI mice are grossly normal, and are fertile with normal reproductive cycles. Using the immature (estrogen naïve) uterus as a model estrogen-responsive tissues, we found using RNA-seq that the repertoire of estrogen-regulated genes in the wild-type and ERαAcKI mice largely overlap, but the estrogen-upregulated genes are turned on with much faster kinetics in the ERαAcKI mice than the wild-type mice. Interestingly, pregnant ERαAcKI mice show an enhanced thrombogenic state in the placenta in a model of placental abruption in response to low-dose estrogen administrated at mid-gestation, leading to the elevated fetal lethality compared to the wild-type mice. We are currently investigating the molecular mechanisms of enhanced E2-responsiveness in ERαAcKI mice, including binding to DNA and coregulators. Collectively, our results suggest that strain-specific polymorphisms and the ERαAcKI are useful models to study the molecular mechanisms regulating ERα enhancer assembly and function in vivo. This work is supported by a grant from the NIH/NIDDK to W.L.K.

Mechanism of Chromium-VI toxicity on the cell death pathways of rat placenta. Wu JZ, Stanley JA, Taylor RJ, Arosh JA, Burghardt RC and Banu SK. Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA Hexavalent chromium (CrVI), one of the most toxic heavy metals, is widely used in more than 50 industries. As one of the world's leading producers of Cr compounds, the U.S. is facing growing challenges in protecting human health against multiple adverse effects of CrVI. Placenta plays a very significant role for the nutrition of the embryo and development of secretory and regulatory functions for the maintenance of pregnancy. It also acts as a filter reducing the passage of harmful substances, protecting the fetus from exposure to endocrine disrupting chemicals (EDCs). However, several EDCs including heavy metals were detected in the placental tissues, amniotic fluid and umbilical cord blood. However, not much studies were carried out to determine the toxic effects of CrVI on the placental structure and function. The current study is focused to identify the molecular mechanism behind the effects of CrVI on the placental survival and/or cell death pathways. Pregnant rats were given 50 ppm CrVI (potassium dichromate) from gestational day (GD) 9.5 to 14.5 through drinking water, placentae were removed on GD18, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased Cr accumulation in the placenta; 2) increased apoptosis in both maternal and fetal compartments by increasing BAX, p53, PUMA and NOXA; and decreasing BCL2; 3) Increased cleaved caspase-3 in maternal compartment but not in the fetal compartment; 4) increased AIF and p27 and decreased XIAP in both the compartments; 5) Interestingly, Beclin-1, a major regulator of autophagy, was not detected either in the maternal or in the fetal compartment. Taken together, our results indicate for the first time that gestational CrVI exposure directly targets the placental cell survival and increases the cell death of the placenta through selective activation of p53-dependent and caspase-3 dependent as well as caspase-3-independent cell death pathways in a spatio-temporal pattern.

Epigenetic Changes in MSH5 Lead to Male Infertility

Boryana Zhelyazkova

Molecular & Cellular Biology Ph.D. Program, Baylor College of Medicine

Advisor: Dolores Lamb, Ph.D.

Infertility affects about 15% of couples of which 30-50% are due to male factors, however there is a severe lack of understanding of the factors involved in this condition. A subset of patients, classified as men with non-obstructive azoospermia (NOA), are unable to produce sperm due to spermatogenic failure at the testicular level. We discovered that defects in the DNA mismatch repair pathway contributes to their condition, thus identifying a genetic defect that can affect both the health of the individual, but also his potential progeny.

Of particular interest is the mismatch repair gene, MSH5 (mutS homolog 5), which is hypermethylated in a cohort of NOA men in its gene body. This hypermethylation leads to a reduction of MSH5 expression levels and contributes to failure of the DNA machinery to recover from DNA damage in human fibroblast cells from NOA patients. Deletion of Msh5 in mouse models results in meiotic failure because double-strand breaks are not repaired, thus resulting in spermatogenic failure as well. Because of this finding further analysis of this aberrant MSH5 hypermethylation is warranted and an in vivo model is needed to confirm the importance of this gene in male fertility. We hypothesize that hypermethylation of Msh5 deleteriously affects its expression in vivo and leads to male infertility.

The CRISPR-Cas9 system is a useful tool for genetic manipulation due to its intrinsic nuclease activity and its use of short RNA guides that target it to a specific loci. Mutations D10A and H840A lead to abolished cleaving capability, but still allow the protein to bind to DNA. This type of mutant is termed “dead” Cas9 (dCas9) and can target the complex to a specific loci in the DNA when fused to another protein. I propose to fuse a DNA methyl transferase to a dCas-9 protein and target it to Msh5 gene body. Efficiency of the system will be tested in mouse testicular fibroblast before proceeding to an in vivo model.