Role of GATA4, GATA6, and SP1 on Luteal Function and LH Receptor Expression

Infertility is defined by the inability to achieve pregnancy after 1 year of trying to conceive. According to the most recent report from the Center for Disease Control approximately 10% of woman ages 15-44 have difficulties getting pregnant or staying pregnant accounting for around 6.1 million women in the United States. An aption for couples seeking infertility treatment is the use of assisted reproductive technologies (ART) of which in vitro fertilization (IVF) accounts for over 99% of the ART procedures. The demand for IVF is increasing every year. In fact, the number of ART cycles has risen by 32% between the years 2006 and 2015. Primarily based on patients age, weight, and ovarian reserve, standardized IVF protocols are selected to maximize the likelihood to achieve pregnancy. Most protocols involved the administration of follicle-stimulating hormone (FSH) to induce the maturation of several ovarian follicles, which it is followed by human chorionic gonadotropin (hCG), an analog of LH, to trigger ovulation and the subsequent collection of eggs for in vitro fertilization. Although fertilization may be successful, inherent obstacles remain with achieving embryo transfer and maintaining pregnancy. Therefore, luteal phase support, in the form of progesterone or hCG supplementation, is required to prime the uterus for implantation and to maintain embryogenesis until the placenta become the main source of progesterone. However, luteal phase treatments increase the risk of a potentially life-threatening condition called ovarian hyperstimulation syndrome (OHSS), which is marked by ovarian vasodilation in response to hCG treatment. Chapters three and four of my thesis explored mechanisms involved with luteal cell function. Within chapter five, I have investigated the interaction between the oocyte and cumulus granulosa in primary human cumulus cells and focuses on the regulation of anti- Anti-Müllerian hormone (AMH), which is an important marker for ovarian reserve and OHSS risk. First, we focused our attention on the role of GATA factors in luteal cell function. Previous studies in our lab showed that GATA4 and GATA6 are essential for granulosa cell steroidogenesis, preovulatory follicle formation, and fertility. Therefore, the lack of preovulatory follicle formation in the granulosa cell GATA knockout mice precluded the investigation of the role of GATA in the corpus luteum. To overcome this problem, we used a Cre-lox mouse model in which protein-coding exons of GATA4 and GATA6 were deleted with progesterone receptor (PR) driven Cre Recombinase expression. PR expression only occurs in the ovary at the time of ovulation. Therefore, we were able to investigate the effect of the knockdown of GATA4 and GATA6 as granulosa cells luteinize and the corpus luteum is formed. We observed that the deletion of either GATA4 or GATA6 alone results in subfertility while the elimination of both GATA factors renders female mice infertile. This experimental approach also revealed that although estrous cyclicity and ovulation are normal, serum progesterone levels were significantly decreased, which seems to be the leading cause of infertility. We then showed a corresponding decrease in the expression of steroidogenic enzymes involved in the synthesis of progesterone. To support our findings, double knockout mice were mated with males of proven fertility and administered subcutaneous progesterone injections to rescue pregnancy. In these mice, we observed embryo implantation in over 50% of the treated mice indicating that low progesterone is the primary cause of infertility. Gene expression analysis revealed that GATA4 and GATA6 play a direct role in the expression of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase/Δ5Δ4 isomerase (HSD3B), and inhibin beta b (INHBB). Finally, we demonstrated that the upregulation of LHCGR by GATA factors might play a crucial role in the maintenance of luteal cell function. Based on the novel effect of GATA on LHCGR, we shifted our focus to investigating the role of GATA on LHCGR expression in primary human luteinized granulosa cells obtained from the University of Illinois IVF clinic. We identified that although GATA deletion inhibits LHCGR expression in mice, the mouse, and human LHCGR promoters do not contain a canonical GATA response element but instead have highly conserved SP1 domains. The role of SP1 in primary human luteal cells has never been examined; therefore, we studied the role of SP1 in human LHCGR expression. We observed that treatment of human luteal cells with hCG increases LHCGR and steroidogenic gene expression. To examine the role of SP1 in the induction of LHCGR, the promoter of this gene was cloned into a reporter vector, and versions of this reporter lacking SP1 binding sites were produced. Primary human luteal cells were infected with these constructs and then treated with hCG. hCG significantly stimulated the activity of the reporter carrying the wild-type LHCGR promoter but was unable to stimulate the activity of constructs lacking SP1 elements. Additionally, treatment with Mithramycin A, a competitive inhibitor of SP1 binding, inhibits both hCG induced promoter activation and gene expression. These findings provide the first evidence of the role of SP1 on LHCGR regulation in primary human luteal cells. To complete my thesis, I investigated the mechanism regulating AMH in primary human cumulus cells. My work shows that only the combination of bone morphogenic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) significantly increases mRNA and protein expression of AMH in a concentration-dependent manner. Our results help to illuminate and support our understanding of the complex crosstalk between the oocyte and the surrounding granulosa cells. Also observed from our studies was that FSH inhibited the induction of AMH by GDF9 and BMP15. These results outline for the first time the mechanism involved in the physiological regulation of AMH in primary human granulosa cells.