| Of the six GATA family members, only GATA4 and GATA6 are expressed in the adult ovary, specifically in granulosa, theca, and luteal cells. Although much work has been done on GATA4 and GATA6 to determine gene interactions through the overexpression of these transcription factors, their specific roles are still unknown in the ovary. Of interest, a number of genes important for ovarian function contain the GATA binding motif, WGATAR, including aromatase (Cyp19a1), cholesterol side chain cleavage enzyme (Cyp11a1), steroidogenic acute regulatory protein (StAR) and inhibin alpha (Inha). As GATA factors are highly expressed in the ovary and have been shown to regulate genes crucial for ovarian function, the aim of this project was to identify the specific roles of GATA factors in female fertility, specifically during folliculogenesis through corpus luteum formation and to provide insight into the gene targets of these factors.;Single GATA6 (G6gcko), GATA4 (G4gcko), and double GATA4/6 (G4/6gcko) granulosa cellspecific knockout mice were generated to investigate the role of GATA transcription factors in ovarian function in vivo. No reproductive defects were found in G6 gcko animals. G4gcko animals were subfertile as indicated by the reduced number of pups per litter and the release of significantly fewer oocytes at ovulation. In contrast, G4/6gcko females fail to ovulate and are infertile. Furthermore, G4/6gcko females had irregular estrous cycles, which correlate with the abnormal ovarian histology found in unstimulated adult G4/6gcko females showing lack of follicular development and increased follicular atresia. Moreover, treatment with exogenous gonadotropins did not rescue folliculogenesis or ovulation in G4/6gcko mice. In addition, ovary weight and estradiol levels were significantly reduced in G4gcko and G4/6gcko animals when compared with control and G6gcko mice. The expression of Cyp19a1, Cyp11a1, and luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) was significantly lower in G4gcko and G4/6gcko mice when compared with control animals. Most prominently, follicle stimulating hormone receptor (FSHR) protein was undetectable in granulosa cells of G4gcko and G4/6gcko. Accordingly, gel shift and reporter assays revealed that GATA4 binds and stimulates the activity of the FSHR promoter. These results demonstrate that GATA4 and GATA6 are needed for normal ovarian function. Our data are consistent with a role for GATA4 in the regulation of the FSHR gene and provide a possible molecular mechanism to explain the fertility defects observed in animals with deficient GATA expression in the ovary.;Knockdown of the transcription factors GATA4 and GATA6 in granulosa cells (GCs) impairs folliculogenesis and induces infertility. To investigate the pathways and genes regulated by these factors, we performed microarray analyses on wildtype (WT) GCs or GCs lacking GATA4, GATA6 or GATA4/6 (G4gcko , G6gcko and G4/6gcko) after in vivo treatment with equine chorionic gonadotropin (eCG). GATA4 deletion affected a greater number of genes than GATA6, which correlates with the subfertility observed in G4gcko mice and the normal reproductive function found in G6 gcko animals. An even greater number of genes were affected by the deletion of both factors. Moreover, the expression of FSHR, Lhcgr, inhibin alpha and beta, versican, pregnancy-associated plasma protein A, and the regulatory unit 2b of protein kinase A, which are known to be crucial for ovarian function, was greatly affected in double GATA4 and GATA6 knockouts when compared with single GATA deficient animals. This suggests that GATA4 and GATA6 functionally compensate for each other in the regulation of key ovarian genes. Functional enrichment revealed that ovulation, growth, intracellular signaling, extracellular structure organization, gonadotropin and growth factor actions, and steroidogenesis were significantly regulated in G4/6gcko mice. The results of this analysis were confirmed using qPCR, IHC, and biological assays. Treatment of GCs with cAMP/IGF1, to bypass FSH and IGF1 signaling defects, revealed that most of the affected genes are direct targets of GATA4/6. The diversity of pathways affected by the knockdown of GATA underscores the important role of these factors in the regulation of GC function.;Lack of follicle development in the GATA4/6gcko precluded studies to examine the role of GATA4 and GATA6 in luteal cells where GATA factors are also expressed. Therefore, it is not known if these factors are involved in the regulation of luteal function in vivo. Our last aim was to determine the effect of the knockdown of GATA4 and GATA6 at ovulation on corpus luteum function and fertility. To delete GATA4/6 in the corpus luteum, mice expressing Cre recombinase driven by the progesterone receptor (PR) promoter, which is highly upregulated in the granulosa cells of the preovulatory follicle, were crossed with mice containing single or combined floxed alleles for GATA4 and GATA6 (G4prko, G6 prko, G4/6prko). G4/6prko females produced no pups while both the G4prko and G6prko animals had a reduced number of pups per litter. G4/6prko animals cycled normally and had normal hormone-induced ovulation rates. However, plasma progesterone levels and the ovarian expression of Cyp11a1 and StAR, both essential for progesterone synthesis, were significantly low in G4/6prko mice treated with eCG/hCG (96 hs) when compared with control animals. Ovarian histology demonstrated that corpora lutea were present in the G4/6 prko animals treated with eCG/hCG (96 hs). Progesterone was administered to the double knockouts animals from day 1.5 to day 8.5 of pregnancy to determine if exogenous progesterone rescues pregnancy. This treatment rescued implantation in one out of two animals. In addition, structural abnormalities in the oviducts of G4/6prko mice suggest the presence of luminal epithelium hypertrophy and tubular occlusion. Additionally, oviductal and uterine genes were significantly altered in the double knockout. Although, additional studies are needed to examine the effects of the deletion of GATA4 and GATA6 in other PR expressing tissues, such as the oviduct and uterus, these findings provide new insights into the roles these transcription factors have in female reproduction and demonstrate that they are not only crucial for the progression of folliculogenesis but also necessary for luteal cell progesterone production. |
