Normal fertility in the female requires the development of one or more follicles to the pre-ovulatory stage, and formation and maintenance of a functional corpus luteum. During these transitions the follicle acquires the ability to synthesize estradiol and progesterone and transforms following ovulation into a predominantly progesterone-secreting structure. Understanding how multiple genes are turned on by the gonadotropins during ovarian cell differentiation will help us understand why some follicles are selected to ovulate and others undergo atresia. The ability to synthesize steroid hormones is a marker of differentiation in granulosa cells and is under the control of pituitary gonadotropins and locally produced ovarian factors, including FSH, LH, and Insulin-like Growth Factor I (IGF-I). IGF-I augments gonadotropin action at several levels of the steroidogenic pathway, including cholesterol uptake, transport, and utilization by the cytochrome P450 cholesterol-side chain cleavage complex in ovarian granulosa cells. Little is known about how gonadotropins and IGF-I act in concert to control the transcription of genes regulating steroidogenic capacity. This proposal will investigate how gonadotropins and IGF-1 interact to regulate two genes critical to steroidogenesis, namely the low density lipoprotein (LDL) receptor and Steroidogenic Acute Regulatory protein (StAR). (Years 1-2): The LDL receptor and StAR promoters will be mapped for putative transcription factors responding to gonadotropins and IGF-I. Deletional fragments of each gene promoter linked to a luciferase reporter gene will be transfected into FSH and LH responsive primary cultures of ovarian cells to identify hormone-responsive enhancer regions of each promoter and repressor regions. Dnase footprinting studies will also be performed to delineate responsive regions. (Years 2-4): Mutations of potential cis-acting elements will be made to determine the relative contributions of each site to hormone mediated transactivation of each gene promoter. (Years 2-5): Granulosa cell nuclear extracts from hormone-treated cells will be subjected to gel shift analyses with hormone responsive regions of each promoter. Trans-acting proteins necessary for transcription of these genes induced by gonadotropins and/or IGF-I will be identified. (Years 2-5: Parallel studies with dated ovarian follicle nuclear proteins will help identify which factors are regulated and expressed concurrently with StAR and LDL receptor mRNA in vivo. These studies will promote our understanding of how multiple genes are coordinated by different trophic factors in the ovary during follicular differentiation and add to our understanding of mechanisms of infertility.
