Insulin-like growth factor I (IGF-I) is an important hormone influencing steroidogenesis in granulosa cells of developing mammalian ovarian follicles. IGF-I affects ovarian steroidogenesis at multiple points in the steroidogenic pathway. One of the most important enzymes is cytochrome P450 cholesterol side-chain cleavage (P450scc), the rate-limiting enzyme in steroidogenesis. I have shown that IGF-I alone causes increased P450scc mRNA concentrations in swine granulosa cells. In contrast, rat granulosa cells only minimally increase P450scc mRNA concentrations when treated with IGF-I alone (bovine and human granulosa cells have not been tested). Moreover, both rat and human cell culture systems use granulosa cells exposed to high concentrations of exogenous hormones in vivo. Thus, the swine granulosa cell culture system affords a unique opportunity to study IGF-I actions singularly in immature granulosa cells without. the influence of exogenous hormones. This proposal will study the mechanisms by which IGF-I increases P450scc mRNA concentrations in swine granulosa cells. I isolated from a pig genomic library a 7-kb genomic DNA fragment, which bound specific DNA probes for the P450scc gene. The fragment was subcloned into pBS, underwent selective restriction enzyme digestion, and a 1-kb fragment was subcloned into pBS. Dideoxy DNA sequencing has identified the porcine P450scc promoter/regulatory region in this 1-kb DNA fragment, and additional sequencing of the fragment is in progress. An IGF-Imediated increase in transcription of P450scc mRNA will be assessed by nuclear run-on transcription assay. Concurrently, chimeric upstream P450scc/reporter gene constructs will be made for transient expression assay in cultures of swine granulosa cells basally and during treatment with IGF-I to identify IGF-1-responsive regions. Stability of mRNA will also be assessed in swine granulosa cells with pulse-chase experiments. The swine granulosa cell system enables the study of IGF-I actions in immature granulosa cells without the variability caused by exogenous hormone treatment. Results from these studies may directly lead to therapeutic approaches to pathophysiologic conditions of ovarian function in women.
