The long-term objective of the present proposal is to understand how progestins regulate the female reproductive axis, with particular emphasis on progesterone receptor (PR) gene regulation in ovarian granulosa cells. Progestins have been proposed to affect many aspects of ovarian function and to participate in the normal process of ovulation and/or luteinization of the follicle. We hypothesized that an intracellular PR molecule may mediate progesterone action in the ovary and therefore, isolated the rat PR cDNA and genomic clones to initiate a multifaceted approach to study the expression and regulation of ovarian PRs. Our preliminary results demonstrating transient PR gene expression in the granulosa cells of preovulatory follicles during the periovulatory period strongly suggest the potential involvement of PRs for mammalian ovulation. The regulatory mechanisms for PR gene expression in these cells appear to be quite different from other progesterone-responsive cells, because gonadotropins, but not estrogen, directly induce PR gene expression in rat granulosa cells. This gonadotropin-induced PR gene expression in ovarian granulosa cells is likely to be mediated by a cAMP- mediated pathway, at least in part, at the level of transcription. Our preliminary results also suggest the likely involvement of protein kinase C in PR gene regulation in ovarian granulosa cells. The central hypothesis in this proposal, then, is that the PR gene in ovarian granulosa cells is regulated predominantly by G protein-coupled signaling pathways, for example cAMP- and DAG-dependent pathways. We now propose to extend our preliminary studies to investigate the cellular and molecular mechanisms by which the PR gene is regulated in rat ovarian granulosa cells. The first goal of this proposal is to examine intracellular signaling pathways which are directly involved in PR gene expression in ovarian granulosa cells; - our studies will focus on protein kinases A and C. The second goal of this proposal is to identify and characterize the cis-DNA regulatory elements mediating the activation of the PR gene by these intracellular signaling pathways; our studies will focus on cAMP- or TPA-induced expression of the PR gene. The final goal of this proposal is to examine the potential role of estrogen in PR gene expression in rat granulosa cells; we will determine the capacity of ovarian granulosa cells in mediating estrogen-induced signals. We expect these experiments to enhance our understanding of PR regulation and function for the process of ovulation and/or luteinization in the mammalian ovary and to provide a framework for determining whether alterations in PR function might participate in reproductive diseases or dysfunction.
