The overall goal of this proposal is to test the hypothesis that progesterone regulates granulosa cell (GC) function by activating a membrane receptor that has some GABAA receptor features. We have recently shown that progesterone's anti-apoptotic and anti-mitotic actions are attenuated by the GABA antagonist, bicuculline. Further, progesterone's actions can be attenuated by an antibody directed against the ligand binding domain of the nuclear progesterone receptor. When used in a western blot analysis, this antibody recognizes a 60 kDa protein within ovarian and GC lysates. This 60 kDa protein is also detected within membrane preparations of ovarian lysates. Finally, the progesterone receptor antibody detects a 60 kDa protein after immunoprecipitation with an antibody against the GABAA receptor. These findings support our hypothesis that progesterone mediates its actions within the ovary through a membrane-type receptor that has a molecular weight of approximately 60 kDa and has some pharmacological and immunological characteristics of a GABAA-receptor. In this proposal we will directly test this hypothesis through a series of experiments which use 1) pharmacological agents to define progesterone's function, 2) competitive progesterone assays and scatchard plot analysis to assess the specificity of progesterone binding to GCs, 3) western blot and immunocytochemistry to detect the expression pattern of this putative 60 kDa membrane progesterone receptor during follicular growth, differentiation and atresia. It is proposed that progesterone acts through this putative 60 kDa membrane progesterone receptor to regulate both GC mitosis and viability within the preovulatory follicle. The concept is in marked contrast to the traditional view that progesterone's biological actions are almost exclusively restricted to non-ovarian tissues. Therefore, the proposed studies will provide new and important insights into the cellular mechanisms through which progesterone regulates GC function. Further characterization of this ovarian mechanism could ultimately lead to the development of antagonist and/or agonist which specifically modify progesterone's actions within the ovary without altering its genomic action within non-ovarian target cells. As such, these putative antagonists/agonist could find applications in the treatment of ovarian cancers, infertility and premature ovarian failure.
