Recent evidence in primates demonstrates that proliferation of granulosa cells is arrested within the first quarter of the periovulatory interval. However, specific markers of cell cycle progression, e.g., cyclin D2, are increased while levels of cell cycle suppressors such as p53 are reduced in concurrence with cell cycle arrest. This application will test the hypothesis that an ovulatory stimulus induces a transient burst of granulosa cell proliferation that is an essential feature of terminal differentiation and luteal formation in the primate. Experiments are planned to examine cell cycle characteristics of granulosa cells at early time points following a luteinizing dose of gonadotropin to granulosa cells in vitro and after an ovulatory stimulus to adult, female rhesus monkeys in vivo (aim 1). The functional consequences of the proliferative burst on terminal differentiation and luteinization of granulosa cells will be determined using in vitro protocols (aim 2). This application will further test the hypothesis that estrogen mediates the proliferative burst via regulation of key cell cycle components, and that early periovulatory estrogen action is essential to the formation of a functional corpus luteum (aim 3). The proposed studies will utilize an in vivo controlled ovarian stimulation model in which the differential effects of gonadotropins and steroids can be determined. Granulosa cells will be obtained from rhesus monkeys undergoing controlled ovarian stimulation prior to an ovulatory stimulus for in vitro experimentation. Levels of mRNA will be determined with real time RT-PCR, and protein levels and activity by western blot, gel shift, and kinase assays. Concentrations of steroid hormones will be measured by RIA. The proposed studies are expected to demonstrate that primate granulosa cells undergo a proliferative burst in response to the ovulatory stimulus that is an essential component of luteinization. It is expected that estrogen plays a heretofore unexpected role in driving the proliferative burst through the regulation of key cell cycle components. These studies will provide insight into the etiologies of ovarian cancer and certain kinds of infertility, as well as provide potential novel avenues for contraceptive agents.
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