Cellular Determinants of Differentiation Versus Apoptosis in Avian Granulosa Cells

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This proposal describes studies of secreted and nonsecreted factors responsible for promoting avian ovarian granulosa cell viability, and facilitating differentiation at the time of ovarian follicle selection into the final stages of development (the preovulatory hierarchy). In most vertebrates, including avian species, the vast majority of growing ovarian follicles ( 90%) are lost via the death of follicles (follicle atresia) at some point during development prior to the preovulatory stage. It is now well established that ovarian follicle atresia in all vertebrate species studied to date is mediated via apoptosis, and that this process is initiated within the granulosa cell layer. The identification of mechanisms that promote the survival of granulosa cells and provide for sufficient numbers of ovarian follicles for optimal follicle development and thus, optimal fertility, remains an active area of investigation. Similarly, cellular processes that precisely control ovarian follicle selection and the establishment of an orderly preovulatory hierarchy in avian species are poorly understood. Throughout the reproductive life span of the avian female, ongoing attrition of developing follicles eventually results in the survival of a small cohort of follicles that are responsive to follicle stimulating hormone. Subsequently, a single follicle from this cohort is selected per day to rapidly grow and undergo final differentiation before ovulation, thus establishing an orderly hierarchy of preovulatory follicles. Significantly, follicle selection into the avian preovulatory hierarchy is reflected by the concomitant expression of anti-apoptotic factors and cell survival signaling pathways that render granulosa cells resistant to apoptosis, and accordingly, results in diminished susceptibility to follicle atresia. Thus, there is a clear relationship between the initiation of differentiation and acquisition of apoptosis resistance within the granulosa cell layer at the time of follicle selection. The present proposal describes experiments, using the ovary of the domestic hen (Gallus gallus) as a model system, which study several secreted and nonsecreted factors, plus a unique Inhibitor of Apoptosis Protein (IAP), which together are proposed to mediate a balance between granulosa cell apoptosis versus cell survival and differentiation. Utilizing a combination of cell culture and gene transfection, in vitro assays and molecular techniques, the principal investigator will approach the study of these processes by achieving the following objectives: 1) establish signaling pathways within the cell that are responsible for epidermal growth factor receptor- (EGF-R-) mediated effects on promoting granulosa cell proliferation and cell survival, while simultaneously inhibiting premature cell differentiation; 2) define the role of transforming growth factor b (TGFb) and the related family member, activin, in promoting granulosa cell differentiation at the time of follicle selection. Related studies will define mechanisms by which EGF-R signaling blocks TGFb/activin-induced differentiation; and 3) investigate the role of the multifunctional IAP family member, survivin, as a regulator of cell proliferation and inhibitor of apoptosis in prehierarchal follicle granulosa cells. An important outcome of these studies will be a better understanding of causative factors and associated cellular events that promote granulosa cell resistance to apoptotic cell death and facilitate granulosa cell differentiation. More broadly, the data generated will contribute to our understanding of mechanisms responsible for the establishment of an ovarian preovulatory follicle hierarchy that is prerequisite for egg laying in a variety of egg-laying (oviparous) species, including reptiles. All studies described will incorporate the training of both undergraduate and graduate students. It is expected that during the course of investigations novel avian genes will be identified and functionally characterized, thus contributing to the further characterization of the avian genome. Accordingly, all results will be promptly submitted to appropriate, publicly accessible databases (e.g., GenBank) and subsequently published in first-tier journals.