It is now widely accepted that ovarian follicle atresia is initiated within the granulosa cell layer via the process of apoptosis. However, the identity of intracellular pathways leading to granulosa cell death remain largely unknown. Similarly, factors that render granulosa cells resistant to apoptosis, and thus permit follicles to proceed through the process of final differentiation, are as yet incompletely studied. Thus, knowledge of extracellular and intracellular mediators of cell viability in granulosa cells will lead to understanding processes related not only to follicle selection and atresia but also to pathologies of the ovary that originate from the lack of normal cell death (e.g., tumors). Experiments outlined herein are designed to further our knowledge related to: 1) intracellular mediators of extracellular death signals; and 2) intracellular checkpoints that provide cellular resistance against the progression of apoptosis within granulosa cells. The avian ovary provides a unique model system to study follicle atresia due to the ability to reliably identify a small population of follicles (eight to ten 6 to 8-mm diameter follicles) from which follicle selection into the preovulatory hierarchy occurs. It has been determined that hen follicle selection is highly correlated with the acquisition of resistance to apoptotic cell death within granulosa cells, and it is proposed that the acquisition of such resistance is a prerequisite for subsequent follicle recruitment and granulosa cell differentiation. A major advantage of this model system is the capacity to readily identify and directly compare two populations of granulosa cells which are apoptosis-susceptible (from prehierarchal, atresia- susceptible follicles) and apoptosis-resistant (from preovulatory follicles). Accordingly, four specific objects are outlined for study using this model system. Objective I: Evaluate mechanisms by which TNFalpha mediates apoptosis in apoptosis-susceptible granulosa cells; Objective II: Determine the relationship of a novel Inhibitor of Apoptosis Protein, Inhibitor of T-Cell Apoptosis (ITA), to apoptosis resistance in preovulatory follicle granulosa; Objective III: Test the ability of the death-suppressing protein, Bcl-xLong, to provide protection against apoptotic cell death; and Objective IV: Evaluate the ability of the death-inducing protein, Bax, to promote apoptotic cell death. Results will provide novel insights into the identity and function of intracellular mechanisms associated with granulosa cell, and thus follicle, viability. Due to the well-documented conservation of mechanisms mediating apoptosis among vertebrate species, such information is predicted to be directly applicable to mammalian granulosa cells.
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