The primary functional unit of the ovary is the follicle, a structure composed of a single germ cell (oocyte) surrounded by a somatic cell lineage, referred to as granulosa cells (GC), that is the source of estrogens produced by the ovaries. A finite number of primordial follicles are endowed in the ovaries at birth from which follicles are selected for growth and differentiation to release an egg at ovulation. Each menstrual cycle in adult life is accompanied by the growth of more follicles than are needed for ovulation, resulting in a selection process that eliminates many follicles by atresia. When the reserve of follicles nears exhaustion, steroid secretion from the ovary declines and then menopause ensues. Apoptotic cell death plays a central role in follicular atresia in all animal species, including humans. It is now known that apoptosis in both oocytes and GC is regulated by a genetic program conserved through evolution, with the pro-apoptotic protein, Bax, standing out as a principal player. In addition to correlating increased bax expression with apoptosis in rat and human GC, evaluations of ovaries from young adult female mice with a targeted disruption in the bax gene (Bax-null) have revealed the presence of numerous follicles that failed to complete atresia. Preliminary data show that young adult Bax-null, as compared with wild-type, female mice possess 3-fold more oocyte-containing primordial follicles, and that this surfeit of follicles extends ovarian lifespan into advanced chronological age. The mechanisms by which Bax is regulated and promotes apoptosis remain elusive. Preliminary data indicate that another pro-apoptotic protein, Bad, which generates more intracellular "free" Bax, also appears to be important for oocyte loss, and that the anti-apoptotic Bax-interacting protein, Bcl-w, is expressed in oocytes and is critical for normal germ cell survival. Data from cell culture models indicate that Bax and Bcl-w may control apoptosis by modulating release of cytochrome c from mitochondria, an essential co-factor needed for activation of a family of cytosolic "executioner" molecules referred to as caspases. Therefore, to test the hypothesis that follicle depletion is determined by a Bax-driven "rheostat" in ovarian cells responsible for controlling cytochrome c release and caspase activation needed for apoptosis, the following Specific Aims are proposed: 1) characterize ovarian follicular dynamics in Bax-null and Bad-null female mice throughout life to advanced chronological age; 2) examine if ovaries removed from aged Bax-null female mice reinitiate ovulation following transplantation to young adult female mice; 3) test oocyte competency in aged Bax-null female mice; 4) determine if Bax-null female reproductive phenotypes can be reversed by crossing these mice with Bcl-w-null mice; and, 5) evaluate if microinjection of recombinant Bax protein into wild-type oocytes induces mitochondrial cytochrome c release, caspase activation and apoptosis, and if this response is blunted or abolished in oocytes with targeted expression of Bcl-2 or targeted disruption of caspase-2 or -3. It is anticipated these data will provide the first unequivocal proof that exhaustion of the follicle reserve can be delayed, if not prevented, by manipulating apoptosis-regulatory genes.