Although depletion of the ovarian follicle pool plays a critical role in reproductive aging in the human female, the events underlying follicular atresia remain to be elucidated. It is well-established that apoptosis, a form of physiological cell death, is important for remodeling of tissues and removal of transient cell populations. Moreover, the penultimate controlling events which initiate or suppress physiological cell death may involve a conserved cohort of """"""""cell death"""""""" proteins, and the fate of cells may be decided by the ratio of death repressors (Bcl-2, Bcl-x/long) to death inducers (Bax, Bcl-x/short). The mechanisms of action of these proteins have not been fully clarified. However, there is evidence that Bcl-2 functions as an antioxidant to protect cells from damage by reactive oxygen species such as superoxide anion, peroxide, and hydroxyl radical. It is possible that these death repressor proteins (Bcl-2, Bcl-x/long) are components of a family of oxidative stress response proteins which also includes superoxide dismutase (SOD; an enzyme which catalyzes conversion of superoxide anion to hydrogen peroxide) and glutathione peroxidase (GSHPx; an enzyme which converts hydrogen peroxide to water). As recent studies conducted by the PI have revealed that granulosa cell death during atresia in all species studied thus far, including humans, is mediated via apoptosis, it is hypothesized that follicular atresia is initiated by a shift in the ratio of death repressor to death inducer gene expression. The altered expression of these cell death genes would then lead to insufficient protection of granulosa cells from the damaging effects of reactive oxygen species. To test this hypothesis, the Specific Aims of this proposal are to: 1) characterize changes in the levels of mRNA encoding bcl-2, bax, bcl-x (long and short), SOD and GSHPx during maturation of rat ovarian follicles from the primordial to antral stage of development; 2) evaluate if gonadotropin-mediated inhibition of apoptosis in granulosa cells of rat antral follicles in vivo and in vitro is associated with enhanced bcl-2, bcl-x/long, SOD and GSHPx, and suppressed bax and bcl-x/short, gene expression; 3) determine if selective destruction of the bcl-2, bcl- x/long, SOD and GSHPx genes by the apoptotic endonuclease contributes to a loss of death repressor gene expression during granulosa cell apoptosis and atresia; 4) investigate if inhibitors of oxidative free radical damage prevent apoptosis in granulosa cells of rat antral follicles incubated in vitro without trophic hormone support; and, 5) determine if inducers of oxidative free radical damage interfere with the ability of gonadotropins to inhibit apoptosis in granulosa cells of rat antral follicles incubated in vitro.
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