Depletion of the human female germ cell pool through follicle atresia accounts for >99% of post-natal germ cell loss. Atresia occurs by apoptosis, a mode of physiological cell death which functions to remove unwanted, senescent or potentially harmful cells. Regardless of species or cell lineage, apoptosis may be triggered by altered expression of several conserved genes. Among these are members of the bcl-2 gene family (Bcl-2: an inhibitor of apoptosis; Bax: a Bcl-2 antagonist and an inducer of apoptosis; Bcl-x(long): a Bcl-2 homolog; a Bax homolog). The death repressor actions of Bcl-2 are likely linked to its antioxidant properties, serving to protect cells from the damaging effects of reactive oxygen species (ROS). Since granulosa cell death during follicle atresia occurs by apoptosis, we hypothesized that similar events involving bcl-2- related factors and oxidative stress would serve as the trigger for atresia. Apoptosis in granulosa cells is prevented by gonadotropins and is restricted to granulosa cells of maturing antral follicles. Based on these findings and data presented herein, it is proposed that gonadotropin- induced differentiation of granulosa cells is associated with elevated levels of ROS. Thus, gonadotropins must simultaneously initiate a sufficient oxidative stress response to protect granulosa cells from damage by ROS. If the oxidative stress response is inadequate, it is hypothesized that the elevated levels of ROS lead to granulosa cell apoptosis and follicle atresia. To test these hypotheses, the Specific Aims of this proposal are: 1) to determine if gonadotropin-mediated follicle survival is associated with enhanced expression of oxidative stress response factors (superoxide dismutase; glutathione peroxidase; catalase; bcl-2; bcl-x(long) and reduced expression of bcl-2 antagonists (bax; bcl-x(short)); 2) to evaluate if apoptosis in granulosa cells is associated with increased levels of ROS, and if inducers of oxidative stress interfere with the ability of gonadotropins to prevent apoptosis; 3) to investigate if DNA collected from follicles exposed to oxidative stress displays evidence of damage (adduct formation) and is more susceptible to DNase-catalyzed internucleosomal cleavage compared to DNA prepared from healthy follicles; and 4) to study expression of interleukin-1beta-converting enzyme (ICE; an protease postulated to activate DNases responsible for internucleosomal DNA cleavage) during follicle maturation and atresia, and to determine if expression of ICE is required for granulosa cell apoptosis. It is anticipated that results from these experiments will elucidate the underlying molecular mechanisms involved in the initiation of granulosa cell apoptosis and ovarian follicular atresia in all species.
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