Opposing effects of glutathione depletion and follicle-stimulating hormone on reactive oxygen species and apoptosis in cultured preovulatory rat follicles

Oxidative stress and depletion of the antioxidant glutathione (GSH) trigger apoptosis in many systems. Previous work showed that antioxidants prevented apoptosis as effectively as FSH in preovulatory follicles. We aimed to test the hypotheses that follicular reactive oxygen species (ROS) initiate apoptosis and that follicular GSH protects against apoptosis. Preovulatory follicles were isolated from ovaries of immature rats primed with pregnant mare serum gonadotropin. Negative control (0-h) follicles were processed immediately. Others were cultured for 2 to 48 h with 1) medium alone, 2) 75 ng/ml ovine FSH, or 3) FSH plus 100 mu M buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Total GSH concentrations declined in follicles cultured without FSH for 48 h, whereas FSH increased GSH levels above those observed at 0 h. BSO suppressed GSH to undetectable levels. Treatment with FSH prevented apoptosis in granulosa cells, measured by terminal dUTP transferase-mediated nick-end-labeling and activated caspase 3 immunohistochemistry. Addition of BSO partially and significantly reversed the antiapoptotic effect of FSH on granulosa cells; supplementation of GSH completely prevented BSO-induced granulosa cell apoptosis. Whole-follicle ROS production, measured as dichlorofluorescein and rhodamine fluorescence using confocal microscopy, was significantly increased by 4 h of culture and increased further thereafter. FSH significantly suppressed ROS production, and the addition of BSO partially overcame this effect of FSH. These findings provide evidence that oxidative stress induces apoptosis in preovulatory follicles and that the antiapoptotic effect of FSH is mediated in part by stimulation of follicular GSH synthesis and suppression of ROS production.