Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis

Apoptotic cell death has recently been suggested to be the underlying mechanism of ovarian follicle atresia. To study the regulation of follicle cell apoptosis by sex steroids, we have analyzed ovarian DNA fragmentation, the hallmark of apoptosis, in rats treated with estrogens and androgens. Immature rats were hypophysectomized and implanted with diethylstilbestrol (DES) capsules. Two days later, DES implants were removed in some animals, followed by treatment with estrogens with or without androgens. The extent of ovarian apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. After DES withdrawal, ovarian weight decreased and DNA fragmentation increased in a time-dependent manner. In granulosa cells, an increase in apoptotic DNA fragmentation was seen 12 h after withdrawal of DES implants, followed by a 25-fold increase at 48 h. In situ analysis of DNA fragmentation on histological sections of ovaries, using a nonisotopic labeling of DNA by digoxigenin-dideoxy-UTP, also demonstrated that apoptosis induced by DES withdrawal is confined to the granulosa cells in early antral and preantral follicles. No increase in DNA breakdown was detected in thecal cells and interstitial tissues or granulosa cells of primordial and primary follicles. In contrast, replacement with DES (0.5 mg twice daily) or estradiol benzoate (3 mg daily) completely prevented the observed ovarian weight loss and increases in granulosa cell apoptosis. Treatment with estradiol benzoate (0.003-3 mg/day) dose dependently suppressed the apoptosis seen 2 days after removal of DES implants. Furthermore, the antiatretogenic effect of estrogen was blocked by treatment with testosterone (0.5 mg twice daily), which increased ovarian apoptotic DNA fragmentation and decreased ovarian weight in DES-treated animals in a time-dependent manner. Also, in situ examination showed that androgen treatment increased apoptosis in the granulosa cells in a subpopulation of early antral and preantral follicles. The specificity of testosterone action was further demonstrated by the lack of effect of progesterone and cortisol on ovarian apoptosis. These data suggest that sex steroids play an important role in the regulation of ovarian apoptotic cell death, with estrogens preventing apoptosis and androgens antagonizing the effect of estrogens. These data provide the basis for future studies on the role of sex steroid hormones in follicular atresia and the regulation of endonuclease activity by steroid hormones.