HORMONAL-REGULATION OF APOPTOSIS IN EARLY ANTRAL FOLLICLES - FOLLICLE-STIMULATING-HORMONE AS A MAJOR SURVIVAL FACTOR

Hormonal regulation of apoptosis has been studied in cultured preovula tory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, t he present studies were designed to investigate the hormonal regulatio n of apoptosis using in vitro culture of early antral follicles. ]Eats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. lifter c ulture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electro phoresis. In situ analysis of apoptotic DNA fragmentation revealed tha t granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12- fold increase in the level of apoptotic DNA fragmentation which was pr evented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED(50) of 30 ng/ml). Similarly, treatment wit h (Bu)(2)cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation ( 35% maximal suppression). Insulin-like growth factor-I (IGF-I) also su ppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding prot ein-3, suggesting a potential mediatory role of endogenous IGF-I. Howe ver, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubatio n of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effe ctiveness of EGF on apoptosis in early antral follicles, as compared w ith its potent action in preovulatory follicles reported earlier, may be due to a 3.5-fold increase in EGF receptor concentration in the mat ure follicles. High doses (150 or 500 ng/ml) of interleukin-1 beta als o suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as e ffectively as that of FSH. Furthermore, treatment with activin resulte d in a dose-related suppression of follicle apoptosis, reaching a maxi mal 40% suppression. In contrast, cotreatment of activin with its bind ing protein, follistatin, abolished this effect. Collectively, these d ata demonstrated a stage-dependent difference in the hormonal regulati on of follicle apoptosis. Although FSH, LH/ human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1 beta are all effecti ve survival factors for preovulatory follicles, FSH is a major surviva l factor for early antral follicles, the stage during which a majority of follicles undergo atresia under physiological conditions.