Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor.

Abstract

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats 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. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that 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 prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP 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 suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation 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 effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.

Nitric oxide influences the meiotic maturation of porcine oocytes cultured in hypoxanthine‐supplemented medium

“…We find that NO suppressed the cumulus expansion and cumulus cells DNA fragmentation, especially at a lower level of NO derived from 0.1 m m SNP, but this function was attenuated by overdose exogenous NO in a dose‐dependent manner when the toxicity of NO became serious. Such result is well consistent with the results of Chun and colleagues (1996). Depending on the specific conditions and cell types, NO induces apoptosis and DNA damage, and NO also displays anti‐apoptotic properties (Dimmeler and Zeiher, 1997; Kim et al., 2001).…”

Effect of androgens on the development of mouse follicles growing in vitro

“…(Baird, 1977) and in vitro using co-cultures of granulosa and theca-interstitial cells (Lui and Hsueh, 1986 (Chun et al, 1996, Spears et al, 1998. In this, and the first experiment, in which androgens were added to the culture medium, concen¬ trations were chosen that reflect concentrations in both human and pig follicular fluid (Ding and Foxcroft, 1992;McNatty et al, 1979) since no comparable data are available for rodents.…”

The Role of Antioxidant Enzymes in the Ovaries

“…After blocking the synthesis of GSH with the inhibitor buthionine sulfoximine (BSO), a statistically significant increase in atretic antral follicles was observed in rat ovaries [135]. Prevention of apoptosis initiation in antral follicles by FSH is commonly accepted [136, 137]. Interestingly, FSH treatment can reportedly stimulate GSH synthesis [138, 139].…”