Sang Y. Chun

Although the majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in apoptotic DNA fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on apoptotic DNA fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

A growing body of evidence suggests that intraovarian interleukin-1 beta (IL-1 beta) may play an intermediary role in the ovulatory process. Furthermore, induction of nitric oxide (NO) by IL-1 beta has been reported in a wide variety of tissues. As the majority of ovarian follicles undergo an atretic degeneration process involving apoptotic cell death, we set out to determine whether IL-1 beta rescues follicles from apoptosis and the possible involvement of NO. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends with [32P]dideoxy-ATP. Follicular NO production was also determined by a colorimetric method. Treatment with IL-1 beta dose-dependently suppressed the spontaneous onset of apoptosis in cultured follicles, but stimulated NO production. In contrast, the addition of IL-1 receptor antagonist eliminated both effects of IL-1 beta, confirming receptor mediation. Follicles treated with sodium nitroprusside, a NO generator or an analog of cGMP, the second messenger for NO, also showed decreased follicle apoptosis. Moreover, the addition of NG-monomethyl-L-arginine, a NO synthase inhibitor, reversed both IL-1 beta stimulation of NO production and suppression of apoptosis, suggesting a mediatory role of NO in these IL-1 beta effects. Gonadotropins also prevent follicle apoptosis. Of interest, treatment with hCG stimulated NO production, and the hCG suppression of follicle apoptosis and stimulation of NO production were partially blocked by cotreatment with IL-1 receptor antagonist, indicating the mediation of endogenous IL-1 beta. Treatment with IL-1 beta also stimulated a small increase in the production of cAMP, estrogen, and progesterone. Taken together, these findings suggest that IL-1 beta is a survival factor for ovarian follicles, and its action is partially mediated via NO and cGMP generation. Moreover, part of the suppressive action of gonadotropins on follicle apoptosis is mediated by endogenously produced IL-1 beta.

Apoptosis is an important cellular process by which superfluous or unwanted cells are deleted from an organism during tissue remodeling and differentiation. Recent studies have demonstrated the role of this programmed cell death or "controlled cell suicide" in the physiological function of an organism. Suppression of apoptosis increases the susceptibility of an individual to malignancy whereas uncontrolled cell death is associated with degenerative diseases. Normal development of both female and male gonads is characterized by massive cell death. More than 99% of ovarian follicles endowed at early life are destined to undergo apoptosis and the exhaustion of these follicles serves as a "clock" for female reproductive senescence. In the testis, up to 75% of male germ cells also undergo apoptosis, perhaps as a mechanism to delete superfluous or defective germ cells. Gonadal cell apoptosis provides valuable models to study hormonal regulation of apoptosis. In the ovary, gonadotropins, estrogens, growth hormone, growth factors (IGFI, EGF/TGF-alpha, basic FGF), cytokine (interleukin-1 beta) and nitric oxide act in concert to ensure the survival of preovulatory follicles. In contrast, androgens, interleukin-6 and gonadal GnRH-like peptide are apoptotic factors. Developmental studies further indicate that fractions of endowed follicles are recruited throughout the reproductive life whereas most of the primordial follicles are "arrested" at the initial stage of development for a prolonged time. Because a transcriptional factor WT1 is expressed in high levels in follicles at early stages of development and because WT1 over-expression represses the promoter activity of inhibin-alpha gene, this nuclear protein may be important in the maintenance of follicles at early stages of development. Once a cohort of follicles is recruited to grow, it is destined to undergo apoptosis unless rescued by survival factors. After puberty onset and under gonadotropin stimulation, some of the growing antral follicles are "selected" to continue their final maturation and secrete high levels of estrogens to trigger ovulation. Following repeated cycles of recruitment, atresia or ovulation, the follicle reserve is exhausted, thus signaling the onset of reproductive senescence. Although the somatic granulosa cell is the major cell type undergoing apoptosis in the ovary, the germ cells in the testis also exhibit signs of apoptotic cell demise. In the testis, gonadotropins and androgens act as survival factors whereas exposure to elevated temperature in cryptorchid testes increases apoptosis. In the seasonally breeding hamster model, photoperiod-entrained regression and recrudescence of testis tissue serves as a unique natural model of apoptosis. With recent advances in our understanding of the cellular mechanism of apoptosis, including the elucidation of the Ced9/bc12 and Ced3/ICE family of proteins, further investigation of gonadal apoptosis may lead to a better understanding of gonadal degenerative disorders (such as premature ovarian failure and oligospermia), reproductive senescence and tumorigenesis. The gonadal model should also be valuable in studying the regulation of intracellular apoptosis genes by external hormonal signals.