GONADOTROPIN SUPPRESSION OF APOPTOSIS IN CULTURED PREOVULATORY FOLLICLES - MEDIATORY ROLE OF ENDOGENOUS INSULIN-LIKE GROWTH-FACTOR-I

Although the majority of ovarian follicles undergo atresia through a m echanism involving apoptotic cell death, in vivo studies concerning th e hormonal regulation of atresia have been difficult due to the presen ce of heterogeneous population of follicles in the ovary. In the prese nt study, the regulation of follicle apoptosis by gonadotropins, insul in-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) w as examined using a serum-free culture of preovulatory follicles. Imma ture rats at 26 days of age received a single dose of PMSG. Two days l ater, the largest preovulatory follicles were collected for in vitro c ulture with or without hormones. After 24 h of culture, follicular apo ptotic DNA fragmentation was analyzed by autoradiography of size-fract ionated DNA labeled at 3'-ends by [P-32]dideoxy-ATP. A spontaneous inc rease 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 mu g/ml causing maximal suppression by 60-62%. Cotreatment with hCG an d FSH had no additional effect. Like gonadotropins, treatment with IGF -I and insulin also suppressed the spontaneous onset of apoptosis, wit h IGF-I being more effective than insulin. Cotreatment with IGFBP-9 an d hCG dose-dependently reversed the suppressive effect of hCG on apopt osis 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-9 alone had no effect on apo ptotic DNA fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment r esulted in a marked stimulation of the production of both steroid prod uctions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment wit h IGFBP-3 alone decreased both estrogen and progesterone production, c otreatment with IGFBP-3 and hCG resulted in a slight decrease in estro gen production but an increase in progesterone production. Furthermore , IGFBP-3 did not affect IGF-I action on steroid production. To furthe r substantiate the hypothesis that IGFBP-3 blocks the suppressive effe ct of hCG on apoptosis by neutralizing endogenously produced IGF-I, so lution hybridization analysis was performed, and hCG treatment was sho wn to increase IGF-I messenger RNA levels in cultured follicles by 1.9 -fold. These data suggest that gonadotropins and IGF-I are survival fa ctors for ovarian follicles, and that part of the suppressive action o f gonadotropins is mediated by endogenously produced IGF-I.