Concerted stimulation of rat granulosa cell deoxyribonucleic acid synthesis by sex steroids and follicle-stimulating hormone.

Although follicle-stimulating hormone (FSH) and estrogens are known to be the main physiological stimuli for the development of the ovarian follicle in mammals, their growth-promoting activity has not been clearly established "in vitro". Furthermore, experimental evidence indicates that FSH and estradiol can independently inhibit granulosa cell proliferation. The present study was aimed at examining the effect of sex steroids in combination with FSH, on DNA synthesis in rat granulosa cells cultured in completely defined medium. Estradiol and FSH, when added separately, produced a significant inhibition of [3H] thymidine incorporation. In contrast, a combination of a low dose of FSH (20 ng/ml) with estradiol (100 ng/ml) produced a shift in the period of maximal DNA synthesis from 96 to 48 h after plating. Dose response studies showed that estradiol effects were produced at physiological intraovarian concentrations (1-100 ng/ml), whereas the effects of FSH were biphasic, with high doses (200 ng/ml) being inhibitory. A similar biphasic dose response curve was observed with increasing concentrations of a cAMP derivative in the presence of maximally effective doses of either an aromatizable steroid (androstenedione), insulin or insulin-like growth factor I. Non-aromatizable androgens (5alpha-dihydrotestosterone, 5alpha-androstane 3alpha-17beta diol and androsterone) showed a potency comparable to that of estradiol. The effect of 5alpha-dihydrotestosterone was completely blocked by a specific antiandrogen (hydroxy-flutamide), indicating that it was mediated by the androgen receptor. The effects of estradiol and androgens were not additive. The interaction between estradiol and FSH was further amplified in the presence of a maximally effective dose of insulin. Data presented herein indicate that both estrogens and androgens are able to elicit a mitogenic response in purified granulosa cells, cultured in a completely defined medium, provided the cells are stimulated by a physiological dose of FSH. These results suggest that, during follicular development, the stimulus for granulosa cell proliferation is given by the concerted action of steroid and peptide hormones acting through different signalling pathways.