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 clea rly established ''in vitro''. Furthermore, experimental evidence indic ates that FSH and estradiol can independently inhibit granulosa cell p roliferation. The present study was aimed at examining the effect of s ex 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 [H-3] 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 maxi mal DNA synthesis from 96 to 48 h after plating. Dose response studies showed that estradiol effects were produced at physiological intraova rian concentrations (1-100 ng/ml), whereas the effects of FSH were bip hasic, with high doses (200 ng/ml) being inhibitory. A similar biphasi c dose response curve was observed with increasing concentrations of a cAMP derivative in the presence of maximally effective doses of eithe r an aromatizable steroid (androstenedione), insulin or insulin-like g rowth factor I. Non-aromatizable androgens (5 alpha-dihydrotestosteron e, 5 alpha-androstane 3 alpha-17 beta diol and androsterone) showed a potency comparable to that of estradiol. The effect of 5 alpha-dihydro testosterone was completely blocked by a specific antiandrogen (hydrox y-flutamide), indicating that it was mediated by the androgen receptor . The effects of estradiol and androgens were not additive. The intera ction between estradiol and FSH was further amplified in the presence of a maximally effective dose of insulin. Data presented herein indica te that both estrogens and androgens are able to elicit a mitogenic re sponse in purified granulosa cells, cultured in a completely defined m edium, provided the cells are stimulated by a physiological dose of FS H. These results suggest that, during follicular development, the stim ulus for granulosa cell proliferation is given by the concerted action of steroid and peptide hormones acting through different signalling p athways. (C) 1997 Elsevier Science Ltd.