Androgen/antiandrogen modulation of cyclic amp-induced steroidogenesis during granulosa cell differentiation in tissue culture

Abstract

FSH stimulation of granulosa cell differentiation is believed to be mediated by the intracellular cyclic AMP (cAMP) level. However, steroidogenic enzyme induction in the differentiating granulosa cell is subject to direct modulation by androgenic steroid: in granulosa cell cultures established from ovaries of oestrogen-pretreated, prepubertal rats, potentiating effects of testosterone (T) on FSH induction of oestrogen synthetase (aromatase) and progesterone (P) biosynthesis can be blocked by including a stoichiometric excess of antiandrogen (hydroxyflutamide, SCH 16423) in the culture medium. In this study we used the same experimental model to determine effects of T and SCH 16423 on the induction of steroidogenesis by endogenous cAMP and an exogenous cAMP analogue, 8-bromo-cAMP (8brcAMP). Granulosa cells were cultured in medium containing variable FSH concentration (3–300 ng/ml) with a fixed (100 μm) dose of 3-isobutylmethylxanthine (MIX), or containing a fixed (minimally effective: 10–15 ng/ml) dose of FSH with MIX concentration variable (50–800 μM). By relating steroidogenic endpoints at 48 h to the acute cAMP response (accumulations in the medium) at 1 h, it was deduced that aromatase induction was saturable under conditions where FSH-sensitive cAMP production and the induction of P biosynthesis showed further, proportionate increases. Although T (0.1μM) did not alter acute FSH-responsive cAMP production, its presence throughout the 48 h culture was required for full expression of FSH-induced steroidogenesis in the cell monolayers. When the aromatase response (but not the P response) was ‘supersaturated’ by endogenous cAMP (i.e. culture with FSH plus MIX), SCH16423 was unable to antagonize the potentiating effect of T on aromatase induction while it continued to block T-potentiated P biosynthesis. Steroidogenic induction by cholera toxin (100 ng/ml) was also subject to similar modulation by T and SCH16423. However, the phosphodiesterase-resistant cAMP analogue 8brcAMP (3 mM) not only induced each response (albeit submaximally in the case of aromatase) in the absence of T, but its effects tended to be refractory to androgen/antiandrogen modulation. Accumulations of cAMP in the medium from 48 h cultures which had been incubated with FSH (100 ng/ml) were increased 2–3-fold by the additional presence of T (0.1 μM). This long-term stimulatory effect of T on FSH-dependent cAMP accumulation was blocked by culture in the presence of SCH16423 (10 μM). Thus, androgen potentiation of steroidogenic enzyme induction during FSH-stimulated granulosa cell differentiation may involve a suppression of cAMP catabolism exerted by way of the androgen-receptor system.