Antigonadal Action of Prolactin: Further Studies on the Mechanism of Inhibition of Follicle-Stimulating Hormone-Induced Aromatase Activity in Rat Granulosa Cell Cultures*

Abstract

The specificity and mechanism of the action by which PRL inhibits FSH-induced aromatase activity have been studied in rat granulosa cells in culture. Granulosa cells isolated from 25-day-old diethylstilbestrol-primed rats and cultured under defined conditions for 48 or 72 h with NIH-FSH-S13 (300 ng/ml) had elevated levels of aromatase activity, as assessed by the release of ³H₂O from [1β-³H]testosterone. PRL (1 μg ovine NIH-P-S7–ml) alone had no effect on aromatase activity, but when added together with FSH, it effectively suppressed the effect of FSH. The effect of PRL was not due to cell death, since the rate of incorporation of [³H]leucine into trichloroacetic acidinsoluble material was unchanged by prior treatment with FSH alone or with FSH and PRL. The effect of PRL on the release of ³H₂O from [1β-³H]testosterone was dose dependent; the doses that caused 50% inhibition of the FSH-induced responses after 48 and 72 h of treatment were 45 and 47 ng/ml, respectively, which are within the physiological range found in human follicular fluid. Homologous rat PRL also effectively inhibited the FSH induction of aromatase activity. The specificity of PRL action was examined by comparison with the effects of GH. Both ovine and rat GHs were without significant effect at 1 μg/ml, but at higher concentrations (5 and 10 μg/ml), they inhibited the FSH response to an extent consistent with an effect of the contaminating PRL. Furthermore, the effects of PRL and GH were additive at lower concentrations, whereas GH did not influence the maximal PRL effect. In addition to inhibiting the FSH-induced response by the addition of hormone at the time of plating, PRL also facilitated a decrease in the aromatase activity subsequent to the initiation by FSH of the induction process. The inhibitory action of PRL on FSH-induced aromatase activity was reversible, and this was apparent 33 h after removal of the PRL from the culture medium. PRL pretreatment of granulosa cells effectively reduced the amount of active aromatase induced by FSH in cell sonicates incubated under optimal conditions. PRL added directly to the cell sonicates did not influence the enzymatic activity. PRL also interfered with dibutyryl cAMP plus 3-isobutyl-1-methylxanthine-stimulated aromatase activity, suggesting that one site of action of PRL may be distal to FSH-induced cAMP production. The studies reported here suggest that one contraceptive action of PRL may reside in its ability to interfere with FSH effects on estrogen biosynthesis.