Aromatase inhibitors prevent granulosa cell differentiation: an obligatory role for estrogens in luteinizing hormone receptor expression.

To determine the role of newly synthesized estrogens in LH receptor expression, granulosa cells from diethylstilbestrol-implanted immature rats were cultured with FSH plus aromatase inhibitors. When present throughout the 48-h culture period, 4-hydroxy-4-androstene-3,17-dione (4-OHA; greater than or equal to 100 microM) and 1,4,6-androstatriene-3,17-dione (greater than or equal to 5 microM) inhibited FSH-induced LH receptor formation by 40% and 90%, respectively. Both aromatase inhibitors caused relatively greater inhibition of LH receptor formation when added from 20-48 h of culture, the period during which FSH-stimulated estrogen synthesis occurs (85% maximal inhibition with 4-OHA and 95% with 1,4,6-androstatriene-3,17-dione). Addition of estradiol, but not androstenedione, reversed the reduction of LH receptor formation by 4-OHA, indicating that the effects of the aromatase inhibitors were specifically related to their blockade of estradiol synthesis. The stimulation of estrogen production by FSH alone (8-fold) or with androstenedione (80-fold) during the 48-h culture period was prevented by 4-OHA. FSH-stimulated cAMP production was initially enhanced by 4-OHA from 0-20 h of culture, but was reduced from 20-48 h. Lower concentrations of 4-OHA (less than or equal to 50 microM) amplified FSH-stimulated cAMP production and LH receptor formation. However, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, indicating that 4-OHA or a metabolite may have partial estrogenic or androgenic properties. The inhibitory effects of higher concentrations of 4-OHA on LH receptor expression were potentiated by keoxifene or flutamide. These results indicate that estrogen production and action are necessary for LH receptor expression in the granulosa cell.