Disparate Effects of Triphenylethylene Antiestrogens on Estrogen and Progestin Biosyntheses by Cultured Rat Granulosa Cells*

Abstract

The influence of triphenylethylene antiestrogens on FSH-stimulated production of estrogen, progesterone and 20.alpha.-hydroxy-4-pregnen-3-one (20.alpha.-OH-P) was examined in cultured rat granulosa cells. The cells were cultured with increasing concentrations of FSH, with or without diethylstilbestrol (DES) or various antiestrogens. After 3 days, medium progesterone and 20.alpha.-OH-P contents were determined. Cells were reincubated for an additional 8 h with .DELTA.4-androstene-3,17-dione, and estrogen formation was measured. Steroid production was negligible by cultures treated with DES or antiestrogens alone. FSH treatment increased estrogen and progestin production, while the addition of DES (10-7 M) further enhanced FSH-stimulated steroidogenesis. Treatment with 10-6 M of the antiestrogens tamoxifen (Tam), nafoxidine (Naf), or clomiphene citrate (CC) also enhanced FSH-stimulated aromatase activity. The antiestrogens each inhibited FSH stimulation of progesterone and 20.alpha.-OH-P production (> 73% inhibition at 300 ng/ml FSH). Increasing concentrations (3 .times. 10-8 to 10-6 M) of the antiestrogens augmented the stimulatory effect of FSH (10 ng/ml) on estrogen production in a dose-related manner (CC > Tam > Naf). Similar doses of these antiestrogens inhibited the stimulatory effect of FSH (30 ng/ml) on progesterone and 20.alpha.-OH-P production (Tam > CC > Naf). The observed inhibition of progestin production is associated with decreases in FSH-stimulated pregnenolone biosynthesis in antiestrogen-treated cells incubated with 10-6 M cyanoketone. The antiestrogens inhibited the binding of [3H]estradiol to ovarian estrogen receptors with binding affinity constants of 364, 437, and 2144 nM for CC, Tam and Naf, respectively. Antiestrogens exert disparate actions on granulosa cell estrogen and progestin biosyntheses. Like estrogens, CC, Tam and Naf enhance FSH-stimulated aromatase activity with potencies comparable with their abilities to interact with ovarian estrogen receptors. Unlike estrogens, the antiestrogens inhibit FSH-stimulated progestin biosynthesis, partially via suppression of pregnenolone biosynthesis. The present granulosa cell culture system provides a valuable model for elucidating the disparate actions of estrogens and antiestrogens on ovarian steroidogenesis.