Phorbol Ester Inhibition of Ovarian and Testicular Steroidogenesis in Vitro¹

Possible influences of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) upon gonadal steroidogenesis were investigated in vitro. Granulosa cells from hypophysectomized, estrogen-treated rats were cultured for 2 days in medium containing 0.1 µm androstenedione. Follicle-stimulating hormone (FSH) treatment increased estrogen, progesterone, and 20α-hydroxypregn-4-en-3-one production. Concomitant TPA treatment inhibited FSH-stimulated estrogen production by up to 95% [concentration that induced 50% inhibition of steroid production (ED₅₀), 1.1 ng/ml]. TPA also inhibited FSH-stimulated progesterone (ED₅₀, ∼0.6 ng/ml) and 20α-hydroxypregn-4-en-3-one (ED₅₀, ∼1.1 ng/ml) production. N⁶O^2′-dibutyryl cyclic adenosine 3′:5′-monophosphate increased steroidogenesis; however, cotreatment with TPA blocked progestin but not estrogen production. The TPA inhibition of progestin biosynthesis was accompanied by decreases in FSH-stimulated pregnenolone biosynthesis and 3β-hydroxysteroid dehydrogenase activity without decreasing the activity of 20α-hydroxysteroid dehydrogenase. In primary cultures of rat testicular cells, human chorionic gonadotropin treatment increased testosterone production 44-fold, whereas concomitant treatment with TPA inhibited testosterone production by up to 86% (ED₅₀, 10 ng/ml). Cholera toxin and N⁶O^2′-dibutyryl cyclic adenosine 3′:5′-monophosphate also increased testosterone production, while the actions of these agents were decreased by TPA. The TPA suppression of testosterone production was associated with a decrease in accumulation of 17α-hydroxyprogesterone and androstenedione and an increase in progesterone production, suggesting a specific inhibition of 17α-hydroxylase and 17,20-lyase activities. These results demonstrate the inhibitory effects of a tumor promoter upon gonadotropin-stimulated steroidogenesis by cultured rat granulosa and Leydig cells through specific regulation of steroidogenic enzymes. Additional studies may assist in further elucidation of cellular mechanisms associated with carcinogenesis and steroidogenesis.