INHIBITION OF THE ACTIVITIES OF P450 CHOLESTEROL SIDE-CHAIN CLEAVAGE AND 3-BETA-HYDROXYSTEROID DEHYDROGENASE AND THE AMOUNT OF P450 CHOLESTEROL SIDE-CHAIN CLEAVAGE BY TESTOSTERONE AND ESTRADIOL-17-BETA IN HEN GRANULOSA-CELLS

We have found that androgens and estradiol-17 β (E2) produced by theca cells suppress progesterone (P4)secretion by granulosa cells of the domestic hen in a dosedependentmanner. Furthermore, testosterone (T) and E2 inhibitedthe conversion of cholesterol to pregnenolone (P6) and of P6to P4, respectively. The aim of this study was to determine if T and E2 suppress P4 biosynthesis by changing activities of thecytochrome P450 cholesterol side chain cleavage (P450SCC) and 30-hydroxysteroid dehydrogenase (3β-HSD) (Exp I) and theamount of P450SCC(Exp II). Granulosa layers of the largest follicle of two to four hens were obtained 22 h before ovulation, pooled, and isolated granulosa cells were prepared. In Exp I, the specific activities of the P450BCC and 3β-HSD were measured in mitochondrial and microsomal proteins of granulosa cells, respectively, in the presence of T or E2 (0-10 µM). Addition of T to mitochondrial proteins increased the Michaelis-Menten constant(Km) with no change in the maximum velocity (Vmax) of the P450JCC, which suggests competitive inhibition (Ki = 30.9µM), whereas E2 had no effect on Km and Vmax of the P450scc.Likewise, addition of E2 to microsomal proteins increased theKm with no change in the Vmax of the 3β -HSD, which suggests competitive inhibition (Ki = 15.1 µM), whereas T had no effecton Km and Vmax of the 3β-HSD. In Exp II, granulosa cells (3 x106β ml · tube) were incubated for 0-12 h in triplicate for eachcombined treatments of 25-OH-cholesterol (8 µM) and cyanoketone(10 µM), T, or E2 (0-10 µM) in the presence or absenceof LH (25 ng). Protein content and P6 secretion were measured and the amount of P450SCC was determined by Western blotanalysis. Incubation of granulosa cells with T decreased the amount of the P450SCC in granulosa cells cultured for 12 h andP6 secretion in granulosa cells cultured for 3 h or longer (P < 0.05), without a change in protein content and cell viability. Our results suggest that P4 production by granulosa cells is suppressed by T and E2 acting as competitive inhibitors of theP450SCC and 3β-HSD, respectively, and by T decreasing the amount of the P450SCC. We conclude that steroid genesis in the follicle of the chicken is regulated through the interaction of theca and granulosa layers. © 1990 by The Endocrine Society.