CYTOCHROME-P450 SIDE-CHAIN CLEAVAGE (P450SCC) IN THE HEN OVARY .1. REGULATION OF P450SCC MESSENGER-RNA LEVELS AND STEROIDOGENESIS IN THECA CELLS OF DEVELOPING FOLLICLES

We have recently shown that granulosa cells from hen ovarian follicles, collected at a stage of development 2-3 wk prior to ovulation (e.g., 6-8 mm in diameter) are steroidogenically inactive. Therefore, the hypothesis tested in the present studies was that theca cells from follicles at this stage of development must contain sufficient levels of functional cytochrome P450 side-chain cleavage (P450scc) enzyme to produce the progestin precursor required for the synthesis of androgens and estrogens. Northern blot analysis of total theca RNA collected from 6-8-mm follicles indicated the presence of a single P450scc mRNA transcript of approximately 2 kb whose expression was increased following an 8-h preincubation with 200 ng/ml ovine LH (oLH) or 10-mu-M forskolin. Western blot analysis of crude mitochondrial protein revealed a band of immunoreactive P450scc protein of approximately 53 kDa that was determined to be capable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. In the second set of studies, conducted to examine the cellular regulation of steroidogenesis in isolated theca cells of 6-8-mm follicles, theca cells were found to produce measurable basal levels of cAMP, progesterone, androstenedione, and estradiol following a 3-h incubation of 5 x 10(5) cells. Furthermore, significant dose-dependent increases in steroidogenesis were observed in response to oLH (0.2-200 ng/ml), chicken FSH (cFSH; 20-200 ng/ml), cholera toxin (0.002-20 ng/ml), and 8-bromo-cAMP (0.1-3.33 mM). Phorbol 12-myristate 13-acetate (PMA; 10-167 nM) also stimulated dose-dependent increases in basal progesterone, androstenedione, and estradiol production. In addition, while PMA had no effect on oLH (200 ng/ml)-promoted cAMP accumulation, or on oLH (20 ng/ml)- or 8-bromo-cAMP (1 mM)-stimulated progesterone production, it attenuated oLH-induced and 8-bromo-cAMP-induced androstenedione and estradiol accumulation. We conclude that theca cells from 6-8-mm follicles possess mRNA and immunoreactive protein coding for functional P450scc. Furthermore, basal steroidogenesis is increased by both the protein kinase A and protein kinase C pathways, whereas evidence suggests that protein kinase C inhibits LH-induced androstenedione production at a site distal to cAMP and progesterone production, most likely by decreasing C-17, C-20-lyase activity.