Expression and regulation of cytochrome P450 17α-hydroxylase messenger ribonucleic acid levels and androstenedione production in hen granulosa cells

Studies were conducted to evaluate the expression and regulation of cytochrome P450 17α-hydroxylase (P450 17α-OH) mRNA levels and androstenedione production in 6-8-mm-diameter follicle granulosa cells from the domestic hen. Although P450 17α-OH mRNA was detected within granulosa cells from follicles at all stages of development (3-12-mm and preovulatory follicles), 8-12-fold higher levels were found in small, less developed follicles (3-12 mm in diameter) compared to the three largest follicles within the preovulatory hierarchy (F₃ to F₁ follicles). By comparison, P450 17α-OH mRNA levels were 25-89-fold higher in theca tissue compared to the granulosa layer at comparable stages of development. Despite detection of P450 17α-OH mRNA in granulosa cells from 6-8-mm follicles, androstenedione production was low to nondetectable when cells were cultured in the presence of exogenous progesterone (10 ng/ml). Treatment with FSH increased levels of P450 17α-OH mRNA (by 6-fold after 16 h of treatment) and induced androstenedione production in cultured granulosa cells; these actions were mimicked by the cAMP analog, 8-bromo-cAMP. By contrast, addition of the growth factors transforming growth factor α (TGFα), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) completely suppressed the stimulatory effects of FSH on both mRNA levels and androstenedione production, while insulin-like growth factor I (IGF-I) inhibited only androstenedione production. We conclude that in the granulosa layer of developing (≤ 12 mm) hen follicles the expression of P450 17α-OH mRNA does not directly reflect P450 17α-OH enzyme activity. Moreover, P450 17α-OH mRNA levels dramatically decrease in granulosa cells from preovulatory (compared to developing) follicles, and in the theca layer from the F₁ preovulatory follicle (compared to remaining follicles). It is proposed that FSH initiates P450 17α-OH enzyme activity (and perhaps to a lesser extent regulates mRNA levels) at the time a follicle is recruited into the preovulatory hierarchy, and that this action is mediated, at least in part, by the adenylyl cyclase/cAMP second messenger pathway. On the other hand, growth factors (including TGFα, EGF, bFGF, and possibly IGF-I) may act to prevent premature expression of P450 17α-OH activity in the granulosa layer of follicles that have yet to enter the rapid growth phase of follicle development.