VASOACTIVE INTESTINAL PEPTIDE-INDUCED EXPRESSION OF CYTOCHROME-P450 CHOLESTEROL SIDE-CHAIN CLEAVAGE AND 17-ALPHA-HYDROXYLASE ENZYME-ACTIVITY IN HEN GRANULOSA-CELLS

Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cl eavage (P450(scc)) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mR NA levels and enzyme activity in granulosa cells from nonhierarchal (6 -8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resti ng (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potenti al paracrine mechanism of action for VIP. While short-term (3 h) incub ation of granulosa cells with VIP (0.001-1.0 mu M) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h c ulture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesteron e synthesis and increased P450(scc) mRNA levels; control levels of eac h endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor a lpha (TGF alpha) in the presence of VIP (1 mu M) prevented increases i n P450(scc) mRNA levels and progesterone production. Similar effects o f VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an add itive effect of VIP (0.1 mu M) plus recombinant human (rh) FSH (100 mI U) on the initiation of progesterone production in cultured 6-8-mm fol licle granulosa cells compared to the addition of VIP or rhFSH alone. The ability of VIP to increase P450(scc) and P450 17 alpha-OH mRNA lev els and induce steroid production in nonhierarchal follicles is simila r to previously published results following culture with rhFSH. Theref ore, we suggest that VIP and FSH may act in concert to initiate steroi d production at the time of follicle selection (proposed to occur at t he 9-12-mm stage of development), possibly via the adenylyl cyclase/cA MP second messenger system. On the other hand, TGF alpha may represent a physiological mechanism to prevent premature expression of these en zymes.