Insulin-like growth factor type I increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells

Insulin-like growth factor type I (IGF-I) is an important intraovarian peptide that stimulates granulosa cell steroidogenesis during follicular development. The cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) that converts cholesterol to pregnenolone is the rate-limiting step in progesterone biosynthesis. Since treatment of primary cultures of immature porcine granulosa cells with IGF-I will increase progesterone production as well as the synthesis of immunoprecipitable P450scc enzyme, we examined possible molecular mechanisms subserving these inductive effects of IGF-I. To this end, cultures of porcine granulosa cells were maintained in serum-free medium with or without IGF-I under various treatment paradigms. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a ³²P-labeled 1-kilobase porcine cDNA clone. Northern blot autoradiogram densitometry data were normalized with a constitutively expressed 1.2-kilobase chicken glyceraldehyde-3-phosphate dehydrogenase cDNA clone. Steroidogenesis was monitored by measuring concomitant progesterone accumulation in the culture medium. Treatment with pure recombinant human IGF-I (100 ng/ml) significantly increased P450scc mRNA concentrations after 18 h, and maximal stimulation (10- to 20-fold) occurred by 48 h for both P450scc mRNA and progesterone accumulation. The IGF-I dose-response curve studied at 48 h showed a significant increase in P450scc mRNA levels at a minimal IGF-I concentration of 1 ng/ml (although progesterone production was not increased). Treatment with equimolar concentrations of epidermal growth factor, IGF-I, or insulin significantly increased P450scc mRNA concentrations, whereas fibroblast growth factor did not. To examine possible mechanisms underlying stimulation of P450scc by IGF-I, immature granulosa cells were treated with aminoglutethimide (a P450scc enzyme inhibitor), low density lipoprotein (to increase cholesterol delivery to granulosa cells), or estradiol in the presence or absence of IGF-I. Aminoglutethimide had no effect, alone or with IGF-I, on P450scc mRNA concentrations, but suppressed progesterone production. Low density lipoprotein alone also did not stimulate P450scc mRNA levels and only slightly increased progesterone accumulation, but acted synergistically with IGF-I to augment P450scc mRNA concentrations and progesterone accumulation. Estradiol alone did not stimulate P450scc mRNA concentrations, but did significantly increase progesterone production. Estradiol cotreatment with IGF-I synergistically enhanced progesterone production, but did not alter IGF-I-stimulated P450scc mRNA concentrations. We conclude that IGF-I significantly increases concentrations of P450scc mRNA in primary cultures of porcine granulosa cells in a specific time- and dose-dependent fashion. This stimulation of P450scc mRNA accumulation is not always directly correlated with concomitant progesterone production by granulosa cells. Further studies are needed to help clarify the exact nature of the molecular actions of IGF-I on the cytodifferentiation of granulosa cells.