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*

Abstract

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.(ABSTRACT TRUNCATED AT 400 WORDS)