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.(ABSTRACT TRUNCATED AT 400 WORDS)
Urban
R J
RJ
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Garmey
J C
JC
Shupnik
M A
MA
Veldhuis
J D
JD
D000818:Animals; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000494:pharmacology; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism*; D013261:Sterols / Q000037:antagonists & inhibitors; D013552:Swine; D013997:Time Factors
D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D013261:Sterols; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D002786:Cholesterol Side-Chain Cleavage Enzyme