Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells.
FSH is the primary hormonal inducer of ovarian follicle maturation and a critically important regulator of steroidogenesis in granulosa cells. We examined possible molecular mechanisms subserving FSH action by assessing concentrations of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA in porcine granulosa cells maintained in serum-free culture. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Specificity was tested by estimating the granulosa cell mRNA content of the constitutively expressed enzyme, glyceraldehyde-3-phosphate dehydrogenase. Steroidogenesis was evaluated by measuring concomitant progesterone accumulation in the culture medium. Treatment with ovine FSH (100 ng/ml) increased P450scc mRNA concentrations in a time-dependent fashion, with significant effects on both P450scc mRNA concentrations and progesterone accumulation by 4 h and a maximal increase (8- to 10-fold) at 48 h. FSH dose-response studies at 48 h revealed a significant stimulatory effect of 30 ng/ml FSH on P450scc mRNA accumulation and progesterone production, with a maximal effect at 100 ng/ml FSH. To examine the role of cAMP in mediating granulosa cell P450scc mRNA accumulation, granulosa cells were treated with forskolin, cholera toxin, 8-bromo-cAMP, 8-bromo-cGMP, 5'AMP, or cAMP analogs that differentially stimulate the two isoenzymes of protein kinase-A. Increased specific P450scc mRNA accumulation and progesterone production occurred in response to each agent except 5'AMP and 8-bromo-cGMP. No effects of these agents were observed on glyceraldehyde-3-phosphate dehydrogenase mRNA. To assess possible feedback effects of steroid or sterol on FSH-stimulated P450scc mRNA concentrations, granulosa cells were treated with aminoglutethimide to block or with low density lipoprotein to stimulate steroid production. Inhibition of sterol utilization by the cholesterol side-chain cleavage enzyme had no effect on basal or FSH-stimulated concentrations of P450scc mRNA, but markedly suppressed progesterone production. Low density lipoprotein, which increases intracellular sterol, also did not alter basal or FSH-stimulated P450scc mRNA accumulation, suggesting that neither the utilization nor the availability of sterol regulates specific P450scc mRNA levels. Estradiol alone did not increase P450scc mRNA accumulation, but did augment progesterone production. Treatment of granulosa cells with estradiol and FSH produced a synergistic increase in progesterone concentrations, but did not affect FSH-stimulated P450scc mRNA accumulation.(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
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000008:administration & dosage / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D013552:Swine
D007527:Isoenzymes; D012333:RNA, Messenger; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004958:Estradiol; D005640:Follicle Stimulating Hormone; D002772:Cholera Toxin; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D011494:Protein Kinases