Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells

Randall Urban, James C. Garmey, Margaret A. Shupnik, Johannes D. Veldhuis

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

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. We conclude that 1) FSH augments specific P450scc mRNA accumulation in swine granulosa cells; 2) the effects of FSH can be mimicked by nonreceptor-mediated activators of the cAMP-dependent protein kinases (types I and II); and 3) FSH action is not mediated solely by increased sterol utilization or availability in granulosa cells.

Original languageEnglish (US)
Pages (from-to)2000-2007
Number of pages8
JournalEndocrinology
Volume128
Issue number4
StatePublished - Apr 1991

Fingerprint

Cholesterol Side-Chain Cleavage Enzyme
Granulosa Cells
Follicle Stimulating Hormone
Cytochrome P-450 Enzyme System
Swine
RNA
Cholesterol
Messenger RNA
Progesterone
Sterols
8-Bromo Cyclic Adenosine Monophosphate
Glyceraldehyde-3-Phosphate Dehydrogenases
Cyclic AMP-Dependent Protein Kinases
LDL Lipoproteins
Estradiol
Steroids
Aminoglutethimide
Ovarian Follicle

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells. / Urban, Randall; Garmey, James C.; Shupnik, Margaret A.; Veldhuis, Johannes D.

In: Endocrinology, Vol. 128, No. 4, 04.1991, p. 2000-2007.

Research output: Contribution to journalArticle

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AB - 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. We conclude that 1) FSH augments specific P450scc mRNA accumulation in swine granulosa cells; 2) the effects of FSH can be mimicked by nonreceptor-mediated activators of the cAMP-dependent protein kinases (types I and II); and 3) FSH action is not mediated solely by increased sterol utilization or availability in granulosa cells.

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