Hormonal regulation of P450scc (20,22-desmolase) and P450cl7 (17α-hydroxylase/17,20-lyase) in cultured human granulosa cells

Raimo Voutilainen, Juha Tapanainen, Bon Chu Chung, Karla Matteson, Walter L. Miller

Research output: Contribution to journalArticle

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Abstract

Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10−6M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin- specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with32P-labeled human P450cl7 cDNA (P450cl7 is the single enzyme mediating both 17α-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450cl7 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.

Original languageEnglish (US)
Pages (from-to)202-207
Number of pages6
JournalJournal of Clinical Endocrinology and Metabolism
Volume63
Issue number1
DOIs
StatePublished - Jan 1 1986

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Cholesterol Side-Chain Cleavage Enzyme
Steroid 17-alpha-Hydroxylase
Granulosa Cells
Mixed Function Oxygenases
Messenger RNA
Progesterone
Estrogens
Complementary DNA
Genes
Pregnenolone
Butyric Acid
Enzymes
Transcription
Gonadotropins
Adrenocorticotropic Hormone
Culture Media
Steroids
Cholesterol

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Endocrinology
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Hormonal regulation of P450scc (20,22-desmolase) and P450cl7 (17α-hydroxylase/17,20-lyase) in cultured human granulosa cells. / Voutilainen, Raimo; Tapanainen, Juha; Chung, Bon Chu; Matteson, Karla; Miller, Walter L.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 63, No. 1, 01.01.1986, p. 202-207.

Research output: Contribution to journalArticle

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abstract = "Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490{\%} and 240{\%} of control values, respectively, with hCG, to 166{\%} and 168{\%} with FSH, and to 495{\%} and 380{\%} with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10−6M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin- specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with32P-labeled human P450cl7 cDNA (P450cl7 is the single enzyme mediating both 17α-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450cl7 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.",
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AB - Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10−6M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin- specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with32P-labeled human P450cl7 cDNA (P450cl7 is the single enzyme mediating both 17α-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450cl7 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.

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