Expression of P450 side-chain cleavage (CYP11A1) and P450 17α-hydroxylase-17/20 lyase (CYP17) messenger ribonucleic acid in hamster primary interstitial cells in vitro: Differential regulation of steroidogenesis by cyclic adenosine monophosphate

J. R. Schwartz, Shyamal K Roy

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYPllA1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.

Original languageEnglish (US)
Pages (from-to)503-507
Number of pages5
JournalBiology of reproduction
Volume63
Issue number2
DOIs
StatePublished - Jan 1 2000

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Cholesterol Side-Chain Cleavage Enzyme
Steroid 17-alpha-Hydroxylase
Mixed Function Oxygenases
Cricetinae
Cyclic AMP
RNA
Androstenedione
Colforsin
Messenger RNA
Progesterone
Cultured Cells
Adenylyl Cyclases
In Vitro Techniques
LH Receptors
Granulosa Cells
Serum-Free Culture Media
Second Messenger Systems
Reverse Transcription
Radioimmunoassay
Up-Regulation

Keywords

  • Ovary
  • Theca cells
  • cAMP

ASJC Scopus subject areas

  • Cell Biology

Cite this

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title = "Expression of P450 side-chain cleavage (CYP11A1) and P450 17α-hydroxylase-17/20 lyase (CYP17) messenger ribonucleic acid in hamster primary interstitial cells in vitro: Differential regulation of steroidogenesis by cyclic adenosine monophosphate",
abstract = "Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5{\%} fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYPllA1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.",
keywords = "Ovary, Theca cells, cAMP",
author = "Schwartz, {J. R.} and Roy, {Shyamal K}",
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doi = "10.1095/biolreprod63.2.503",
language = "English (US)",
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T1 - Expression of P450 side-chain cleavage (CYP11A1) and P450 17α-hydroxylase-17/20 lyase (CYP17) messenger ribonucleic acid in hamster primary interstitial cells in vitro

T2 - Differential regulation of steroidogenesis by cyclic adenosine monophosphate

AU - Schwartz, J. R.

AU - Roy, Shyamal K

PY - 2000/1/1

Y1 - 2000/1/1

N2 - Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYPllA1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.

AB - Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYPllA1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.

KW - Ovary

KW - Theca cells

KW - cAMP

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