Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis

J. L. Tilly, A. L. Johnson

Research output: Contribution to journalArticle

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Abstract

We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3′,5′-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3β-hydroxysteroid dehydrogenase (3β-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 μM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 μM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 μM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 μM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 81 and 162 nM) or OAG (2.5, 25 and 63 μM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.

Original languageEnglish (US)
Pages (from-to)155-166
Number of pages12
JournalDomestic Animal Endocrinology
Volume6
Issue number2
DOIs
StatePublished - Apr 1989

Fingerprint

steroidogenesis
Granulosa Cells
Diglycerides
granulosa cells
Phorbol Esters
diacylglycerols
hens
esters
cyclic AMP
Adenosine Monophosphate
pregnenolone
Pregnenolone
adenylate cyclase
Adenylyl Cyclases
1-Methyl-3-isobutylxanthine
Acetates
acetates
protein kinase C
luteinizing hormone
Luteinizing Hormone

All Science Journal Classification (ASJC) codes

  • Food Animals
  • Animal Science and Zoology
  • Endocrinology

Cite this

@article{f2f58ab61f6a404f93d430741e13502d,
title = "Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis",
abstract = "We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3′,5′-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3β-hydroxysteroid dehydrogenase (3β-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 μM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 μM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 μM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 μM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 81 and 162 nM) or OAG (2.5, 25 and 63 μM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.",
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Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis. / Tilly, J. L.; Johnson, A. L.

In: Domestic Animal Endocrinology, Vol. 6, No. 2, 04.1989, p. 155-166.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis

AU - Tilly, J. L.

AU - Johnson, A. L.

PY - 1989/4

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N2 - We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3′,5′-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3β-hydroxysteroid dehydrogenase (3β-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 μM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 μM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 μM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 μM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 81 and 162 nM) or OAG (2.5, 25 and 63 μM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.

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