Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II: In vitro studies with swine granulosa cells

James C. Garmey, Richard N. Day, Kathleen H. Day, Johannes D Veldhuis

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 ± 1.2 ng/ml (0.61 ± 0.16 nM) between 0-48 h of culture and 27 ± 5.7 ng/ml (3.6 ± 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 ± 0.70 ng/ml (0.78 ± 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 μg/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interaction also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays. In summary, immature swine granulosa cells are highly responsive to the actions of IGF-II in vitro. Nanomolar concentrations of IGF-II can enhance progesterone biosynthesis by 12-fold. IGF-II's mechanisms of action include facilitation of sterol delivery via increased LDL binding and metabolism and concomitantly higher steady state cellular concentrations of LDL receptor mRNA. Moreover, IGF-II augments sterol utilization in progesterone biosynthesis by increasing levels of cholesterol side-chain cleavage enzyme mRNA. Given the evidence for intrafollicular production of IGF-II in human and porcine ovaries, paracrine and autocrine effects of IGF-II may be physiologically significant in the developing Graafian follicle.

Original languageEnglish (US)
Pages (from-to)800-808
Number of pages9
JournalEndocrinology
Volume133
Issue number2
StatePublished - Aug 1993
Externally publishedYes

Fingerprint

Insulin-Like Growth Factor II
Granulosa Cells
Sterols
Swine
Progesterone
Cholesterol Side-Chain Cleavage Enzyme
LDL Lipoproteins
LDL Receptors
In Vitro Techniques
Messenger RNA
Cytochrome P-450 Enzyme System
8-Bromo Cyclic Adenosine Monophosphate

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II : In vitro studies with swine granulosa cells. / Garmey, James C.; Day, Richard N.; Day, Kathleen H.; Veldhuis, Johannes D.

In: Endocrinology, Vol. 133, No. 2, 08.1993, p. 800-808.

Research output: Contribution to journalArticle

Garmey, James C. ; Day, Richard N. ; Day, Kathleen H. ; Veldhuis, Johannes D. / Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II : In vitro studies with swine granulosa cells. In: Endocrinology. 1993 ; Vol. 133, No. 2. pp. 800-808.
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T1 - Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II

T2 - In vitro studies with swine granulosa cells

AU - Garmey, James C.

AU - Day, Richard N.

AU - Day, Kathleen H.

AU - Veldhuis, Johannes D

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N2 - The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 ± 1.2 ng/ml (0.61 ± 0.16 nM) between 0-48 h of culture and 27 ± 5.7 ng/ml (3.6 ± 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 ± 0.70 ng/ml (0.78 ± 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 μg/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interaction also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays. In summary, immature swine granulosa cells are highly responsive to the actions of IGF-II in vitro. Nanomolar concentrations of IGF-II can enhance progesterone biosynthesis by 12-fold. IGF-II's mechanisms of action include facilitation of sterol delivery via increased LDL binding and metabolism and concomitantly higher steady state cellular concentrations of LDL receptor mRNA. Moreover, IGF-II augments sterol utilization in progesterone biosynthesis by increasing levels of cholesterol side-chain cleavage enzyme mRNA. Given the evidence for intrafollicular production of IGF-II in human and porcine ovaries, paracrine and autocrine effects of IGF-II may be physiologically significant in the developing Graafian follicle.

AB - The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 ± 1.2 ng/ml (0.61 ± 0.16 nM) between 0-48 h of culture and 27 ± 5.7 ng/ml (3.6 ± 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 ± 0.70 ng/ml (0.78 ± 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 μg/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interaction also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays. In summary, immature swine granulosa cells are highly responsive to the actions of IGF-II in vitro. Nanomolar concentrations of IGF-II can enhance progesterone biosynthesis by 12-fold. IGF-II's mechanisms of action include facilitation of sterol delivery via increased LDL binding and metabolism and concomitantly higher steady state cellular concentrations of LDL receptor mRNA. Moreover, IGF-II augments sterol utilization in progesterone biosynthesis by increasing levels of cholesterol side-chain cleavage enzyme mRNA. Given the evidence for intrafollicular production of IGF-II in human and porcine ovaries, paracrine and autocrine effects of IGF-II may be physiologically significant in the developing Graafian follicle.

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