In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert

Todd A. Winters, Jeffery A. Hanten, Johannes D Veldhuis

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

To investigate the cellular mechanisms and cell-cell heterogeneity of the actions of insulin-like growth factor-1 (IGF-I) and follicle-stimulating hormone (FSH) exerted alone and in combination on ovarian cholesterol side- chain cleavage gene expression (P450scc mRNA) in (pig) granulosa cells, we implemented semiquantitative in situ molecular hybridization at the single target-cell level. To this end, a 1-kb cDNA specific to the catalytic region of porcine p450scc gene was subcloned into pGEM-3 and directionally transcribed in vitro in the presence of 35S-dUTP to yield radiolabeled antisense (and sense, negative control) cRNA hybridization probes. Swine granulosa cells harvested nonenzymatically from immature (1-5 mm) Graafian follicles were anchored on eight-chamber multiwell slides and treated with control solvent, human recombinant IGF-1 (10 nM), ovine FSH (10 nM), or both hormones, for 48 h to stimulate progestin biosynthesis maximally. After appropriate cellular permeabilization, cRNA hybridization, and solvent washes, granulosa cells were exposed to Kodak NTB-2 emulsion for 6 wk. Semiquantitative automated image analysis software (NIH IMAGE 1.5) was used to evaluate the number of silver grains deposited/20,000 square pixels. Specificity controls included labeled sense riboprobe, pretreatment with RNase, and 100-fold molar excess unlabeled cRNA. Grain counts and their distributions were examined by ANOVA and the Wilcoxon nonparametric test. The mean number of silver grains deposited per granulosa cell increased over control (reflecting specific P450scc mRNA expression) in granulosa cells pretreated with IGF-1, FSH, or IGF-I + FSH (p < 0.05 by ANOVA). The rank order of abundance of expression of P450scc mRNA (grains/ovarian cell) was (IGF-1 + FSH) > FSH > IGF-1 > control treatment. Distributional analysis showed that each treatment introduced skewed distributions toward granulosa cells expressing more P450scc per cell than controls (p < 0.01). The median grain count of granulosa cells treated with FSH was significantly increased over that of IGF-1 treatment (p < 0.05). Treatment with both IGF-1 and FSH further shifted the grain count distribution per cell to favor granulosa cells expressing more P450scc mRNA compared to IGF-1 or FSH treatment alone (p < 0.05). Accordingly, a demonstrable mechanism of IGF-1 and FSH's regulation of specific P450scc gene expression at the single granulosa cell level is amplification in the number of target ovarian cells expressing this enzymatically rate-determining gene transcript. Interestingly, the induction of P450scc mRNA is not sufficient to explain fully the synergistic increases in progesterone accumulation driven by combined treatment with IGF-1 and FSH, thus suggesting that other steroidogenic control points are also targets of IGF-1/FSH action.

Original languageEnglish (US)
Pages (from-to)57-63
Number of pages7
JournalEndocrine
Volume9
Issue number1
DOIs
StatePublished - 1998
Externally publishedYes

Fingerprint

Cholesterol Side-Chain Cleavage Enzyme
Granulosa Cells
Follicle Stimulating Hormone
Insulin-Like Growth Factor I
Cytochrome P-450 Enzyme System
Swine
Messenger RNA
Complementary RNA
Silver
Gene Expression
Ovarian Follicle
Somatomedins
Progestins
Ribonucleases
Emulsions
Genes
In Situ Hybridization
Progesterone
Catalytic Domain
Sheep

Keywords

  • Granulosa cells
  • Ovary
  • P450scc
  • Porcine

ASJC Scopus subject areas

  • Endocrinology

Cite this

In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert. / Winters, Todd A.; Hanten, Jeffery A.; Veldhuis, Johannes D.

In: Endocrine, Vol. 9, No. 1, 1998, p. 57-63.

Research output: Contribution to journalArticle

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AU - Hanten, Jeffery A.

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N2 - To investigate the cellular mechanisms and cell-cell heterogeneity of the actions of insulin-like growth factor-1 (IGF-I) and follicle-stimulating hormone (FSH) exerted alone and in combination on ovarian cholesterol side- chain cleavage gene expression (P450scc mRNA) in (pig) granulosa cells, we implemented semiquantitative in situ molecular hybridization at the single target-cell level. To this end, a 1-kb cDNA specific to the catalytic region of porcine p450scc gene was subcloned into pGEM-3 and directionally transcribed in vitro in the presence of 35S-dUTP to yield radiolabeled antisense (and sense, negative control) cRNA hybridization probes. Swine granulosa cells harvested nonenzymatically from immature (1-5 mm) Graafian follicles were anchored on eight-chamber multiwell slides and treated with control solvent, human recombinant IGF-1 (10 nM), ovine FSH (10 nM), or both hormones, for 48 h to stimulate progestin biosynthesis maximally. After appropriate cellular permeabilization, cRNA hybridization, and solvent washes, granulosa cells were exposed to Kodak NTB-2 emulsion for 6 wk. Semiquantitative automated image analysis software (NIH IMAGE 1.5) was used to evaluate the number of silver grains deposited/20,000 square pixels. Specificity controls included labeled sense riboprobe, pretreatment with RNase, and 100-fold molar excess unlabeled cRNA. Grain counts and their distributions were examined by ANOVA and the Wilcoxon nonparametric test. The mean number of silver grains deposited per granulosa cell increased over control (reflecting specific P450scc mRNA expression) in granulosa cells pretreated with IGF-1, FSH, or IGF-I + FSH (p < 0.05 by ANOVA). The rank order of abundance of expression of P450scc mRNA (grains/ovarian cell) was (IGF-1 + FSH) > FSH > IGF-1 > control treatment. Distributional analysis showed that each treatment introduced skewed distributions toward granulosa cells expressing more P450scc per cell than controls (p < 0.01). The median grain count of granulosa cells treated with FSH was significantly increased over that of IGF-1 treatment (p < 0.05). Treatment with both IGF-1 and FSH further shifted the grain count distribution per cell to favor granulosa cells expressing more P450scc mRNA compared to IGF-1 or FSH treatment alone (p < 0.05). Accordingly, a demonstrable mechanism of IGF-1 and FSH's regulation of specific P450scc gene expression at the single granulosa cell level is amplification in the number of target ovarian cells expressing this enzymatically rate-determining gene transcript. Interestingly, the induction of P450scc mRNA is not sufficient to explain fully the synergistic increases in progesterone accumulation driven by combined treatment with IGF-1 and FSH, thus suggesting that other steroidogenic control points are also targets of IGF-1/FSH action.

AB - To investigate the cellular mechanisms and cell-cell heterogeneity of the actions of insulin-like growth factor-1 (IGF-I) and follicle-stimulating hormone (FSH) exerted alone and in combination on ovarian cholesterol side- chain cleavage gene expression (P450scc mRNA) in (pig) granulosa cells, we implemented semiquantitative in situ molecular hybridization at the single target-cell level. To this end, a 1-kb cDNA specific to the catalytic region of porcine p450scc gene was subcloned into pGEM-3 and directionally transcribed in vitro in the presence of 35S-dUTP to yield radiolabeled antisense (and sense, negative control) cRNA hybridization probes. Swine granulosa cells harvested nonenzymatically from immature (1-5 mm) Graafian follicles were anchored on eight-chamber multiwell slides and treated with control solvent, human recombinant IGF-1 (10 nM), ovine FSH (10 nM), or both hormones, for 48 h to stimulate progestin biosynthesis maximally. After appropriate cellular permeabilization, cRNA hybridization, and solvent washes, granulosa cells were exposed to Kodak NTB-2 emulsion for 6 wk. Semiquantitative automated image analysis software (NIH IMAGE 1.5) was used to evaluate the number of silver grains deposited/20,000 square pixels. Specificity controls included labeled sense riboprobe, pretreatment with RNase, and 100-fold molar excess unlabeled cRNA. Grain counts and their distributions were examined by ANOVA and the Wilcoxon nonparametric test. The mean number of silver grains deposited per granulosa cell increased over control (reflecting specific P450scc mRNA expression) in granulosa cells pretreated with IGF-1, FSH, or IGF-I + FSH (p < 0.05 by ANOVA). The rank order of abundance of expression of P450scc mRNA (grains/ovarian cell) was (IGF-1 + FSH) > FSH > IGF-1 > control treatment. Distributional analysis showed that each treatment introduced skewed distributions toward granulosa cells expressing more P450scc per cell than controls (p < 0.01). The median grain count of granulosa cells treated with FSH was significantly increased over that of IGF-1 treatment (p < 0.05). Treatment with both IGF-1 and FSH further shifted the grain count distribution per cell to favor granulosa cells expressing more P450scc mRNA compared to IGF-1 or FSH treatment alone (p < 0.05). Accordingly, a demonstrable mechanism of IGF-1 and FSH's regulation of specific P450scc gene expression at the single granulosa cell level is amplification in the number of target ovarian cells expressing this enzymatically rate-determining gene transcript. Interestingly, the induction of P450scc mRNA is not sufficient to explain fully the synergistic increases in progesterone accumulation driven by combined treatment with IGF-1 and FSH, thus suggesting that other steroidogenic control points are also targets of IGF-1/FSH action.

KW - Granulosa cells

KW - Ovary

KW - P450scc

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