Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells

Abstract Studies were conducted to determine the importance of de novo cholesterol synthesis and cholesterol side-chain cleavage enzyme in the action of IGF-I in bovine granulosa and thecal cells. Granulosa and thecal cells from bovine follicles were cultured for 2 days in 10% fetal calf serum and then treated with luteinizing hormone (100 ng/ml) and IGF-I (0 or 100 ng/ml) for an additional 2 days in serum-free medium. During the last 24 h of treatment, cells were concomitantly treated with simvastatin (0, 0·5 or 5 μg/ml) or 25-hydroxycholesterol (0 or 10 μg/ml). Simvastatin, a potent inhibitor of the key enzyme controlling de novo cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, completely inhibited (P<0·05) progesterone production by granulosa cells and progesterone and androstenedione production by thecal cells. Simvastatin also inhibited (P<0·05) granulosa cell and thecal cell proliferation. Concomitant treatment with mevalonate, an immediate product of HMG-CoA reductase, attenuated the inhibitory effect of simvastatin on progesterone and androstenedione production by thecal cells and blocked the inhibitory effect of simvastatin on cell proliferation. The addition of 25-hydroxycholesterol, a substrate for cholesterol side-chain cleavage enzyme, had no effect (P>0·10) on IGF-I-stimulated progesterone or androstenedione production by thecal cells and actually inhibited (P<0·05) IGF-I-stimulated progesterone production by granulosa cells. These results provide indirect evidence indicating that stimulation of HMG-CoA reductase is an important locus of IGF-I action in bovine granulosa and thecal cells, whereas IGF-I has little or no effect on side-chain cleavage enzyme activity in these same cell types under the culture conditions employed. Journal of Endocrinology (1996) 151, 365–373

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Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro

Journal of Endocrinology ◽

10.1677/joe.0.1740499 ◽

2002 ◽

Vol 174 (3) ◽

pp. 499-507 ◽

Cited By ~ 36

Author(s):

JM Silva ◽

CA Price

Keyword(s):

Gene Expression ◽

Cytochrome P450 ◽

Granulosa Cells ◽

Side Chain ◽

Mrna Levels ◽

Side Chain Cleavage ◽

P450 Aromatase ◽

Chain Cleavage ◽

Igf I

The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence.

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Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model

Journal of Endocrinology ◽

10.1677/joe.0.1760151 ◽

2003 ◽

Vol 176 (1) ◽

pp. 151-161 ◽

Cited By ~ 26

Author(s):

V Sriraman ◽

MR Sairam ◽

AJ Rao

Keyword(s):

Leydig Cells ◽

Serum Testosterone ◽

Side Chain ◽

Mrna Levels ◽

Rt Pcr ◽

Lh Receptor ◽

Side Chain Cleavage ◽

Chain Cleavage ◽

Cleavage Enzyme

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.

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Notch signalling regulates steroidogenesis in mouse ovarian granulosa cells

Reproduction Fertility and Development ◽

10.1071/rd18281 ◽

2019 ◽

Vol 31 (6) ◽

pp. 1091 ◽

Cited By ~ 2

Author(s):

Yishu Wang ◽

Enhang Lu ◽

Riqiang Bao ◽

Ping Xu ◽

Fen Feng ◽

...

Keyword(s):

Transcription Factors ◽

Granulosa Cells ◽

Notch Signalling ◽

Side Chain ◽

Notch Signalling Pathway ◽

Steroidogenic Factor 1 ◽

Ovarian Granulosa Cells ◽

Chain Cleavage ◽

Cleavage Enzyme ◽

Stimulation Of

The Notch signalling pathway in the mammalian ovary regulates granulosa cell proliferation. However, the effects of Notch signalling on steroidogenesis are unclear. In this study we cultured mouse ovarian granulosa cells from preantral follicles invitro and observed the effect of Notch signalling on steroidogenesis through overexpression, knockdown and inhibition of Notch signalling. Activation of Notch signalling decreased progesterone and oestrogen secretion. In contrast, inhibition of Notch signalling increased the production of progesterone and oestrogen. Expression of the genes for steroidogenic-related enzymes, including 3β-hydroxysteroid dehydrogenase, p450 cholesterol side-chain cleavage enzyme and aromatase, was repressed after stimulation of Notch signalling. The expression of upstream transcription factors, including steroidogenic factor 1 (SF1), Wilms’ tumour 1 (Wt1), GATA-binding protein 4 (Gata4) and Gata6, was also inhibited after stimulation of Notch signalling. Production of interleukin (IL)-6 was positively correlated with Notch signalling and negatively correlated with the expression of these transcription factors and enzymes. In conclusion, Notch signalling regulated progesterone and oestrogen secretion by affecting the expression of upstream transcription factors SF1, Wt1, Gata4 and Gata6, as well as downstream steroidogenic-related enzymes. IL-6, which may be regulated directly by Notch signalling, may contribute to this process. Our findings add to the understanding of the diverse functions of Notch signalling in the mammalian ovary.

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