316. Regulation of the cholesterol side chain cleavage (CSCC) in human term placenta mitochondria by naturally occuring steroids in vitro

1982 ◽  
Vol 17 (3) ◽  
pp. cvi
Author(s):  
T. Rabe ◽  
K. Weidenhammer ◽  
L. Kiesel ◽  
B. Runnebaum
Keyword(s):  
Side Chain ◽  
Human Term Placenta ◽  
Side Chain Cleavage ◽  
Term Placenta ◽  
Chain Cleavage

Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal

1990 ◽  
Vol 4 (3) ◽  
pp. 239-245 ◽  
Author(s):  
T. Imai ◽  
H. Seo ◽  
Y. Murata ◽  
M. Ohno ◽  
Y. Satoh ◽  
...  
Keyword(s):  
Steady State ◽  
Adrenal Weight ◽  
Side Chain ◽  
Total Rna ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Acth Treatment ◽  
Steady State Levels ◽  
Cytochrome P 450

ABSTRACT The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.

scholarly journals 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

2002 ◽  
Vol 174 (3) ◽  
pp. 499-507 ◽  
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.

scholarly journals 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

2003 ◽  
Vol 176 (1) ◽  
pp. 151-161 ◽  
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.

scholarly journals The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1971 ◽  
Vol 123 (2) ◽  
pp. 143-152 ◽  
Author(s):  
A. P. F. Flint ◽  
D. T. Armstrong
Keyword(s):  
Microsomal Fraction ◽  
Specific Radioactivity ◽  
Subcellular Fractions ◽  
Side Chain ◽  
Electron Microscopic ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Rat Ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-14C]acetate in vitro for various times. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [14C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./μmol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./μmol. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20α-hydroxypregn-4-en-3-one was 6150d.p.m./μmol. 3. By using glutamate dehydrogenase and cytochrome (a+a3) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-14C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [14C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.

scholarly journals Effect of copper and thiomolybdates on bovine theca cell differentiation in vitro

2006 ◽  
Vol 189 (3) ◽  
pp. 455-463 ◽  
Author(s):  
N R Kendall ◽  
P Marsters ◽  
L Guo ◽  
R J Scaramuzzi ◽  
B K Campbell
Keyword(s):  
Cytochrome P450 ◽  
Mrna Expression ◽  
Lysyl Oxidase ◽  
Side Chain ◽  
Side Chain Cleavage ◽  
Theca Cells ◽  
Chain Cleavage ◽  
Copper Supplementation ◽  
Effect Of Copper

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the national dairy herd. The aims of this study were to determine the effect of copper and/or copper chelating thiomolybdates on LH-induced differentiation by looking at the effects on androgen production, steroidogenic enzymes (cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage) and lysyl oxidase mRNA expression in cultured theca cells maintained under serum-free conditions. The effect of thiomolybdates and copper on LH differentiation was investigated by supplementing (ammonium) tetrathiomolybdate to optimum theca cell culture media at 0–100 μg/ml, copper (chloride) at equimolar concentrations (0–51.6 μg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage mRNA was examined using real time PCR. Both PCRs used bovine specific primers and cell lysates obtained from bovine theca cells cultured for 6 days and in the presence or absence of the 100 μg/ml dose of thiomolybdate and equimolar copper thiomolybdate. Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 μg/ml at 96 h (P<0.05); doses above 20 μg/ml were all greatly reduced at all time points and at 192 h when related to numbers of cells (P<0.001). Copper alone had no effect at physiological doses, but the use of the equimolar copper thiomolybdate reversed the effect of tetrathiomolybdates on androstenedione production at the 20 μg/ml dose. Thiomolybdate supplementation, with and without copper, had no significant effect on the level of lysyl oxidase or cytochrome P450 side-chain cleavage expression. However, cytochrome P450 17α-hydroxylase expression was significantly increased (P<0.05) by tetrathiomolybdate, possibly due to a local regulatory system. In conclusion, these results demonstrate that thiomolybdates can prevent LH-induced differentiation of bovine theca cells in vitro and that these effects can be ameliorated by copper supplementation. Our results also indicate that it is unlikely that the effects of thiomolybdate are mediated at the transcriptional level and further work is required to determine if thiomolybdate exerts its effects through post-translation processing or some other unrelated mechanism. Overall, these data support the hypothesis that copper responsive subfertility results from perturbation of the normal pattern of ovarian steroidogenesis.

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