Determination of Nonconjugated and Conjugated Steroid Levels in Plasma and Prostate after Separation on C‐18 Columns

Bélanger, Alain; Couture, Jean; Caron, Serge; Roy, R

doi:10.1111/j.1749-6632.1990.tb34299.x

Cited by 94 publications

(33 citation statements)

References 13 publications

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“…Culture medium samples were assayed for E 2 as previously reported (Bélanger et al 1990) but without C-18 column extraction. Cross-reaction of A 4 and E 1 with the E 2 assay was !0.1% (Bélanger et al 1980).…”

Section: Steroid Assaysmentioning

confidence: 99%

Transforming growth factor-β1 inhibits luteinization and promotes apoptosis in bovine granulosa cells

Zheng¹,

Boerboom²,

Carrière³

2009

Reproduction

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We have previously shown that TGFB1 inhibits estradiol (E 2 ) and progesterone (P 4 ) biosynthesis in FSH-stimulated bovine granulosa cells by selective inhibition of steroidogenic enzymes. The objective of this study was to assess the effects of TGFB1 on E 2 and P 4 steroidogenesis in bovine granulosa cells cultured in the absence of FSH and to measure the effects of TGFB1 on cell proliferation and apoptosis in the presence and absence of FSH. Bovine granulosa cells from 2 to 5 mm follicles were cultured in serum-free medium for 2-6 days. In the absence of FSH, the secretion of P 4 increased with time in culture (P!0.05). Addition of TGFB1 for 6 days decreased P 4 secretion and mRNA levels of the P 4 synthesis-associated genes STAR, CYP11A1, HSD3B1, and GSTA (P!0.05). In the absence of FSH, the secretion of E 2 decreased and addition of TGFB1 for 6 days partially reversed this decline and stimulated E 2 biosynthesis, CYP19A1 and HSD17B1 mRNA levels and CYP19A1 activity (P!0.05). Conversely, TGFB1 did not affect HSD17B7 expression and HSD17B-reducing activity. TGFB1 decreased the proportion of cells in the G0/G1 and SCG2/M phases in FSHstimulated and unstimulated granulosa cells (P!0.05). Furthermore, in the presence or absence of FSH, TGFB1 increased the proportion of cells in apoptosis measured by propidium iodide staining and flow cytometry and confirmed by increased levels of cleaved caspase-3 (P!0.05). Our results therefore indicate that TGFB1 inhibits luteinization in cultured bovine granulosa cells while maintaining an estrogenic phenotype, and this effect was associated with increased apoptosis.

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Section: Steroid Assaysmentioning

confidence: 99%

Transforming growth factor-β1 inhibits luteinization and promotes apoptosis in bovine granulosa cells

Zheng¹,

Boerboom²,

Carrière³

2009

Reproduction

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show abstract

“…Culture medium samples were assayed for E 2 as reported previously (Bélanger et al 1990), but without C-18 column extraction. Cross-reaction of A 4 and E 1 with the E 2 assay was less than 0.1% (Bélanger et al 1980).…”

Section: Steroid Assaysmentioning

confidence: 99%

Role of transforming growth factor-β1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

Zheng

Price²,

Tremblay³

et al. 2008

Reproduction

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Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-b1 (TGFB1) in the regulation of estradiol-17b (E 2 ) and progesterone (P 4 ) secretion in FSHstimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E 2 secretion and mRNA expression of E 2 -related enzymes cytochrome P450 aromatase (CYP19A1) and 17b-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E 2 secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P 4 secretion and mRNA expression of 3b-hydroxysteroid dehydrogenase (HSD3B) and a-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P 4 and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P 4 secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E 2 , but did not decrease the conversion of estrone (E 1 ) to E 2 and pregnenolone to P 4 . Overall, these results indicate that TGFB1 counteracts stimulation of E 2 and P 4 synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E 2 and cholesterol to P 4 without shutting down HSD17B reducing activity and HSD3B activity.

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“…Oestradiol was measured in conditioned medium without extraction with the assay described by Bélanger et al (1990), with modifications (Price et al 1995). Inter-and intra-assay coefficients of variation (CV) were 11 and 9% repectively.…”

Section: Steroid Assaysmentioning

confidence: 99%

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

Price

2002

Journal of Endocrinology

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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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Determination of Nonconjugated and Conjugated Steroid Levels in Plasma and Prostate after Separation on C‐18 Columns

Cited by 94 publications

References 13 publications

Transforming growth factor-β1 inhibits luteinization and promotes apoptosis in bovine granulosa cells

Transforming growth factor-β1 inhibits luteinization and promotes apoptosis in bovine granulosa cells

Role of transforming growth factor-β1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

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

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