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

Abstract

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small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days.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.

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro

“…Therefore, the addition of PKC and PI3K inhibitors could potentially alter Cyp19 expression and oestradiol secretion in FSH-stimulated cells by inhibiting IGF-I rather than FSH signalling. However, withdrawal of IGF-I from culture did not affect Cyp19 expression or oestradiol secretion in a previous study (Silva & Price 2002). Furthermore, withdrawal of both IGF-I and insulin caused a decrease in Cyp19 mRNA levels but did not affect oestradiol secretion (Silva & Price 2002), which is inconsistent with the present results (decrease in oestradiol but no change in Cyp19 mRNA).…”

“…Cells were stimulated with FSH (USDA bFSH-17; 1 ng/ml) or a higher dose of insulin (100 ng/ml) in the absence of FSH. This dose of FSH stimulates Cyp19 mRNA abundance and oestradiol secretion without affecting cytochrome P450 cholesterol side-chain cleavage (Cyp11a) or progesterone secretion, and the concentrations of insulin and IGF-I (10 ng/ml) in the medium are insufficient to support Cyp19 expression in the absence of FSH (Silva & Price 2000, 2002. The higher dose of insulin used stimulates Cyp19 mRNA levels and aromatase activity in the absence of FSH (Silva & Price 2000).…”

Effects of PI3K and FSH on steroidogenesis, viability and embryo development of the cumulus–oocyte complex after in vitro culture

“…Culture medium that did not contain FSH sustained higher levels of LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1 expression. Steroid production depends on gonadotropins and enzymes, and previous studies have demonstrated that FSH controls the expression levels of CYP11A1, CYP19A1, HSD17B1, LHR and FSHR in COCs and granulosa cells (Silva & Price, 2002;Calder et al, 2003;Sahmi et al, 2006). However, the gene expression levels of gonadotropins and enzymes are not directly related to hormone production.…”

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

“…However, in cultured bovine granulosa cells harvested from small bovine antral follicles, the addition of low dose of FSH maintains follicle characteristics by stimulating E 2 and CYP19A1 for several days (Gutierrez et al 1997, Silva & Price 2002, Sahmi et al 2004) and appears to counteract the process of luteinization by inhibiting STAR mRNA expression (Zheng et al 2008). Our previous study showed that TGFB1 inhibits stimulation of E 2 and P 4 synthesis in bovine granulosa cells cultured in the presence of FSH (Zheng et al 2008).…”