Expression of 17β- and 3β-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro

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.

Section: Discussionsupporting

confidence: 94%

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

“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Zheng¹,

Boerboom²,

Carrière³

2009

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.

“…Alternatively, other enzymes involved in oestrogen biosynthesis, such as NADPH-cytochrome P450 reductase and 17bHSD, may be regulated through PKA-independent pathways. Phorbol ester increased 17bHSD type 1 expression in JET-3 choriocarcinoma cells (Piao et al 1997), although 17bHSD is regulated by FSH in bovine granulosa cells (Sahmi et al 2004).…”

Section: Figure 4 Effect Of Inhibitors Of Intracellular Signalling Pamentioning

confidence: 91%

“…They express Cyp19 mRNA predominantly from the ovarian-specific Cyp19 promoter (Hamel et al 2005) and do not luteinize (Sahmi et al 2004). The present data show that under these culture conditions, FSH stimulates Cyp19 mRNA and oestradiol secretion predominantly through cAMP signalling, most likely to PKA.…”

Section: Figure 4 Effect Of Inhibitors Of Intracellular Signalling Pamentioning

confidence: 99%

“…The emphasis on 'oestrogenic' granulosa cells is important, as granulosa cells undergo a change in responsiveness to FSH/cAMP during luteinization (Gonzalez-Robayna et al 1999), thus potentially changing the physiological response of cells to various ligands. The aim of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate Cyp19 expression and oestradiol accumulation in an established non-luteinizing bovine granulosa cell culture model (Gutiérrez et al 1997, Sahmi et al 2004). …”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Silva¹,

Hamel²,

Sahmi³

et al. 2006

Self Cite

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P!0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin-and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway. Reproduction (2006) 132 909-917