The role of transforming growth factor-beta 1 in steroidogenesis, cell proliferation, and apoptosis in cultured bovine granulosa cells.

摘要

Infertility in dairy cattle restrains the economic success of this industry. Abnormal follicle growth, anoestrus and anovulation are among the principal causes of declining fertility in high-yielding dairy cattle. To resolve these problems, more basic knowledge is required to understand the endocrine mechanisms that control normal growth of the ovarian follicle. Gonadotropin stimulation of responsive follicles is associated with increased synthesis of estradiol-17beta (E2) and progesterone (P4). E2 is a key marker of growth and selection of the dominant follicle, and P4 synthesis is necessary for the induction of ovulation. Granulosa cells play an important role in determining the fate of the developing follicle towards ovulation or atresia because these cells produce E2 and P4 and are the main target for cell proliferation and apoptosis signals.;Keywords: TGF-beta1, bovine, ovary, follicle, granulosa cell, steroidogenesis, FSH, steroids, cell cycle, apoptosis, caspase-3.;Transforming growth factor-beta1 (TGF-beta1) is an important factor produced by the follicle cells and is known to modify E2 and P4 secretion, ovarian function and fertility. The physiological effects of TGF-beta1 on granulosa cells is unclear. In rodent granulosa cells of E2-treated immature rats, TGF-beta1 stimulates E 2 and P4 in vitro, but in cultured granulosa cells of domestic animals, TGF-beta1 inhibits E2 and P4. The first objective of this study was to determine the mechanism of action of TGF-beta1 on the steroidogenic enzymes that transform androgens, estrone (E1) and cholesterol to E2 and P4 in FSH-stimulated and quiescent bovine granulosa cells. The second objective was to study the effect of TGF-beta1 on granulosa cell differentiation, proliferation and apoptosis. Bovine granulosa cells were obtained from 2-5 mm follicles and were cultured in serum-free medium in the presence or absence of FSH. Quiescent granulosa cells spontaneously luteinized by increasing P4 secretion and the mRNA expression of P4-related enzymes: StAR, CYP11A1, HSD3B and GSTA. The addition of FSH slowed down this process by increasing E2 and E2-synthetic enzymes CYP19A1 and HSD17B1 and by inhibiting StAR mRNA. In FSH-stimulated granulosa cells, TGF-beta1 inhibited the rise in E 2 and P4 secretion and inhibited the expression of the corresponding steroidogenic enzymes. TGF-beta1 inhibited expression of FSH receptor ( FSHr) mRNA and inhibited FSH-induced expression of CYP19A1 and HSD17B1 (but not HSD17B) and StAR, CYP11A1, HSD3B and GSTA. In quiescent granulosa cells, TGF-beta1 also inhibited luteinization of granulosa cells but preserved estrogenic capacity. Furthermore, this study shows that HSD178 reducing activity that transforms E1 to E2 is very high in bovine granulosa cells, is unaffected by FSH and TGF-beta1 and appears to be correlated with HSD17B7 but not HSD17B1. TGF-beta1 treatment significantly decreased the proportion of granulosa cells in the proliferative phase of the cell cycle (S, G2 and M) and increased cell death by apoptosis as indicated by an increase of cleaved caspase-3. Overall, these results demonstrate that TGF-beta1 inhibits luteinization and keeps granulosa cells in a more immature state by opposing FSH action. By acting in this manner, TGF-beta1 may have a physiological role to limit proliferation and differentiation of granulosa cells in growing antral follicles and may be involved in the process of selection of the dominant follicle.