Transforming growth factor beta1 regulates follistatin mRNA expression during in vitro bovine granulosa cell differentiation.

In order to test the hypothesis that transforming growth factor beta (TGF-beta) acts by FS regulation on bovine granulosa cells in in vitro differentiation, we analyzed the effect of TGF-beta1 on follistatin mRNA expression in three differentiation states of bovine granulosa cells. We showed a positive regulation of FS mRNA after TGF-beta1 (1 ng/ml) treatment of freshly isolated granulosa cells from small-medium antral follicles (2-8 mm). This effect was abolished by the addition of exogenous follistatin (100 ng/ml), suggesting that this effect could be mediated by activin. Although these cells showed a similar effect on FS mRNA expression after treatment with activin-A, a soluble form of activin receptor type IIA was unable to inactivate the TGF-beta effect. When we tested the TGF-beta effect on FS mRNA in different granulosa cell states, TGF-beta1 regulation was associated with progesterone production only in freshly isolated cells. The amount of total activin-A produced by first passage cells (dedifferentiated cells), was ten times smaller than the one measured in a conditioned medium from freshly isolated cells (mature cells). The TGF-beta1-dependent FS mRNA expression persisted in first passage cells without changes with FS addition. On the other hand, the BGC-1 granulosa cell line (immature cells) produced large amounts of activin-A regulated by TGF-beta1 and an invariable steady state of FS mRNAs. In summary, our results showed that FS mRNA expression is regulated by TGF-beta1 independently of activin effects in differentiated granulosa cells.

Paradoxical effects of tumour necrosis factor-alpha on rat granulosa cell DNA synthesis.

Tumour necrosis factor-alpha (TNF-alpha) has been proposed as an intraovarian modulator of granulosa cell function. The effect of TNF-alpha on DNA synthesis in cultured rat granulosa cells was examined. Tumour necrosis factor-alpha stimulated thymidine incorporation when added in the presence of transforming growth factor-beta (TGF-beta). In contrast, the co-mitogenic effect of follicle-stimulating hormone (FSH) and TGF-beta was inhibited in a dose-dependent manner by TNF-alpha. Inhibition of FSH-dependent DNA synthesis by TNF-alpha was also found when cultures were co-stimulated with activin A. The inhibitory action of TNF-alpha on FSH-treated cultures was not associated with changes in cell viability. The inhibitory effects of TNF-alpha could not be solely explained by a decrease in cAMP levels, since TNF-alpha was also able to inhibit the stimulation by dibutyryl-cAMP and TGF-beta on granulosa cell DNA synthesis. These results suggest that TNF-alpha regulation of granulosa cell growth is elicited either independently or downstream from gonadotrophin-induced cAMP production. The actions of TNF-alpha could be only partially mimicked by a cell-permeable analogue of ceramide, thus indicating that actions of this cytokine can not be fully ascribed to an activation of sphingomyelinase. Data presented here indicate that, in addition to its previously demonstrated inhibitory effects on gonadotrophin-induced cell differentiation, TNF-alpha may also exert a marked inhibition on hormonally stimulated immature granulosa cell proliferation. In contrast to this inhibitory action, this cytokine could amplify the mitogenic action of putative intraovarian growth regulators such as TGF-beta. These observations add further support to the notion that TNF-alpha plays a physiological role as a paracrine modulator of follicle development and may be also relevant to the alteration of ovarian function during physiopathological processes.

Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation.

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.