The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNFα) and transforming growth factor alpha (TGFα) have been suggested. TGFα stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNFα and its possible interaction with TGFα in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNFα and TGFα interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGFα (0.1 or 10 ng/ml), but not TNFα (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin β3 subunit was also stimulated by TGFα in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNFα in the presence of maximal stimulatory concentrations of TGFα potentiated this response. TGFα increased both F5,6 and F1 granulosa cell [3H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNFα had no effect on [3H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNFα and TGFα than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGFα-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNFα alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGFα but not of TNFα. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNFα and TGFα was necrosis and not apoptosis. The current study demonstrates that TNFα and TGFα interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNFα; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.
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