Follicular stage-dependent tumor necrosis factor alpha-induced hen granulosa cell integrin production and survival in the presence of transforming growth factor alpha in vitro

The link between cell adhesion to extracellular matrix and integrin-mediate d survival signals has been established in several physiological systems, a nd roles for the cytokines tumor necrosis factor alpha (TNF alpha) and tran sforming growth factor alpha (TGF alpha) have been suggested. TGF alpha sti mulates fibronectin production in hen granulosa cells and is an important s urvival factor during follicular maturation. In contrast, the role of TNF a lpha and its possible interaction with TGF in the regulation of granulosa c ell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determin e if TNF alpha and TGF alpha interact in the regulation of hen granulosa ce ll fibronectin and integrin content in the context of cell death and surviv al during follicular development. TGF alpha (0.1 or 10 ng/ml), but not TNF alpha (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin c ontent in granulosa cell cultures of F5,6 but not F1 follicles. The express ion of integrin beta (3) subunit was also stimulated by TGF alpha in a foll icular stage-dependent manner, and culture of F5,6 granulosa cells with TNF alpha in the presence of maximal stimulatory concentrations of TGF alpha p otentiated this response. TGF alpha increased both F5,6 and F1 granulosa ce ll [H-3]thymidine incorporation but not 3-(4,3-dimethylthiazol-2-yl)3,5-dip henyl tetrazolium bromide (MTT) metabolism. Although TNF alpha had no effec t 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 TNF alpha and TGF alpha than with either cytokine alone. Incuba tion of F5,6 granulosa cells for 48 and 72 h resulted in a TGF alpha -inhib ited loss of cellular adhesion and detachment of granulosa cells from the g rowth surface. Although TNF alpha alone had no effect on cell morphology, i t facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradat ion significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF alpha but not of TN F alpha. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and at tenuated by the presence of TNF alpha and TGF alpha was necrosis and not ap optosis. The current study demonstrates that TNF alpha and TGF alpha intera ct in the regulation of granulosa cell integrin content and cell survival i n vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF alpha; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.