Acyl chain length-specific ceramide-induced changes in intracellular Ca2+ concentration and progesterone production are not regulated by tumor necrosis factor alpha in hen granulosa cells

Although tumor necrosis factor alpha (TNF-alpha) has long been known to be a potent inhibitor of gonadotropin-induced cytodifferentiation in the ovaries of a variety of mammalian species, its early signal transduction events are poorly understood. We previously demonstrated that TNF-alpha induces a small, delayed follicular stage-dependent increase in intracellular Ca2+ concentration (Ca2+]i) in hen granulosa cells and promotes carbachol (Cch)-induced mobilization of Ca2+ from intracellular stores in cells otherwise unresponsive to the cytokine. The focus of the current study was to examine the role of ceramide in TNF-alpha-induced Ca2+ regulation. Treatment with exogenous sphingomyelinase (SMase; 50 mU/ml) failed to influence basal Ca2+]i but increased the magnitude of Cch-induced Ca2+ transients. While C8-ceramide (0.03-30 microM), but not C2-ceramide (0.03-30 microM), mimicked this effect of SMase, challenge with sphingosine (3 microM) resulted in a slow and delayed increase in basal Ca2+]i. In order to determine whether SMase is activated by TNF-alpha action, changes in sphingomyelin and ceramide concentrations in F1 and F5,6 granulosa cells were determined. SMase activation was not observed after 1-, 5-, 15-, and 60-min incubations with TNF-alpha (1-50 ng/ml) in either F1 or F5,6 cells. Exogenous SMase and C2-ceramide both inhibited LH-induced progesterone production in F1 and F5,6 cells; however, incubation with C8-ceramide resulted in increases in both basal and LH-induced progesterone. In contrast, incubation with TNF-alpha had no effect on either basal or LH-induced steroidogenesis. In conclusion, our findings indicate that although ceramide regulates Ca2+]i and progesterone secretion, the sphingolipid does not appear to play a role in the action of TNF-alpha in avian granulosa cells. Furthermore, ceramide-mediated responses are highly dependent on acyl chain length, potentially reflecting differences in the abilities of these ceramides to access, bind to, and/or activate ceramide-dependent signal transduction mechanisms. Nonetheless, since TNF-alpha did not increase the production of ceramide, the physiological regulator(s) of these responses remain unknown.