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

Although tumor necrosis factor α (TNF-α) 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-α 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-α-induced Ca²⁺ regulation. Treatment with exogenous sphingo-myelinase (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 μM), but not C2-ceramide (0.03-30 μM), mimicked this effect of SMase, challenge with sphingosine (3 μM) resulted in a slow and delayed increase in basal [Ca²⁺](i). In order to determine whether SMase is activated by TNF-α 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-α (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-α 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-α 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-α did not increase the production of ceramide, the physiological regulator(s) of these responses remain unknown.