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

• Published in: Biology of reproduction 1999-02, Vol.60 (2), p.262
• Main Authors: J. Soboloff, A. Sorisky, M. Désilets, B.K. Tsang
• Format: Article
• Language: English
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod60.2.262

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 Ca 2+ concentration ([Ca 2+ ] i ) in hen granulosa cells and promotes carbachol (Cch)-induced mobilization of Ca 2+ 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 Ca 2+ regulation. Treatment with exogenous sphingomyelinase (SMase; 50 mU/ml) failed to influence basal [Ca 2+ ] i but increased the magnitude of Cch-induced Ca 2+ 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 [Ca 2+ ] 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 [Ca 2+ ] 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.