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Acyl Chain Length-Specific Ceramide-Induced Changes in Intracellular Ca²⁺ Concentration and Progesterone Production Are Not Regulated by Tumor Necrosis Factor in Hen Granulosa Cells¹

^a Reproductive Biology Unit, Departments of Obstetrics and Gynaecology, ^b Cellular&Molecular Medicine, and ^c Biochemistry, Microbiology and Immunology, University of Ottawa, ^d The Ottawa Hospital (Civic Campus) Loeb Research Institute, Ottawa, Ontario, Canada K1Y 4E9

Although tumor necrosis factor (TNF-) 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- 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--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 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- 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- (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- 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- 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- did not increase the production of ceramide, the physiological regulator(s) of these responses remain unknown.

¹ This work was supported by the Medical Research Council of Canada (MRC grants MT-10369 to B.K.T. and MA-9690 to M.D.). J.S. is a recipient of a Natural Science and Engineering Research Council Studentship.

² Correspondence: Benjamin K. Tsang, Reproductive Biology Unit, Department of Obstetrics&Gynaecology, The Ottawa Hospital (Civic Campus), 1053 Carling Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365; ben{at}civich.ottawa.on.ca

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