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 Cells1

Abstract

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.

Effect of TNF-alpha on LH and IGF-I modulated chicken granulosa cell proliferation and progesterone production during follicular development

“…However, Bryan et al (1997) did not study the effects of IGF-I or gonadotrophins. Soboloff et al (1999) did not find any effect of different doses (1-50 ng ml -1 ) of rhTNF-α on basal or LH-stimulated progesterone production by granulosa cells of White Leghorn hens. However, these studies were conducted under short-term 3 h incubations.…”

“…Recombinant human TNF-α (rhTNF-α) induces apoptosis in the granulosa cells of large white follicles and has a role in follicular atresia (Witty et al, 1996). TNF-α increased intracellular calcium in granulosa cells of chickens without a concomitant increase in progesterone production (Soboloff et al, 1995(Soboloff et al, , 1999. Bryan et al (1997) showed that 5 ng TNF-α ml -1 had an inhibitory effect on progesterone production by granulosa cells of two lines of chicken, whereas 250 ng ml -1 had either no effect or was stimulatory.…”

Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library

“…It has been previously shown that the metabolism of membrane sphingolipids (such as sphingomyelin or ceramide) may be an important regulatory pathway in the control of steroid metabolism and steroid hormone synthesis (Sender Baum & Ahren 1988, Hattori & Horiuchi 1992, Degnan et al 1996, Budnik et al 1999, Soboloff et al 1999. It has also been shown that in cultured fibroblasts, exogenous sphingomyelinase decreases cholesterol synthesis (Degnan et al 1996).…”

The interplay between bioactive sphingolipids and steroid hormones

“…A growing number of sphingolipid species, including ceramide (cer), sphingosine-1-phosphate (S1P), sphingosine (SPH), and sphingomyelin (SM), have been reported to modulate various steps of the steroidogenic pathway. They stimulate steroid hormone secretion by regulating steroidogenic gene transcription [23-26], acting as intracellular second messengers [27-32] and/or extracellular paracrine/autocrine regulators [25, 33-35], and by serving as ligands for nuclear receptors [26]. In this review, a summary of studies focused on the various roles of sphingolipids in the regulation of steroidogenesis will be presented.…”