Influence of Tumor Necrosis Factor Alpha on Intracellular Ca2+ in Hen Granulosa Cells in Vitro during Follicular Development1

Abstract

In the present study, we have determined the influence of tumor necrosis factor alpha (TNF alpha) on both basal and carbamylcholine chloride (Cch)-induced [Ca2+]i in granulosa cells from the largest (F1) and smallest (F5,6) preovulatory follicles. TNF alpha (10 ng/ml) induced a small (51-63 nM) and delayed (approximately 1 min) transient increase in [Ca2+]i. The percentage of cells that responded to the cytokine was greater in F5,6 granulosa cells (48%; n = 48) than F1 granulosa cells (24%; n = 41). These responses were completely abolished in Ca(2+)-free media containing 5 mM EGTA and 2.5 mM Mg2+ or 1 mM Mn2+. Cch induced large increases (> 250 nM) in [Ca2+]i via mobilization of Ca2+ from intracellular stores in approximately 50% of Cch-responsive F1 granulosa cells but only about 15% of Cch-responsive F5,6 cells. Pretreatment with TNF alpha (4-5 min) increased the magnitude of the Cch response in both F1 and F5,6 granulosa cells previously incapable of producing large Cch-induced changes in [Ca2+]i. In F1 cells, the effects of TNF alpha on Cch-induced [Ca2+]i were far more extensive, such that the Cch response in the presence of TNF alpha was indistinguishable from the fast Cch-induced Ca2+ transient reported previously. Furthermore, the TNF alpha effect was reversible, as subsequent challenge with Cch in the absence of TNF alpha failed to produce the large Ca(2+)-transients observed earlier with the cytokine present. In conclusion, TNF alpha induces transient increases in [Ca2+]i by transmembrane Ca2+ flux, which are suppressed during cytodifferentiation. In addition, TNF alpha appeared to enhance Cch-induced mobilization of Ca2+ from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…Since TNF-␣ did not activate SMase (Fig. 2), the similarities between these observations and our previous findings of TNF-␣-induced Ca 2ϩ transients [25] may merely be coincidental. Nonetheless, although sphingosine-induced Ca 2ϩ transients have been previously reported to occur via mobilization of Ca 2ϩ from intracellular stores [33,34], this study represents the first demonstration that ceramide regulates [Ca 2ϩ ] i .…”

“…As indicated by our prior studies [25], TNF-␣ increases [Ca 2ϩ ] i in hen granulosa cells in the presence and absence of Cch. Whereas TNF-␣ challenge increased extracellular Ca 2ϩ -dependent [Ca 2ϩ ] i in approximately 50% of the undifferentiated F5,6 granulosa cells and 25% of the highly differentiated F1 cells, the cytokine facilitated slow Cchinduced Ca 2ϩ transients in both F1 and F5,6 cells, with a more pronounced response in F1 cells.…”

“…Nonetheless, a prior study from this laboratory showed that granulosa cells from both developmental stages respond to TNF-␣ with increases in both basal and Cch-induced [Ca 2ϩ ] i [25]. Moreover, additional studies of TNF-␣-transforming growth factor ␣ interactions in the regulation of F5,6 granulosa cell survival and integrin production have been performed (unpublished results).…”

“…Consequently, the similarities between sphingosine-induced Ca 2ϩ transients (Fig. 1E) and TNF-␣-induced Ca 2ϩ transients [25] are likely only coincidental.…”

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

“…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.…”