Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.