TUMOR-NECROSIS-FACTOR-ALPHA AND ITS 2ND MESSENGER, CERAMIDE, STIMULATE APOPTOSIS IN CULTURED OVARIAN FOLLICLES

In the mammalian ovary, only a small fraction of follicles fully matur e and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian med iators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell ty pes, and the apoptotic effect of TNF alpha is, partially, coupled to t he 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 th e effect of treatment with TNF alpha and ceramide on follicle apoptosi s. Immature rats were implanted with diethylstilbestrol to stimulate t he development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treat ments. During culture, spontaneous follicle apoptosis occurred (10-fol d increase in DNA fragmentation), which was partially blocked by 100 n g/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 t hat the granulosa cell is the follicle cell type undergoing DNA fragme ntation. A membrane-permeable ceramide analog, C-2-ceramide N-acetyl s phingosine, mimicked the effect of TNF alpha and was able to completel y abolish the action of FSH at 50 mu M, In contrast, another ceramide analog, C-2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To s tudy the mechanism of TNF alpha and ceramide action, the effect of sod ium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-convertin g enzyme/ced-9-related cysteine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with AT M (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating folli cle apoptosis. Treatment with either TNF alpha or ceramide increased b oth basal and FSH-stimulated progesterone production by cultured folli cles. Concomitant treatment by ATM did not alter the stimulatory effec t of TNF alpha or ceramide on progesterone production, ruling out nons pecific toxic effect of the inhibitor and indicating that the apoptoti c and steroidogenic pathways are independent. In summary, treatment wi th TNF alpha or its second messenger, ceramide, stimulates apoptosis o f early antral follicles in culture, suggesting a potential role for T NF alpha as an intraovarian regulator of follicle atresia by acting th rough the ceramide signaling pathway.