Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.
Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.
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DOI：
10.1210/endo.137.11.8895358
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发表时间：
1996-11
期刊：
影响因子：
4.8
通讯作者：
Antti Kaipia;S. Chun;K. Eisenhauer;Aaron J. W. Hsueh
中科院分区：
文献类型：
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作者：
Antti Kaipia;S. Chun;K. Eisenhauer;Aaron J. W. Hsueh
在哺乳动物卵巢中，只有一小部分卵泡完全成熟并排卵，而大多数卵泡通过细胞凋亡死亡。促进卵泡存活的多种因素已被确定，但卵巢内细胞凋亡介质知之甚少。肿瘤坏死因子-α（TNF α）是一种能够在不同细胞类型中诱导细胞凋亡的细胞因子，TNF α的细胞凋亡作用部分与神经酰胺作为第二信使的鞘磷脂信号传导途径偶联。因为TNF α已经定位于大鼠卵巢，并且TNF α处理增加颗粒细胞神经酰胺的产生，我们研究了TNF α和神经酰胺处理对卵泡凋亡的影响。用己烯雌酚刺激未成熟大鼠早期有腔卵泡的发育。分离卵泡，并在无血清培养基中培养24小时，有或没有激素处理。在培养过程中，发生自发性卵泡凋亡（DNA片段增加10倍），100 ng/ml FSH部分阻断（60%抑制）。FSH的作用以剂量依赖性方式被TNF α抵消，在100 ng/ml TNF α时效果最大（FSH作用逆转90%）。原位分析表明，颗粒细胞是DNA断裂的卵泡细胞类型。一种膜渗透性神经酰胺类似物，C2-神经酰胺N-乙酰鞘氨醇，模拟TNF α的作用，能够完全消除50 μ M FSH的作用。相反，另一种神经酰胺类似物，C2-二氢神经酰胺N-乙酰二氢鞘氨醇，没有改变FSH的作用，验证了神经酰胺作用的特异性。为了研究TNF α和神经酰胺作用的机制，研究了金硫代苹果酸钠（ATM）的作用，金硫代苹果酸钠（ATM）是已知在哺乳动物细胞凋亡的执行中必不可少的白细胞介素-1 β转化酶/ced-3相关胱氨酸蛋白酶的抑制剂。ATM（1 mM）治疗阻止了TNF α和神经酰胺的促卵泡凋亡诱导作用，表明半胱氨酸蛋白酶在介导卵泡凋亡中的作用。用TNF α或神经酰胺治疗增加了培养卵泡的基础和FSH刺激的孕酮产生。ATM的伴随治疗没有改变TNF α或神经酰胺对孕酮产生的刺激作用，排除了抑制剂的非特异性毒性作用，并表明细胞凋亡和类固醇生成途径是独立的。总之，用TNF α或其第二信使神经酰胺治疗，刺激培养中早期有腔卵泡的凋亡，表明TNF α通过神经酰胺信号通路作为卵泡闭锁的卵巢内调节剂的潜在作用。
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.