Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries
Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries
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DOI：
10.1002/mrd.20349
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发表时间：
2005-10-01
影响因子：
2.5
通讯作者：
Manabe, N
中科院分区：
文献类型：
--
作者：
Matsuda-Minehata, F;Goto, Y;Manabe, N
卵泡选择在哺乳动物卵巢中进行，因为大多数卵泡在卵泡发育和生长期间经历闭锁。提示卵泡退化开始于颗粒细胞凋亡。为了揭示选择的分子机制，我们检测了猪颗粒细胞中细胞Flice样抑制蛋白（cFLIP）表达水平的变化。cFLIP是细胞内凋亡诱导剂（procaspase-8/Flice）的同源物，并且具有两种选择性剪接异构体：cFLIP短型（cFLIP（S））和cFLIP长型（cFLIP（L））。通过与caspase-8竞争，cFLIP抑制由死亡受体引发的细胞凋亡。采用RT-PCR和Western blotting方法检测卵巢颗粒细胞cFLIP（S）和cFLIP（L）mRNA和蛋白表达水平的变化。cFLIP（L）mRNA和蛋白在健康卵泡的颗粒细胞中高表达，闭锁时表达下降。cFLIP（S）mRNA在颗粒细胞中的表达水平较低，且在卵泡发育的不同阶段无明显变化，其蛋白表达水平极低。我们通过原位杂交检测了猪卵巢中cFLIP mRNA定位的变化，发现与闭锁卵泡的颗粒细胞相比，健康卵泡的颗粒细胞中cFLIP（L）含量丰富。免疫组化结果显示，cFLIP蛋白在健康卵泡的颗粒细胞中高表达，而在闭锁卵泡的颗粒细胞中表达较弱。我们推测cFLIP，尤其是cFLIP（L），在猪卵巢健康卵泡的颗粒细胞中起抗凋亡作用，并且cFLIP可能是决定猪卵泡是否生长或闭锁的主要存活因子。
Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.