Matsuda-Minehata Fuko, Goto Yasufumi, Inoue Naoko, Manabe Noboru
Laboratory of Animal Breeding, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Iwama, 319-0206 Tokyo, Japan.
Mol Reprod Dev. 2005 Oct;72(2):145-51. doi: 10.1002/mrd.20349.
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.
在哺乳动物卵巢中会进行卵泡选择，因为大多数卵泡在卵泡发育和生长过程中会发生闭锁。卵泡闭锁被认为始于颗粒细胞凋亡。为了揭示这种选择的分子机制，我们检测了猪颗粒细胞中细胞型类Fas相关死亡结构域样抑制蛋白（cFLIP）表达水平的变化。cFLIP是细胞内凋亡诱导剂（procaspase-8/Fas相关死亡结构域蛋白）的同源物，有两种可变剪接异构体：cFLIP短型（cFLIP(S)）和长型（cFLIP(L)）。通过与caspase-8竞争，cFLIP抑制由死亡受体引发的凋亡。分别通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法测定颗粒细胞中cFLIP(S)和cFLIP(L) mRNA及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，在闭锁过程中降低。颗粒细胞中cFLIP(S) mRNA水平较低，在卵泡发育各阶段无变化，其蛋白水平极低。我们通过原位杂交检测了猪卵巢中cFLIP mRNA定位的变化，发现与闭锁卵泡相比，cFLIP(L)在健康卵泡的颗粒细胞中含量丰富。免疫组织化学分析表明，cFLIP蛋白在健康卵泡的颗粒细胞中高表达，但在闭锁卵泡的颗粒细胞中弱表达。我们推测cFLIP，尤其是cFLIP(L)，在猪卵巢健康卵泡的颗粒细胞中发挥抗凋亡作用，并且cFLIP可能是决定猪卵泡生长或闭锁的主要存活因子。
