新生大鼠卵母细胞凋亡信号通路SCF-FOXO3a的体外实验研究

目的:研究FOXO3a(forkhead box group O)转录因子是否参与调控哺乳动物卵巢中裸露卵母细胞和原始卵泡内卵母细胞的凋亡。方法:体外分离培养新生大鼠卵母细胞巢和原始卵泡内的卵母细胞,干细胞因子(stem cell factor,SCF)单独或与磷脂酰肌醇-3激酶(phosphoinositide 3-kinase,PI3K)抑制剂LY 294002联合作用于卵母细胞,TUNEL法凋亡染色观察卵母细胞凋亡状况;RT-PCR、Western blotting检测FOXO3a及其下游靶分子与卵母细胞凋亡的关系。结果:SCF不影响卵母细胞FOXO3a总蛋白表达水平,但使其磷酸化水平增加,从而抑制了卵母细胞凋亡;LY 294002可完全阻断上述效果。SCF使FOXO3a的靶基因Bim和p27kip1表达下调,此作用同样可被LY 294002逆转。结论:FOXO3a和其下游靶基因Bim及p27kip1可能参与了SCF信号通路对新生大鼠卵母细胞的凋亡调控。

SCF-FOXO3a Signaling Pathway Involved in Controlling Oocyte Apoptosis in Neonatal Rat Ovaries in vitro

Objective: To investigate whether FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles in mammalian ovaries. Methods: Oocytes from ovarian nests and primordial follicles of neonatal rat ovaries were cultured, and treated with stem cell factor (SCF) alone or SCF combined with the phosphoinositide 3-kinase (PI3K) inhibitor, LY 294002. Oocyte apoptosis was examined using the TUNEL technique. The expression of FOXO3a and its target genes Bim and p27kip1 were examined by RT-PCR and Western blotting. Results: SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of its phosphorylation. As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. LY 294002 abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27kip1 and Bim, and this activity was reversed by LY 294002. Conclusion: FOXO3a and its targets Bim and p27kip1 are involved in oocyte apoptosis in neonatal rat ovaries controlled by SCF signaling pathway.