卵丘细胞上AIFM2和FDX1的表达及其与小鼠卵母细胞成熟的关系

摘要

目的 检测小鼠卵母细胞体外、体内成熟过程中卵丘细胞(CCs)上线粒体相关凋亡因子2(AIFM2)和铁氧化还原蛋白1(FDX1)的表达,探讨其在卵母细胞成熟中的作用. 方法 从3周龄ICR雌性小鼠获取体内成熟卵丘-卵母细胞复合体(IVV-COCs)以及GV期卵丘-卵母细胞复合体(GV-COCs),并将GV期体外培养至MⅡ期卵丘-卵母细胞复合体(IVM-COCs).收集对应的CCs,分成GV-CCs、IVM-CCs和IVV-CCs 3组.实时定量逆转录聚合酶链反应检测AIFM2和FDX1 mRNA水平的表达,以GAPDH为参照;激光共聚焦免疫荧光技术观察COCs的AIFM2和FDX1的亚细胞定位. 结果 IVM-CCs和IVV-CCs的AIFM2 mRNA相对表达量[分别为(0.72±0.01)和(0.67±0.02)]显著高于GV-CCs组[(0.10±0.06)](P<0.01);同样,IVM-CCs和IVV-CCs的FDX1 mRNA相对表达量[分别为(1.58±0.35)和(1.82±0.34)]也显著高于GV-CCs组[(0.10±0.03)](P<0.01);而IVM-CCs和IVV-CCs的AIFM2和FDX1表达水平均无显著性差异(P>0.05).免疫荧光显示,AIFM2和FDX1主要定位于CCs胞质中,在GV-COCs中各层均有表达,且GV期卵母细胞中也有表达;IVM-COCs和IVV-COCs中表达阳性的细胞数目较GV期增加. 结论 卵母细胞成熟后,其CCs上AIFM2和FDX1的表达升高,表明AIFM2和FDX1参与了卵母细胞成熟过程,有望作为预测卵母细胞成熟的生物标记物.%Objective: To detect the expressions of apoptosis-inducing factor mitochondrion-associated 2 (AIFM2) and ferredoxin 1 (FDX1) mRNA in the cumulus cells (CCs) around the cumulus oocyte complex (COCs) undergoing in vitro maturation (IVM) or in vivo maturation,and investigate their potential functions in regulating oocyte maturation. Methods: In vivo maturation COCs (IVV-COCs) and germinal vesicle COCs (GV-COCs) were isolated from 3 weeks old mice,and then GV-COCs were cultured to metaphase Ⅱ stage (IVM-COCs) by in vitro maturation. The CCs were collected from GV-COCs,IVM-COCs and IVV-COCs respectively,and defined as GV-CCs group,IVM-CCs group and IVV-CCs group. The expression of AIFM2 and FDX1 mRNA were detected by RT-qRCR. The sub-cellular location of FDX1 protein and AIFM2 protein in GV-COCs,IVM-COCs and IVV-COCs were observed by laser scanning confocal microscope. Results: The expression of AIFM2 mRNA in IVM-CCs group [(0.72±0.01)] and IVV-CCs group [(0.67±0.02)] was significantly higher than that in GV-CCs group [(0.10±0.06)] (P<0.01). The expression of FDX1 mRNA in IVM-CCs group [(1.58±0.35)] and IVV-CCs group [(1.82±0.34)] was also significantly higher than that in GV-CCs group [(0.10±0.03)] (P<0.01). However,there was no significantly difference in the expression of AIFM2 and FDX1 between IVV-CCs group and IVM-CCs group (P>0.05). The immunofluorescence results showed that both AIFM2 and FDX1 were mainly located in the cytoplasm of CCs and expressed in each layer of GV-COCs,which were also expressed in oocytes at GV stage. The number of positive cells in IVM-COCs and IVV-COCs increased compared with GV. Conclusions: After oocyte maturation,the expression of AIFM2 and FDX1 is increased in CCs,indicating that AIFM2 and FDX1 are involved in the process of oocyte maturation. It is expected to be a biomarker for predicting oocyte maturation.