猪卵母细胞c-mos基因表达以及RNA干扰

Expression of c-mos gene and its RNAi in porcine oocyte

c-mos基因在动物卵母细胞减数分裂调控中起作用,但其作用机制目前仍不清楚。本实验通过RT-PCR、免疫荧光激光共聚焦检测方法检测了猪卵母细胞在体外成熟培养过程中c-mos基因在转录水平、翻译水平上的表达以及蛋白的分布,并应用注射小干扰RNA(siRNA)方法对其进行了RNA干扰(RNAi)研究。结果显示,猪卵母细胞在体外成熟培养过程中c-mos基因mRNA量逐渐增高,电激活后6h接近完全降解;MOS(c-mos基因蛋白产物)在GV卵母细胞生发泡中有一定量的表达,生发泡破裂(GVBD)前表达量增加且开始向卵母细胞胞质弥散,成熟培养44h未成熟卵母细胞中的MOS表达量要高于成熟卵母细胞,激活后6h核区MOS明显减少,但仍然有少量MOS分布于胞质中;成熟培养前干扰c-mos基因,所用三个siRNA都能成功敲低mRNA量,分别是同时期对照组mRNA量的0.08±0.03,0.11±0.06和0.20±0.06倍,干扰后虽然没有完全剔除MOS,但MOS量比同期卵母细胞有明显下降,仍可以引发成熟卵母细胞染色体解凝集。研究结果揭示了猪卵母细胞体外成熟及发育进程中c-mos基因在转录和翻译水平上的动态表达规律,建立了猪卵母细胞c-mos基因RNAi体系,为MOS在猪卵母细胞发育过程中的功能研究建立了重要的基础。

c-mos involves in the regulating of oocyte meiosis process, but the mechanism is still not clear, c-mos transcriptional and translational expression pattern during in vitro maturation （IVM） and early parthenogenetic development were studied by realtime PCR and immuno-fluorescence confocal microscopy, c-mos RNA interference （RNAi） system was studied by microinjection of short interference RNA （siRNA） . The results showed that c-mos mRNA increased during IVM, and its degradation happened around 6 h after electric activation. At germinal vesicle （GV） stage, MOS （protein product of c-mos gene） was found inside GV. There was an surge expression of MOS before germinal vesicle breakdown （GVBD） and at the same time began to disperse towards cytoplasm. After 44 h IVM, MOS level in the immatured ooeytes was higher than matured oocytes, and there was less MOS in nucleus than in the cytoplasm at 6 h after matured oocytes were activated; RNAi was performed before IVM, and all the three siRNA applied could successfully knockdown the level of c-mos mRNA, which were 0.08 ± 0.03, 0.11 ± 0.06 and 0.20± 0.06 times of the control group respectively, and the quantity of MOS was obvious decreased compared with the control group at the same stage. Although MOS is not depleted completely, knockdowned MOS by RNAi could induce chromosomes ofmatured oocytes to de-condensation without activation. The study illuminated a dynamic c-rnos transcriptional and translational expression pattern during the process of IVM and early in vitro parthenogenetic development of porcine ooeyte, and established a RNAi system on c-rnos gene in porcine oocyte. These results provided an important base for the study of MOS function in porcine ooeyte .