绵羊卵母细胞超低温冷冻对四跨膜蛋白CD9表达的影响

试验旨在探讨不同发育时期卵泡上四跨膜蛋白CD9的表达,以及体外成熟和超低温冷冻对绵羊卵母细胞CD9的影响。采用免疫组织化学技术检测CD9蛋白在卵泡上的表达部位,实时荧光定量PCR技术检测CD9 mRNA的表达量,Western blotting技术检测CD9蛋白的含量。免疫组织化学结果发现,在绵羊原始卵泡上就开始检测到CD9荧光信号,且随着卵泡的成熟荧光信号逐渐增强,成熟卵泡时荧光信号达到最强;实时荧光定量PCR检测新鲜MⅡ期卵母细胞中CD9 mRNA的表达量显著增高（P<0.05）,冷冻GV期卵母细胞CD9 mRNA的表达量最少;Western blotting检测得出新鲜MⅡ期卵母细胞中CD9蛋白表达最多,冷冻GV期卵母细胞上表达最少,与实时荧光定量PCR结果一致。卵母细胞的冷冻保存要比胚胎保存应用前景广泛,而四跨膜蛋白CD9在精卵融合中更有着重要的作用,以上结果均显示,冷冻保存降低卵母细胞四跨膜蛋白CD9含量,而CD9的损伤是受精率下降的重要原因之一。     

Effect of Cryopreservation on the Expression of Tetraspanin CD9 in Sheep Oocytes

The experiment was aimed to explore the expression of CD9 in the follicles at different developmental stages, and investigate the effect of in vitro maturation and cryopreservation on CD9. The expression of CD9 protein in the follicle was detected by immunohistochemistry technique, the expression of CD9 mRNA was detected by Real-time quantitative PCR, CD9 protein was detected by Western blotting. The results showed that the CD9 fluorescence signal was detected by immunohistochemistry technique, and the fluorescence signal gradually increased with the maturation of the follicle, and fluorescence signal was the strongest in mature follicle;Real-time quantitative PCR showed that the expression of CD9 mRNA in fresh MⅡ oocytes was significantly increase (P<0.05), and was the least in frozen GV oocytes;The result of Western blotting indicated that CD9 protein was the most in fresh MⅡ oocytes and the expression of frozen GV oocytes was the least, consistent with the results of Real-time quantitative PCR. Cryopreservation of oocytes was more extensive than embryo cryopreservation. Tetraspanins CD9 played a very important role in sperm egg fusion. The above experimental results showed that the cryopreservation made damage to oocytes, decrease content of tetraspanins CD9 protein, and CD9 injury was one of the important reasons for the decline of fertilization rate.