牛生发泡期裸卵体外成熟培养体系的建立  

Establishment of Maturation System for Bovine Denuded Germinal Vesicle Oocytes in vitro

作　　者：隋进强[1] 云彦[1] 刘文强[1] 殷吉庆[1] 胡勇策[1] 雷安民[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2010年第1期198-204,共7页Journal of Agricultural Biotechnology

基　　金：西北农林科技大学留学回国人员科研启动项目(No.Z111020723)资助

摘　　要：本研究通过牛(Bos taurus)卵母细胞体外成熟培养不同时间(0和10h),剥除卵母细胞外周的颗粒细胞后继续培养,观察其与不脱颗粒细胞正常体外成熟培养卵母细胞之间的差异。研究结果显示:成熟培养0脱颗粒细胞的卵母细胞,与正常成熟培养的卵母细胞在成熟率(19.51%vs.80.14%,P<0.05)、卵裂率(27.08%vs.82.61%,P<0.05)和孤雌激活胚胎囊胚率(7.69%vs.21.71%,P<0.05)差异显著;成熟培养10h后脱颗粒细胞的牛卵母细胞,与正常体外成熟培养的卵母细胞相比,在成熟率(82.71%vs.80.14%,P>0.05)、卵裂率(83.01%vs.82.61%,P>0.05)和孤雌激活胚胎囊胚率(19.30%vs.21.71%,P>0.05)无显著差异。向成熟培养10h脱颗粒细胞的卵母细胞内注射pVenus-H1foo mRNA,pVenus,pDsRed1-N1和重组质粒pDsRed1-H1e都能够得到表达。非功能标记基因显微注射组与对照组在卵母细胞成熟率(81.42%vs.82.03%,P>0.05)、卵裂率(75.24%vs.78.15%,P>0.05)和孤雌激活胚胎囊胚率(17.42%vs.18.82%,P>0.05)上差异不显著。研究结果提示:在成熟培养10h,剥离颗粒细胞不会影响牛卵母细胞的发育潜能,这一技术平台可以用于牛卵母细胞体外成熟分子机制的研究。Peripheral granulosa cells around Bos taurus oocytes were removed at different time（0 and 10 h） during in vitro maturation（IVM）,and the development capacity of these oocytes was compared with that of normal IVM oocytes.The results showed significant differences between 0 h treated oocytes and the normal maturation of oocytes on maturating rate（19.51% vs.80.14%,P0.05）,cleavage rate（27.08% vs.82.61%,P0.05）,and parthenogenetic blastocyst rate（7.69% vs.21.71%,P0.05）;But there were no significant differences between 10 h trained group and normal group on maturating rate（82.71% vs.80.14%,P0.05）,cleavage rate（83.01% vs.82.61%,P0.05）,and parthenogenetic blastocyst rate（19.30% vs.21.71%,P0.05）.At subsequent experiment,the mRNA of recombinant plasmid pVenus-H1foo,vacancy plasmid of pVenus,pDsRed1-N1,and recombinant plasmid pDsRed1-H1e were injected into the denuded oocytes at 10 h IVM,and the results showed that all of them can be expressed normally.There were no significant differences between microinjection non-functional marker gene group and control group on maturating rate（81.42% vs.82.03%,P0.05）,cleavage rate（75.24% vs.78.15%,P0.05）,and parthenogenetic blastocyst rate（17.42% vs.18.82%,P0.05）.Results demonstrate that denuded granule cells in 10 h maturation did not affect the oocyte development potential,and the technologial platform can be used for molecular mechanism research on bovine oocytes maturation in vitro.

关 键 词：牛 卵母细胞 显微注射 MRNA 质粒 

分 类 号：S823.3[农业科学—畜牧学]