卵母细胞来源对水牛体细胞核移植胚胎发育潜能的影响

Effects of Oocyte Origin on the Developmental Potential of Buffalo Somatic Cell Nuclear Transfer Embryos

【目的】探索不同来源卵母细胞对体细胞核移植(SCNT)重构胚的发育能力及发育潜能关键蛋白表达水平的影响。【方法】试验分为活体采卵(OPU)和屠宰场(SLH)卵巢2组,OPU组用超声波活体采卵仪穿刺抽吸10头非泌乳期经产水牛卵巢的卵泡采卵,SLH组从屠宰场卵巢抽吸卵泡采卵。获得的卵母细胞分别进行体外成熟,体外成熟22~24 h后,吹打去除卵丘细胞,挑选具有第一极体的卵母细胞,去核后与水牛耳部成纤维细胞进行SCNT,分别统计SCNT重构胚的融合率、分裂率和囊胚率,用免疫荧光检测2种SCNT重构胚的E-钙黏蛋白(E-cadherin)和转录因子Sox2蛋白的表达水平。【结果】OPU组卵母细胞成熟率及其SCNT重构胚的囊胚率均显著高于SLH组(P<0.05),但2组SCNT重构胚的融合率和分裂率均无显著差异(P>0.05);免疫荧光结果显示,E-cadherin蛋白定位于细胞膜上,Sox2蛋白分布在细胞核膜及细胞质中,OPU组SCNT重构胚中E-cadherin和Sox2的表达水平均显著高于SLH组(P<0.05)。【结论】活体采集的水牛卵母细胞更适合用于SCNT重构胚的构建。

【Objective】This study was to explore the effects of different oocyte’s origin on developmental ability of reconstructed embryos after somatic cell nuclear transfer(SCNT)and the expression levels of key development related proteins.【Method】The experiment was divided into two groups:Ovum pick up(OPU)and slaughterhouse(SLH)groups.In the OPU group,the follicles of 10 non-lactating buffalo were punctured and aspirated by ultrasonic in vivo to collect oocytes.In SLH group,follicles were drawn from the ovaries of slaughterhouses to collect oocytes.All the COCs were matured in vitro for 22-24 h,respectively.The cumulus cells were removed by blowing,the oocytes with the first polar body were selected and enuvleated for SCNT using ear fibroblasts as donor cells,and the fusion rate,cleavage rate and blastocyst rate of SCNT reconstructed embryos were recorded respectively.Furthermore,the protein expression levels of E-cadherin and transcription factor Sox2 in two SCNT reconstructed embryos were determinated by immunofluorescence.【Result】The maturation rate of oocytes and the blastocyst rate of reconstructed embryos in OPU group was significantly higher than that of SLH group(P<0.05),but there were no significant differences in fusion rate and blastocyst rate of two SCNT reconstructed embryos(P>0.05).According to the analysis of immunofluorescence images,E-cadherin protein was located on the cell membrane,Sox2 protein was distributed in the cell nuclear membrane and cytoplasm,and the expression level of E-cadherin and Sox2 protein in OPU group SCNT embryos was significantly higher than that of SLH-SCNT embryos(P<0.05).【Conlusion】Buffalo oocytes collected in vivo were more suitable for the construction of SCNT reconstructed embryos.