线粒体移植对水牛卵母细胞发育潜能的影响

Effects of Mitochondrial Transplant on the Developmental Potential of Buffalo Oocytes

本研究以水牛卵泡颗粒细胞作为线粒体的来源细胞,初步探讨水牛卵母细胞进行线粒体移植(MIT)后对其发育潜能的影响。试验比较了不同级别水牛卵母细胞mtDNA的拷贝数,并研究了水牛卵母细胞进行MIT后,其后续早期胚胎发育及胚胎线粒体膜电位(ΔΨm)的变化情况。结果显示:一级卵母细胞组的平均mtDNA拷贝数极显著高于二、三级卵母细胞组((202 101±74 432)vs(118 483±17 028),(39 177±7 938),P<0.01),二级卵母细胞组的平均mtDNA拷贝数极显著高于三级卵母细胞组((118 483±17 028)vs(39 177±7 938),P<0.01);孤雌激活处理后发现:一级卵母细胞组的卵裂率和囊胚率也都极显著高于二、三级卵母细胞组(P<0.01),而二级卵母细胞组激活后胚胎的卵裂率和囊胚率亦极显著高于三级卵母细胞组(P<0.01)。用Mito-Tracker探针标记的外源线粒体经移植后,会随着胚胎的发育而发生移动,分布到各个卵裂球中;且发现二级卵母细胞MIT组孤雌激活后的囊胚率显著高于对照组和空注组(27.3%vs 17.4%,7.84%,P<0.05),而三级卵母细胞MIT组激活后的囊胚率与对照组和空注组差异不显著(P>0.05);对胚胎ΔΨm检测发现,水牛植入前各时期胚胎ΔΨm总体呈上升趋势,线粒体移植后胚胎各时期的ΔΨm均显著高于对照组(P<0.05)。综上表明:不同质量的水牛卵母细胞其mtDNA拷贝数存在显著差异,且mtDNA拷贝数与卵母细胞质量和发育能力成正相关;通过移植外源线粒体可以提高二级水牛卵母细胞的发育潜能。

The aim of present study was to investigate the effects of mitochondrial transplant(MIT)on the developmental potential of buffalo oocytes.The mitochondrial DNA(mtDNA)copy number in the oocyte,and the developmental competence,exogenous mitochondria distribution,as well as mitochondrial membrane potential(ΔΨm)in the preimplantation embryos after the MIT were examined.The result showed that the average mtDNA copy number of buffalo oocytes in the first level of oocytes was evidently higher than the second and the third level of oocytes((202 101±74 432)vs(118 483±17 028),(39 177±7 938),P<0.01),and the average mtDNA copy number in the second level of oocytes was significantly higher than that of the third level of oocytes((118 483±17 028)vs(39 177±7 938),P<0.01).Furthermore,the cleavage rates and blastocyst rates of the first level of oocytes were significantly higher than those of the second and the third level of oocytes after parthenogenetic activation(P<0.01),and the cleavage rates and blas-tocyst rates of the second level of oocytes were remarkably higher than those of the third level of oocytes(P<0.01).The exogenetic mitochondria labeled with Mito-Tracker fluorescent probe distributed into each of blastomeres with the embryonic development after MIT.Moreover,more the second level of oocytes that received MIT developed to the blastocyst stage after parthenogenetic activation in comparison with oocytes that did not received MIT or were injected with solution(27.3% vs 17.4%,7.84%,P<0.05),however,there were no significant difference in the blastocyst rates of the third level of oocytes than the control group and the nothing injection group after MIT.TheΔΨm of buffalo preimplantation embryo presented an increased trend during embryonic development,and theΔΨm of embryo derived from MIT group was significantly higher than that of the control group(P<0.05).These results showed that the mtDNA copy numbers in different quality buffalo oocytes have significant differences,and the mtDNA copy numbers of buffalo oocytes are positively related with the quality of oocytes and their developmental potential,and the developmental ability of the second level of oocytes can be improve by MIT.