猪克隆胚早期发育过程中供体线粒体的动态变化及胞吐样排出对其清除的影响

Dynamic changes of donor mitochondria during the early development of porcine cloned embryos and the effect of cytoplasmic discharge on their clearance

为观察猪克隆胚早期发育过程中供体线粒体的动态变化,并初步探究2细胞时期卵裂球间胞质排出对供体线粒体清除的特异性,以孤雌胚为对照,并使用预先标记线粒体的供核体细胞生产克隆胚。分别收集特定时期的胚胎进行共聚焦显微镜以及透射电镜的观察。结果显示:电击活后0~4 h,供体线粒体逐渐从核周扩散到胞质中。与孤雌胚相比,4 h克隆胚中的线粒体具有更多异常的形态。在2细胞(28 h)以及4细胞(52 h)阶段,根据在各个卵裂球中存在的数量,我们分别观察到3种供体线粒体的分布形式。其中在2细胞阶段,以三种形式存在供体线粒体的胚胎的比例分别为51.22%(All)、26.83%(Half)以及21.95%(No)。而在克隆囊胚中,仍可观察到少量红色荧光。此外,在多数2细胞克隆胚(87.93%)卵裂球间有胞质排出,并且在部分胚胎排出的胞质中可以观察到供体线粒体的存在。5μg/mL细胞松弛素B(cytochalasin B,CB)的添加没有明显改变携带供体线粒体的2细胞克隆胚的比例。同时2细胞孤雌胚中也存在相似的胞质排出现象,并且排出区域也可以观察到母源线粒体的存在。结果提示:供体线粒体在2细胞以及4细胞克隆胚中存在不均等分布、并于部分2细胞克隆胚两个卵裂球间随胞质排出的现象,而这种排出对于克隆胚中供体线粒体的清除不具有特异性。

To observe the dynamic distribution of donor mitochondria in cloned embryos during their early development and provide the preliminary inquisition to the specificity of cytoplasmic discharge on the clearance of donor mitochondria,cloned embryos were produced by using pig fetal fibroblasts with pre-labeled mitochondria as donor cells while parthenogenetic activation（ PA） embryos were used as control.The immunostaining with the confocal microscopy and transmission electron microscopy observation were performed on the embryos which were at different developmental stages. Our results showed that donor mitochondria were gradually scattered to part of the cytoplasm（ 4 h）from perinuclear area（ 0 h） after electric activation. Compared with PA embryos,mitochondria of cloned embryos had more abnormal morphology. No matter at 2-cell（ 28 h） or 4-cell（ 52 h） stages,we observed 3 distribution forms of donor mitochondria. At 2-cell stage of pig cloned embryos,donor mitochondria were not observed in both blastomeres of 21. 95% cloned embryos,and 26. 83% cloned embryos had donor mitochondria in one blastomere,while 51. 22% cloned embryos still had donor mitochondria in both two blastomeres. Even to the blastocyst stage,donor mitochondria still could be observed in few cloned blastocysts. In addition,about 87. 93% 2-cell embryos showed＂redundant＂cytoplasm between the blastomeres,and donor mitochondria could be observed in this area of certain such embryos. The addition of 5μg/mL cytochalasin B（ CB） did not significantly change the proportion of cloned embryos which owned donor mitochondria. Meanwhile,a similar phenomenon of cytoplasmic discharge was noticed in 2-cell PA embryos,and the maternal mitochondria could be also observed in the discharge area. Our results suggested that donor mitochondria distributed unequally in 2-cell and 4-cell cloned embryos,and could be extruded with cytoplasmic discharge in some 2-cell cloned embryos,but the discharge was not specific for the removal of donor mitochondria.