Abstract：
It is widely accepted that cytochalasin B (CB) is required in enucleation of the oocyte in order to stabilize the cytoplasm. However, CB treatment results in the uneven distribution of mitochondria, with aggregation towards the nucleus, which might compromise the efficiency and safety of a three-parent embryo. Here, we demonstrated that CB treatment affected mitochondrial dynamics, spindle morphology and mitochondrial DNA carryover in a concentration-dependent manner. Our results showed that mouse oocytes treated with over 1 μg/ml CB exhibited a more aggregated pattern of mitochondria and diminished filamentous actin expression. Abnormal fission of mitochondria together with changes in spindle morphology increased as CB concentration escalated. Based on the results of mouse experiments, we further revealed the practical value of these findings in human oocytes. Chip-based digital PCR and pyrosequencing revealed that the mitochondrial carryover in reconstituted human embryos was significantly reduced by modifying the concentration of CB from the standard 5 μg/ml to 1 μg/ml before spindle transfer and pronuclear transfer. In conclusion, our findings provide an optimal manipulation for improving the efficiency and safety of mitochondrial replacement therapy.
摘要：
广泛接受的是,在卵母细胞的去核中需要细胞松弛素B(CB)以稳定细胞质。然而,CB处理导致线粒体分布不均，向原子核聚集，这可能会损害三亲胚胎的效率和安全性。这里,我们证明CB治疗影响线粒体动力学，纺锤体形态和线粒体DNA携带呈浓度依赖性。我们的结果表明，用超过1μg/mlCB处理的小鼠卵母细胞表现出更多的线粒体聚集模式和减少的丝状肌动蛋白表达。线粒体的异常裂变以及纺锤体形态的变化随着CB浓度的升高而增加。根据老鼠实验的结果,我们进一步揭示了这些发现在人类卵母细胞中的实用价值。基于芯片的数字PCR和焦磷酸测序表明，通过在纺锤体转移和原核转移之前将CB的浓度从标准的5μg/ml修改为1μg/ml，可以显着减少重组人胚胎中的线粒体残留。总之,我们的研究结果为提高线粒体替代疗法的效率和安全性提供了最佳方法.
