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.
Embryo, Mammalian , Mitochondrial Replacement Therapy , Humans , Mice , Animals , Cytochalasin B/pharmacology , Cytochalasin B/metabolism , Oocytes/metabolism , DNA, Mitochondrial/genetics
Mitochondrial DNA (mtDNA) segregation associated with donor-recipient mtDNA mismatch in mitochondria replacement therapy leads to unknown risks. Here, to explore whether matching mtDNA haplotypes contributes to ameliorating segregation, we reproduced various degrees of heteroplasmic mice with three single nucleotide polymorphisms to monitor segregation severity. "Segregation" presented in tissues of heteroplasmic mice containing low-level donor mtDNA heteroplasmy, and disappeared as donor mtDNA heteroplasmy levels ascended. Meanwhile, we found that distribution of donor mtDNA among the blastomeres of preimplantation embryos from the heteroplasmic mice shared the same tendency as that in adult tissues. Statistical analysis showed that no selective replication of donor mtDNA occurred during lifespan. Tracking donor mtDNA distribution showed that uneven distribution of donor mtDNA among embryonic blastomeres gradually became even as donor mtDNA heteroplasmy increased, indicating that the "segregation" in tissues was inherited from the uneven distribution. Our finding suggested that donor-recipient mtDNA matching could circumvent segregation in mitochondria replacement therapy.
