Studies On Equine-Bovine Interspecies Cloned Embryos And Mitochondrial Heteroplasmy

In this study, we explored the impact factors on the in vitro development of equine-bovine interspecies somatic cell nuclear transfer embryos reconstructed by fusion equine ear fibroblasts with enucleated bovine oocytes and established the iSCNT embryos in vitro culture system. In addition, we quantitatively analysed mitochondrial composition of the equine-bovine iSCNT implantation embryos by Taqman quantitative PCR method. The results show that bovine oocytes can support the equine somatic cell reprogramming, and the mitochondria of equine-bovine iSCNT display heterogeneity. The study lays a foundation for producting good race horse and precious horse and exploring the mechanism of interspecies clone.1. The establishment of equine somatic cell lines:We established ear fibroblast cell lines, fetal fibroblasts cell lines and cumulus cell lines. As well as, we observed the biocharacteristics and analyzed the chromosome karyotype and ploidy of this three cell lines, which morphous uniform, ploidy stability, and fully meet the requirements of equine iSCNT.2. The establishment of equine-bovine zone free iSCNT and in vitro culture system:①The SCNT method which combine micro-extrusion and zone free fusion was created. By comparingdifferent activation methods, different in vitro culture medium, the different treatment of somatic cells and micro-injection methods impact on equine-bovine iSCNT fusion rate and embryonic invitro development. The results indicate that the regulation of contact inhibition of ear fibroblast donor cells, zona-free fusion construct iSCNT embryos have a high fusion rate (90.33%), and activated by A23187 5min+6-DMAP 6h with mCR1aa in vitro culture, obtain a higher cleavage rate (83.65%), high-quality embryos which blastomere size uniformity and less debris, and obtain blastocyst (2.72% ).②By comparing equine-bovine iSCNT with bovine–bovine SCNT, the results show that the bovine oocytes can support the horse somatic cell differentiation and reprogramming. However, due to the distance between the two species, the capacity of development of interspecies cloned embryos is far lower than the same species cloned embryos, blastocyst rate (2.71% vs 37.48%).3. Quantitative analysised mitochondrial composition of the equine-bovine iSCNT implantation embryos:①In the detection of seven early equine-bovine iSCNT development embryos, the mitochondria display heterogeneity, and content fewer horses mitochondrial, only account for the bovine 0.08% (morual) ~ 0.42% (blastocyst).②The bovine mtDNA copy number showed a gradual increase trends from 1-cell to 4-cell stage, and from the 8 - cell stage to blastocyst period gradually decreased, while the horse mtDNA copy number in the 1- cell stage to blastocyst period showed a gradual increase trends, and they all decreased in 8-cell stage. We presume the equine-bovine iSCNT embryos occurred maternal embryonic transiton, and embryonic genome support donor equine mitochondrial duplication.③In the morual stage, the equine mtDNAs and bovine mtDNAs copy number were reduced to 159 and 1.84×105the ratio also reduced to 0.09%.,significantly lower than the 1 - cell stage. We presume the interaction between the mitochondrial and nuclear genome emergence disorder, and lead to the cloned embryos arrested development, and its specific mechanisms have yet to be further study.