Study Of Mitochondrial DNA Haplotype And Bovine Somatic Cell Nuclear Transfer Efficiency

Nuclear transfer (NT) technique provide an effective method to evaluate the development competence of nucleus and analyze the relationship between donor cell and oocyte cytoplasm. Despite recent successes in cloning animals from somatic cells, the low efficiency and anomalies remain yet. It is essential for somatic nucleus reprograms appropriately in order to generate more embryos and get normal developmental offspring. During nuclear reprogramming, the change of biological function is the outcome of the interaction between the donor nucleus and recipient cytoplasm, which have become the important part of SCNT study. Mitochondrium is an appropriate material to study the interaction between karyoplast and cytoplast, because mitochondrial DNA(MtDNA) is genetic material outside the nucleus and controlled by nuclear genes .MtDNAs have many haplotypes due to its high mutations and polymorphisms. A lot of studies prove that mitochodria from different materal lineages have distinct effects on embryonic development, which plays important roles on bovine embryogenesis in vivo production.We divided the mitochondria patterns of experimental animals into four haploytpes, named A,B,C and D, respectively by PCR-RFLP method. Then, we collected oocytes recovered by ovum pick-up (OPU) and cultured the donor cells to operate SCNT. Our results showed that the blastocyst rate was apparently higher in the same haplotype than others(45.0%vs.29.0%,p<0.05). It demonstrated that whether the match between hapltype of donor nucleus and that of cytoplasm or not was important for the embryogenesis. In addition, we also found the blastocyst rate of cloned embryos was the highest when the oocytes and donor cells were all from haplotype A, which was statistically higher than other combinations (C-C, A-C, C-A, 53.4% vs. 41.8%, 34.3% and 24.4%, respectively, p<0.05). This implies that the matched haplotype A is the best in all combinations.Furthermore, in order to explain why homo-haplotype is better than heter-haplotype for nuclear transfer, we analyzed the histone H3 lysine 9 (H3-K9) methylation levels of different types of nuclear transfer embryos, and the result showed that the expression model of homo-haplotypes nuclear transfer was much more similar to normal IVF embryos(40% vs. 75%, p>0.05). This study aims directly at the interaction of donor nucleus and recipient cytoplasm that is the key point of SCNT. therefore, it should be important theoretical significance and implicational value.