Studies On Epigenetic Modification And Development Potential Of Porcine Scnt Embryos

The first cloned pigs have been born over ten years, however, the efficiency of nuclear transfer has been low, limiting the application of cloned pigs. Differentiated somatic cells and matured oocytes were used in somatic cell nuclear transfer, thus their qualities directly determine the developmental competence of cloned embryos. At present, the main reason for low cloning efficiency is that donor nucleus is not completely reprogrammed, showing genome highly methylated. Epigenetic modification in somatic cell nuclear transfer is the key factor affecting on the development of cloned embryos, thus regulation of epigenetic modification in cloned embryos may improve the cloning efficiency. This study aimed to analyze the role of cytoplasmic maturation and donor cells in somatic cell nuclear transfer in order to evaluate the effect of cytoplasmic maturation and donor cells on the development of cloned embryos and apply epigenetic modification drugs and knockdown of DNA methyltransferase1to explore and elucidate the molecular mechanism of key genes in the somatic cell nuclear reprogramming, and thus provides a theoretical basis to improve the efficiency of porcine somatic cell nuclear transfer. The main results are as follows.(1) During the progress of oocytes in vitro maturation, we analyzed the effect of cytoplasmic maturation on the developmental competence of cloned embryos, and the results showed that oocytes cultured for33h have achieved nuclear maturation and the time between33h and42h was cytoplasmic maturation. The results also showed cytoplasmic maturation could significantly enhance the developmental competence of porcine cloned embryos (p<0.05), though cytoplasmic maturation was not conducive to remove nuclear materials. These results indicate that full cytoplasmic maturation is the key factor in the development of cloned embryos.(2) Donor cell is a determining factor in somatic cell nuclear transfer, thus the types, sexes and methylation levels of donor cells were analyzed. It was shown that compared with cumulus cells and adult fibroblasts, fetal fibroblasts was more suitable for somatic cell nuclear transfer in term of the fusion rate and cell proliferation. As for sexes, there were not significant differences in the cloned embryo development between male and female fetal fibroblasts, while female fetal fibroblasts are more suitable for the research on the mechanism of X chromosome inactivation. The results also showed that5-aza-dC or5-methyl-dC could significantly decrease or increase the methylation levels of fetal fibroblast cells (p<0.05), however, the changes of methylation levels could not enhance the cloning efficiency. It indicates that female fetal fibroblasts are more suitable as donor cells, while th change in the methylation level is not conducive to improve nuclear reprogramming. (3) In order to enhance the cloned embryo development,5-aza-dC was applied to treat donor cells and cloned embryos, and cell proliferation, cloned embryo development, nuclear remodeling and gene expression were studied. When donor cells were treated, the cell proliferation was inhibited, and the expression levels of DNA methyltransferase1and3a were significantly reduced while those of Igf2and Xist significantly increased (p<0.05), furthermore, the development of cloned embryos was not enhanced, showing that the ability of nuclear remodeling and cleavage rates were low and the expression levels of DNA methyltransferase and pluripotent genes did not improve. When cloned embryos were treated, the development competence was significantly enhance (p<0.05), and the treatment manner with25nmol for24h was best. Treatment cloned embryos significantly (p<0.05) accelerated the reprogramming of donor cell nuclear genes, promoted the pronucleus-like formation and early cleavage, and improved the expression levels of DNA methyltransferase, imprinting, pluripotent and demethylation-related genes, similar to those in IVF embryos. It was surprised that when both donor cells and cloned embryos were treated, the developmental competence was more significantly enhanced (p<0.05), indicating that double treatment is more suitable for the reprogramming of donor nuclei.(4) We analyzed the developmental competence of cloned embryos in vitro and in vivo after treatment cloned embryos with TSA and gene expression levels during TSA treatment, and found that TSA could significantly enhance the developmental competence of cloned embryos in vitro (p<0.05), Furthermore, after embryo transfer, compared with normal cloned embryos, TSA treatment improved pregnancy and birth rates, and prevented the occurrence of large tongue disease and stillbirth effectively. After TSA treatment, the reprogramming of donor cell nuclear and DNA methyltransferase genes was significantly accelerated (p<0.05). These results above indicates that TSA treatment could enhance the donor nuclear remodeling and correct embryo development related gene expression, and thus enhance the developmental competence of cloned embryos in vitro and in vivo.(5) Knockdown of Dnmtlby RNA interference may lead to effectiveDNA demethylation, and thus improve the developmental competence of cloned embryos. We analyzed the state of RNAi system, and oocyte maturation and cloned embryo development after Dnmtl interference, and found that RNAi system was steadily set up, as mural granulosa cells could enhance the maturation quality of deduded oocytes and the maturation rate of oocytes after Dnmtl knockdown did not change, however, it was surprised to find that the developmental competence of cloned embryos derived from oocytes of Dnmtl knockdown significantly decreased (p<0.05), and many cloned embryos were arrested at4and8cell stage. These results suggests that Dnmtl plays an important role in the early developmental process of cloned embryos, and appropriate expression level is the determining factor for embryo development.