Effects Of Methyl Transferase Inhibitors On The Early Development Of Cloned Pig Embryos And Study Of Cloned Pigs Cryptorchidism

Although it has been years since the first smatic cell nuclear transfer cloned pig was born, the efficiency of the nuclear transfer still needs to be promoted. Currently, MⅡ-phase deuncleated oocytes matured in vitro are mostly used as receptor eggs in SCNT. Pig oocytes in vitro maturation system is still immature and has yet to be further improved. Meanwhile, incomplete reprogramming of the reconstructed embryos leads to the unnormal development of preimplantation embryos. Even if few survivals, some developmental defects and abnormal phenotypes are usually accompanied to different degrees in the offspring. Compared with preimplantation embryos and cloning animals fetus, the studies on the methylationpatterns in phenotypic abnormalities cloned pigs are still insufficient, the molecular biology mechanism remains unclear. In this study, we added rhodiola sachalinensis polysaccaride(RSP) in oocyte maturation medium to obtain high quality oocytes matured in vitro. And tried to apply the new methylation inhibitor RG108to correct abnormal preimplantation embryo methylation status, in order to improve the efficiency of nuclear transfer. Four cloned piglets were born in the subsequent production of cloning pigs in the laboratory, one died after it was born, and two cloned piglets occurred in cryptorchidism. We studied the related gene expressions in testis development and the changes of the methylation in the two cryptorchidism cloned pigs. The main results are as follows:(1) Immature oocytes were matured in vitro and supplemented with different concentrations of RSP, the extrusion of first polar body was treated as a symbol of nuclear maturation, then the nuclear maturation rates, oocyte intracellular GSH content and ROS level, Cleavage and blastocyst rates of IVF and SCNT were conducted. The results showed that treated with6and60mg·L-1RSP in the vitro maturation medium could significantly increase the GSH content (P<0.05), and60mg·L-1group of mature oocytes can significantly improve the cleavage and blastocyst rate of IVF and SCNT.(2) This experiment was conducted to study the effect of RG108treated PFF as donor cells on their methylation levels and nuclear transplantation efficiency. Four concentrations,0.05,0.5, Sand50μmol·L-1RG108were selected. PFF was treated for24,48,72h respectively, and the cells methylation levels was determined by HPLC and BSP method; Observed the situation of cell growth and apoptosis by using the MTT method and fluorescent microscope observation method; Using Giemsa staining detected cells’ chromosome and detected nuclear transfer efficiency after treated with four concentrations RG108for72h. The results showed that:The results indicated that PFF incubated with50μmol·L-1RG108RG108for72h reduced the cells’ overall methylation levels and it was time-dependent and5and50μmol·L-1RG108for72h reduced the regional rate of methylation of the imprinted gene DMR, And RG108had no significant impact on normal cell activity.(3) Four concentrations of RG108were selected to treat donor cell or/and SCNT embryon After fusion for72h,studied the effect of different treatments on the efficiency of nuclear transfer.The results showed that blastocyst rate of embryos obtained from donor cells which treated by5μmol·L-1RG108was significantly improved. Subsequently, immunofluorescence was used to detect the Methylation levels of IVF blastocysts, SCNT blastocysts and blastocysts obtained from donor cells which treated by5μmol·L-1RG108. The results showed that blastocyst of the RG108treated group and IVF group had similar methylation level.(4) In order to identify the possible causes of the cryptorchidism in cloning pigs which producted by our research group, The relative fluorescence quantitative PCR techniques and bisulfite salt sequencing analysis are used to analysis the expression and promoter methylation status of the promoter CpG sites of SOX9and INSL3genes in the cloned pig’s cryptorchidism. The results showed that, The expression of SOX9and INSL3genes in the cloned pigs cryptorchidism were abnormal.But analysis of the methylation status showed that INSL3gene was hypermethylation and the SOX9gene methylation status had no significant change.In summary, added RSP in IVM medium improved the quality of matured oocytes in vitro. Oocyte maturation in60mg·L-1RSP used to IVF and SCNT can significantly enhance the viability of the embryos. RG108reduced the levels of the cell methylation and it was Time-dependent. Meanwhile the DMR methylation of H19was also significantly reduced. Nuclear transfer efficiency was significantly improved by PFF treated with5μmol·L-1RG108for72h. The levels of the blastocyst methylation were lower than the control group, but closed to the IVF group.had an abnormal DNA methylation patterns, it causesed abnormal expression.The aberrant DNA methylation patterns occured in INSL3gene and infulenced gene expression, which could be one of the important factors leading to the occurrence of cryptorchidism in cloned pigs.