Effect Of TRIM28 On The Methylation Maintenance In Bovine SCNT Preimplantation Embryos

Despite the rapid development of somatic cell nuclear transfer(SCNT)technology,the efficiency is still far from ideal and animals treated by SCNT often developed abnormally.Imprinting problem due to aberrant methylation patterns is one of the most important reasons for the low success rate of mammalian cloning in preimplantation cloned embryos.Genomic imprinting is an epigenetic regulation phenomenon,restricting monoallelic expression to either the maternally or paternally inherited copy of the gene,which is important for the development of preimplantation embryo.Recent studies show that a maternal TRIM28 can prevent imprinted gene demethylation and resist abnormal development of preimplantation embryos.TRIM28 derived from the oocyte is essential for maintenaning of imprinted DNA methylation during preimplantation embryos.The first step of SCNT is enucleation,during this step,the nucleus and a small amount of the surrounding cytoplasm are removed from MII oocytes,maternal TRIM28 is localized in the nucleus,the result of enucleation is the loss of maternal nuclear TRIM28,which may further lead to subsequent failure to maintain methylation imprints in the cloned embryos.Based on these findings,we analyzed the expression and distribution of TRIM28 and methylation imprints(H19,Mest,and Peg10)in different stages of bovine IVF embryos,SCNT embryos,maternal Trim28-RNAi IVF embryos,using molecular biology and embryo engineering technology,such as RNAi,in vitro fertilization(IVF),SCNT,and etc.The results are summarized below.1.For the first time,the TRIM28 gene was obtained by reverse transcription PCR(RT-PCR).2.The normal oocyte maturation needed 18-22 h to mature,and the mature rate was 74.38±3.77%.The oocyte maturation needed 23-24 h to mature,and the maturation rate was 62.52±2.13 after injecting siRNA.The oocyte maturation rate shows no significant difference between normal group and injected group.The normal IVF embryos begin to cleavage around 16-22 h after fertilization,with a cleavage rate of 63.84±2.22%.The IVF embryos with TRIM28 knocked down began to cleavage at about 28-32 h after fertilization,with a cleavage rate of 49.28±2.73%,which shows significant difference.3.The normal IVF embryos begin to cleavage around 16-22 h after fertilization,with a cleavage rate of 63.84±2.22%.The SCNT embryos begin to cleavage around 27-30 h after chemical activation,with a cleavage rate of 40.33±5.72%.The difference between them is significant.4.TRIM28 was highly expressed during oocyte maturation,reaching the peak level at the 2-cell stage in the SCNT embryos,then significantly decreased at the 4-cell stage(P < 0.05),followed by persisted in the following pre-implantation embryo development stage.Compared to IVF embryos,TRIM28 expression quantity decreased significantly in 2 cell stage of SCNT embryos(P < 0.05),whereas the 4 cells of SCNT embryos were increased but has no significant difference(P > 0.05).5.The paternal imprinting genes H19 DMRs methylation levels in the different stages(2 cell,4 cell,8 cell and blastocyst)of SCNT embryos were31.4%,23.7%,41.1%,and 36.0%,respectively.The maternal imprinting genes Mest were 77.8%,83.3%,64.4%,and 71.3%,respectively.Peg10 were 89.2%,94.2%,74.7%,and 72.0% respectively.Compared to IVF embryos,imprinting gene H19 DMRs methylation level decreased significantly in 2 cell and 4 cell of SCNT embryos(P < 0.05),while the Mest and the Peg10 methylation level in SCNT pre-implantation embryos were similar to IVF embryos,showing no significant difference(P > 0.05).6.H3K9me3 and H3K9 ac were distributed in the nucleus.H3K9me3 and H3K9 ac expressed in GV stage oocytes but not in the MII oocytes.There were strong H3K9me3 and H3K9 ac signals in 2 cells,however,almost no signals were found in 8 cell in the IVF embryos.The H3K9me3 expression level decreased significantly in the 2 cell stage of TRIM28 knock down embryos,compared to IVF group(P < 0.05),but there were no significant differences in 4 cell and 8 cell(P > 0.05).The H3K9 ac expression level increased in 2 cells and 4 stages of TRIM28 knock down embryos.7.Compared to IVF embryos,the H3K9me3 fluorescence signal increased significantly in 8 cell,but decreased significantly in blastocyst of SCNT embryos(P < 0.05).H3K9 ac immunofluorescence signals were significantly decreased in 2 cell,4 cell,and blastocyst of SCNT embryos(P < 0.05),while the H3K9 ac in 8 cell of SCNT embryos showed no significant difference compared to IVF embryos(P > 0.05).