Research On The Effect Of TRIM28 Knock Down On The Methylation Maintenance In Bovine Preimplantation Embryos

Genomic imprinting is an epigenetic process that distinguishes parental alleles and results in specific expression of paternal and maternal genes. During the development process of mammalian, there are two important genome-wide epigenetic reprogrammings, the period of germ cell development and the period of after fertilization to the early embryonic development.Imprinted methylation undergoes a process of erasure, acquisition, and maintenance during gametogenesis and early embryogenesis. Disruptions in any of these steps may lead to imprinting disorders, resulting in the aberrant development of embryogenesis, placentation and postnatal growth. Therefore, the recognition and maintenance of imprints are very important during early embryonic development. The maternal-effect protein TRIM28 is important for the regulation of imprinted gene during this process.In this study, we chose the cattle as the research object, to explore the methylation maintenance function of TRIM28 in bovine preimplantation embryo, the methylation status of imprintings DMRs were examined in preimplantation normal IVF embryos and TRIM28 knock down embryos by the molecular biology and the embryo engineering technology. The results showed that:1. TRIM28 m RNA expression were analysed by using Immunohistochemistry and RT-PCR. The results showed that TRIM28 m RNA espressed in both oocytes and granulosa cells and mainly located in the nucleus of them;2. Three si RNA against TRIM28(1, 6, and 7, respectively), and one nonsense si RNA(+)were chemosynthesised by Shanghai gemma company. The si RNA interference effect was selected using the method of cell transfection. The results showed that the mixture of si RNA 1,6, 7(si M) had the best interference effect, TRIM28 was down regulated to 84.47%, indicated that RNAi technology is an effective way to study gene function.3. The effect of RNAi interference on oocyte maturation and embryo development shown that the mature time of normal oocyte was 18-22 h, mature rate was 80.38±3.92%, the mature time of TRIM28 knock-down oocyte was 23-24 h, mature rate was 75.44± 2.30%, and the difference between them was not significant. The time of normal IVF embryos began to cleavage was about 16-20 h after fertilization, cleavage rate was 63.84±2.22%, the time of IVF embryos with female parent TRIM28 knocked down began to cleavage was about 32-40 h afterfertilization, cleavage rate was 49.28±2.73%, the difference between them was significant.4. The effect of TRIM28 knock-down on methylation levels in the DMRs of imprinting genes in the different stages of preimplantation embryos shown that the paternal imprinting genes H19 DMRs methylation levels in the different stages(2 cells, 4 cells, 8 cells, blastocysts)of preimplantation normal IVF embryos were 74.0%, 68.40%, 68.30% and 74.0% respectively,and the maternal imprinting genes Mest were 88.1%, 81.90%, 78.50% and 75.60% respectively,and the Peg10 were 88.5%, 76.20%, 85.00% and 88.5% respectively. The paternal imprinting genes H19 DMRs methylation levels in the different stages(2 cells, 4 cell and 8 cells) of TRIM28 knock-down embryos were 1.9%, 22.50% and 39.70% respectively, and the maternal imprinting genes Mest were 89.6%, 29.50% and 54.50% respectively, and the Peg10 were81.0%, 67.10% and 70.80% respectively.In conclusion, TRIM28 expressed in both oocytes and granulosa cells. The effect of TRIM28 knock-down was little on oocyte maturation but it had a significant effect on embryonic development. The maternal-effect protein TRIM28 knocked down to the paternal imprinting genes H19 DMRs methylation levels were the most significantly. The methylation in H19 DMR of 2-cell was almost completely lost, though there were certain degree rise in the stages of 4-cell and 8-cell, it also significantly lower than IVF embryos. But the TRIM28 knocked down to the matrilineal imprinting genes Mest and Peg10 DMRs methylation levels was not significantly.