Effects Of Tet3 On Early In Vitro Fertilization Embryo Development In Cattle

The early embryogenesis is the progress from the zygotes to embryonic tissues stretch without establishing contact with the uterus.During the early embryonic development,a variety of genes begin to express while regulated by a regulatory networks,such as histone modifications,DNA methylation,ubiquitylation and phosphorylation and other associated epigenetic modifications.It will also be regulated after transcription by other factors like autophagy and MicroRNA.DNA methylation is a central epigenetic event that regulates cellular differentiation,reprogramming,and pathogenesis.DNA demethylation occurs in preimplantation embryos and primordial germ cells.Recent studies suggest that Tet3-mediated oxidation of 5-methylcytosine(5-mC)contributes to genome-wide loss of DNA methylation,yet the mechanism of this process in bovine preimplanted embryos has remained unknown.Therefore,BSP-PCR,qPCR,Immunofluorescence staining and RNAi techniques were used to explore the expression of Tet3 gene in early preimplantation embryos of bovine and its influence on the development of early preimplantation embryos,so as to provide a basis for the functional research of Tet3 gene in bovine and provide a direction for the research of this gene in other species.The main results of this experiment are as follows:1.We examined the expression of Tet gene family at different stages during bovine IVF embryo development.The q-PCR data indicate that Tet3 had higher expression than Tet1 and Tet2 in bovine oocytes and IVF preimplantation embryos.The levels of Tet3 gradually increased until 8-cell embryos were formed,and then decreased in the blastocyst stage during the development of bovine IVF preimplantation embryos.Then,we examined the expression profile of the TET3 protein in bovine IVF preimplantation embryos by immunofluorescent(IF)staining analysis.IF staining showed that TET3 signals became stronger and localized to the nucleus of IVF embryos from the 2-cell stage to the 8-cell stage.After the 8-cell stage,they gradually became weaker in IVF embryos at the blastocyst stage.2.To explore the transcript?s function of Tet3 in bovine preimplantation development,we chemically synthesized three candidate siRNAs(si-TET3-1,si-TET3-2,and si-TET3-3)to target bovine Tet3,and then injected three specific siRNA into bovine GV oocytes.Real-time qPCR was used to detect the mRNA level of Tet3 after 24 hr of in vitro maturation.Control siRNA-injected and noninjected oocytes were also analyzed.Si-TET3-1 had higher interference efficiency than the other groups at the mRNA level.Then,we chose si-TET3-1 as the optimal siRNA and used in the following experiments.3.To determine the developmental potential of Tet3 siRNA-injected oocytes,we observed changes in their cleavage and blastocyst rates after IVF.The rate of embryo cleavage in the Tet3-siRNA group was significantly lower than that of the noninjected and control siRNA-injected groups.The proportion of IVF that developed to the blastocyst stage was significantly lower in the Tet3-siRNA group compared with the noninjected and control siRNA-injected groups4.We determined the total cell number of per blastocyst and the percentage of apoptotic cells in blastocysts from each experiment.The results indicate that the average total cell number of blastocysts treated with Tet3 siRNA was significantly lower than that of control siRNA-injected and noninjected embryos.The percentage of apoptotic cells in blastocysts was significantly higher in those treated with Tet3 siRNA than in control siRNA-injected and noninjected embryos.We examined the mRNA levels of the apoptosis-related genes BAX and BCL-2 in the 8-cell and blastocyst stage embryos among the three groups by using qRT-PCR.The results show that antiapoptotic gene BCL-2 and proapoptotic gene BAX mRNA levels had no significant difference at the 8-cell stage.However,the mRNA expression levels of BCL-2 were significantly lower,whereas the BAX transcription level was significantly higher in Tet3 siRNA treated embryos at the blastocyst stage.5.We used 5-mC/5-hmC antibodies to detect genome-wide DNA methylation reprogramming.The results show that 5-mC signals significantly increased in bovine 2-cell to 8-cell stages embryos treated with Tet3 siRNA,whereas the staining intensity for 5-hmC slightly declined.However 5-mC and 5-hmC signals showed similar intensities in the blastocyst stage among the three groups.We performed qRT-PCR of bovine 8-cell embryos to analyze the expression levels of the pluripotency genes POU5F1,NANOG,and the imprinted genes H19.We found that the mRNA transcription levels of POU5F1 and NANOG were significantly lower in Tet3 siRNA-treated embryos at the 8-cell stage,whereas the paternally imprinted gene H19 did not change.In addition,there were no significant differences in POU5F1,NANOG,and H19 mRNA levels among the three groups at the blastocyst stage.We examined the methylation status of the promoters for the pluripotency-related genes(POU5F1,NANOG)and imprinted genes(H19)by BSP after oocytes were treated with Tet3 siRNA.The results demonstrate that methylation levels in the promoters of POU5F1 and NANOG were obviously increased in Tet3 siRNA-treated embryos at the 8-cell stage,while there was no evident change in the promoters of H19 among the three groups.We chose the repeat elements,satellite I and α-satellite to test DNA methylation status.The results showed that satellite I and α-satellite had a high DNA methylation level in Tet3 siRNA-treated groups at the 8-cell stage.However,the DNA methylation levels of the pluripotency-related genes(POU5F1,NANOG),imprinted genes(H19),and repeated elements(satellite I and α-satellite)were similar among the three groups in blastocyst stages.Our results indicated that Tet3 could influence the expression level of the pluripotency genes through regulating the methylation status of the promoter region,thus affect embryonic development.In summary,our results indicated that Tet3 could influence the expression level of the pluripotency genes through regulating the methylation status of the promoter region,thus affect embryonic development.This result will provide theoretical for the study of Tet3 gene in bovine.