Studies Of MiR-29b Expression And Its Influence On Porcine Early Embryo Development

The early embryogenesis also called preimplantation)is the progress from the zygotes toestablishing contact with the uterus.During the early embryonic development,gene expression is regulated through DNA methylation,histone modification and other epigenetic modification,in addition,post-transcriptional regulation such as micro RNA also play an important role.Micro RNA29-b(miR-29b)is a member of mir R-29 family,which targets DNA methyltransferases(Dnmts)and regulates DNA demethylation,leading to the downregulation of global DNA methylation in malignant cells.The study in mice suggested that interference of miR-29 b expression could cause the disorder of global DNA methylation,downregulation of genes encoded factors with known essential function during early embryonic development,including the pluripotent stem cell factor.The pig is not only a kind of economic animal but also an important experimental animal,which has an important application value on translational medicine.Therefore,the pig is selected as the laboratory animal to explore the expression of miR-29 b in in vitro fertilization(IVF)and parthenogenetic activation(PA)embryos,and its effect on embryonic development using q PCR and RNAi technologies.The main results of this experiment are listed as follows:1.Factors affecting the IVF including sperm concentration,capacitation treatment,co-incubation system,and hormone combination of in vitro maturation,were investigated in order to improve porcine IVF efficiency.Our results indicated that the best cleavage rate(61.33±0.77)% and blastocyst rate(28.33±1.08)% could be gained by the modified IVF system.2.The early embryos of different stage were obtained by in vitro fertilization and parthenogenetic activation technique,respectively.Then the dynamic expression pattern of miR-29 b and the dynamic m RNA expression levels of Dnmt3 a,Dnmt3b and Dnmt1 m RNA weredetected by q-PCR.The results showed that only in IVF embryos,the expression level of miR-29 b changed signally with the embryo development,which is the highest in 8-cell stage,next to blastocyst stage,and in 4-cell is the lowest.And Dnmt3 a and Dnmt3 b m RNA expression levels were negatively correlated with miR-29 b.These results indicated that the expression and functioning of miR-29 b required activation of the fertilization process.3.The expression of miR-29b is interfered by injecting the micro RNA inhibitor into cytoplasm of IVF embryos to investigate the function of miR-29 b in embryonic development and regulation of gene expression.Then the m RNA expression of Dnmts in blastocyst stage was detected by q-PCR,the global methylation level and the DNA methylation level in the promoter of pluripotency gene Nanog were detected by immunofluorescence staining and bisulfite sequencing PCR(BSP)respectively.The results showed that after the interference of miR-29 b expression,blastocyst rate and total cell number of blastocysts decreased significantly,but there was no significant difference in the cleavage rate.Dnmt3 a,Dnmt3b m RNA expression levels were significantly upregulated,while the global methylation level and Nanog promoter DNA methylation level also increased.These results indicated that miR-29 b expression was associated with embryonic development.,it regulated DNA methylation levels by targeting Dnmt3 a,Dnmt3b,and further regulated embryonic development in blastocysts stage.4.The mRNA expression levels of pluripotency genes Nanog,Sox2,Oct4 and apoptosis genes Bax,Casp3 and anti-apoptosis gene Bcl-xl were detected by q-PCR.And the number of apoptotic cells was detected by immunofluorescence staining.The results showed that the expression of pluripotency genes Nanog and Sox2 was significantly down-regulated,and the expression of apoptosis genes were up-regulated,while the expression of anti-apoptosis gene was down-regulated,and the number of apoptotic cells also increased.The results indicated that miR-29 b could affect the early development of embryos by regulating the expression of pluripotent and apoptotic genes in blastocyst stage of porcine early embryos.