| The early embryo develops into blastocyst after early cleavage(EC),4-cell stage,8-cell stage,morula stage and morula densifying.EC affects the ability of embryo development during pre-implantation and post-implantation.It has been observed about symmetrical cleavage(Sym)and asymmetrical cleavage(Asy)in human and mouse embryos,but the molecular mechanism is still unclear.At present,few studies on the mode and mechanism of EC in large livestock were reported.In this study,the developmental capacity of Sym and Asy embryos was compared in porcine,subsequently transcriptomic data using Smart-Seq2 technology was analyzed,and finally,the expression pattern and function of CEP55 were verified to elucidate the role and molecular mechanism of CEP55 gene in the process of embryo cleavage,and to lay a theoretical foundation for improving the level of early embryonic development of porcine.1.Influence of cleavage modes on early embryonic development and transcriptomic analysisFirstly,embryonic development in vitro fertilization(IVF)and parthenogenesis activation(PA)were compared.The results showed that in IVF embryos,Sym embryos accounted for 62.55% and Asy embryos accounted for 37.45% of total cleavage rate.The 2-cell rate,blastocyst rate and total number of blastocyst cells in Sym embryos were significantly higher than those in Asy embryos(P < 0.05).In the PA embryos,Sym embryos accounted for 63.21% and Asy embryos accounted for 36.79% of total cleavage rate.The 2-cell rate,blastocyst rate and total number of blastocyst cells in Sym embryos were significantly higher than those in Asy embryos(P < 0.05).According to the transcriptomic analysis of Smart-Seq2,a total of 22050 genes were identified from embryos of Sym and Asy groups,and 216 differentially expressed genes(DEGs)were screened,of which 147 genes were significantly up-regulated and 69 genes were significantly down-regulated.CEP55 gene associated with cell mitosis was significantly down-regulated in Asy embryos.Ontology(GO)analysis showed that these DEGs were related to cell cycle,chromosome segregation and microtubule cytoskeleton.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed that the DEGs were mainly enriched in oocyte meiosis,cell cycle and p53 signaling pathways.2.Cloning and expression pattern analysis of porcine CEP55 geneFirstly,the CDS region of porcine CEP55 gene was cloned and the biological information was analyzed.Secondly,the expression and localization of CEP55 were detected in main tissues,oocytes during the in vitro maturation stages and the early embryos of IVF and PA.The results showed that the CDS region of porcine CEP55 gene was 1395 bp.CEP55 gene was expressed in porcine heart,liver,spleen,lung,kidney,ovary and testis,and the expression of CEP55 gene was higher in liver,spleen,and testis compared with the heart(P < 0.001).qRT-PCR results showed that CEP55 gene was significantly up-regulated in MII oocytes compared with GV stage(P < 0.001).The results of immunofluorescence showed that CEP55 protein mainly located on the nucleus at GV and MI phases,and on both sides of the nucleus at MII phase.Compared with zygotic stage,the expression of CEP55 gene in IVF embryos was significantly increased at 2-cell stage(P < 0.05),and significantly decreased at 4-cell stage(P < 0.05),while sharply up-regulated at 8-cell stage,then significantly down-regulated at morula stage,and almost did not express at blastocyst stage(P < 0.01).At the protein level,CEP55 protein was located near the bipolar body in zygote,and near the bipolar body and the nucleus at 2-cell phase,and on both sides of the nucleus at other phases.In PA embryos,the expression tended of CEP55 gene was to increase first in 2-cell stage and then decrease from 4-cell to blastocyst stage.Compared with 1-cell stage,the expression of CEP55 gene was significantly increased at 2-cell,4-cell and 8-cell stages(P < 0.05),and obviously decreased in blastocyst stage(P < 0.05).3.Study on the effects and mechanism of CEP55 gene on embryo cleavageThe influences of CEP55 gene on the early embryo development,cleavage modes,spindle,nucleus and related genes were analyzed by positive and negative regulation of CEP55 gene expression.The results showed that the cleavage rate,4-cell,8-cell,blastocyst rates and total blastocyst cell number significantly decreased when down-regulated the expression of CEP55 gene compared with NC group(P < 0.05).The2-cell embryos were divided into Sym and Asy modes.When down-regulating the expression of CEP55 gene,the 2-cell,4-cell,8-cell and blastocyst rates of Sym group significantly decreased(P < 0.01),and the 2-cell rate of Asy group significantly increased(P < 0.05)compared with NC group.Meanwhile,the blastocyst quality of Sym and Asy groups was significantly decreased(P < 0.05).The immunofluorescence results showed that,with the down-regulation of CEP55 gene expression,the proportion of embryos with normal spindle and normal nucleus significantly decreased(P < 0.001),and the number of embryos with abnormal spindle obviously increased(P < 0.05).Compared with the NC group,the gene expressions of p53,PLK1,and the expressions of genes related to mitosis,cell cycle,spindle formation and assembly significantly decreased(P < 0.05)with the down-regulation of CEP55 gene expression.In order to understand whether the embryo development and cleavage modes influenced by CEP55 gene were through Aurora B-CEP55-p53-PLK1 axis,oocytes were treated by Aurora B inhibitor Barasertib(Bara).The embryos were divided into NC group,Bara group and Bara+CEP55 group.The results showed that the 2-cell rate,4-cell rate,8-cell rate and blastocyst rate in Bara group were significantly decreased compared with NC group(P < 0.05).Compared with Bara group,the 8-cell rate in Bara+CEP55 group was obviously increased(P <0.05),as well as the 2-cell rate and 8-cell rate in Sym embryos(P < 0.05),while the 2-cell rate,4-cell rate and 8-cell rate in Asy group were significantly decreased(P < 0.05).Meanwhile,the blastocyst quality of Sym and Asy groups was significantly increased in the Bara+CEP55group(P < 0.05).Results of immunofluorescence showed that the number of embryos with normal spindle and normal nucleus in the Bara+CEP55 group was significantly increased(P < 0.001),while the number of embryos with abnormal spindle was significantly decreased compared with the Bara group(P < 0.001).Simultaneously,the gene expressions of Aurora B,p53,PLK1,CEP55 and genes related to mitosis,cell cycle,spindle formation and assembly in Bara+CEP55 group were significantly up-regulated compared with Bara group(P < 0.05).The above results demonstrated that the developmental potential of symmetrical cleavage embryos was better than that of asymmetrical cleavage embryos,and CEP55 gene was significantly down-regulated in asymmetric cleavage embryos.The number of asymmetric cleavage embryos increased with the down-regulation of CEP55 gene expression,which was unfavorable to the development of early embryo.Overexpression of CEP55 gene could repaire the embryo damage by Barasertib treatment.The CEP55 gene was regulated by the upstream genes Aurora B,and it also affected the modes of embryo cleavage by regulating the expression levels of p53 and PLK1 genes.CEP55 gene might affect spindle formation and assembly through Aurora B-CEP55-p53-PLK1 axis,sequentially affecting the early embryonic development and embryo cleavage modes in pigs. |
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