Transcriptome Analysis And Comparsion Of Pig Early Embryos Derived By ICSI And SCNT Echnology

The early mammalian embryo starts from zygote and eventually develops into implantable balstocyst after several rounds of cell division.The pattern of cell cleavage during early embryogenesis is highly conserved among different species,while the transcriptomic regulation is quite different.Now,researches about early embryo development mainly focus on human and mouse but barely involve pig.Pig is a kind of irreplaceable economic animal as well as promising medical model for human.In order to dissect the pattern of transcriptomic regulation,identify key regulatory genes and assess the conservation with different species in the field of early embryo development,and explore the molecular mechanisms contributing to lower effeciency of SCNT technology,single cell transcriptomes of two groups of embryos derived by SCNT and ICSI technology were systematically compared.In this study,enbryos in the ICSI group were used to dissect transcriptome change during embryogenesis and to make comparsions with three other species.Embryos in the SCNT group were compared to that in the ICSI group to explore underlying molecular mechanisms contributing to the lower effeciency of SCNT technology.The main findings are listed as follows:(1)In the ICSI group,ZGA was successive from zygote to blastocyst with the biggest change occurred at the stage of four cells.The up-regulated genes at the stage of eight cells were related to transcription initation,regulation of transcription factor and mRNA processing.(2)9931 DEGs were identified between each two adjacent stages in the ICSI group,and most of DEGs were stage-specific expressed,indicating big changes of transcriptome during embryogenesis.Many genes that up-regulated from the stage of eight cell were related to transcription initation and regulation,while genes specifically expressed in balstocyst were related to material and energy metabolism.(3)Alternative splicing genes were identified at all stages in both ICSI and SCNT group with variable number.And those genes expressed at all stages played roles in RNA splicing and cell cycle.Expression of most of genes with multiple transcripts was dominated by one or two transcripts.During the major ZGA in the ICSI group,differentially expressed transcripts were related to RNA transcription.(4)88711 PASs were identified at all stages with variable number in both ICSI and SCNT group.PAS number had positive correlation with both expression and transcript number of gene.Genes with PASs that were differentially used between the four and eight cells stage in the ICSI group were related to mRNA transcription and processing.(5)66619 lincRNAs with high reliability were identified and 18612 of them were novel.Many lincRNAs were differentially expressed between each two adjacent stages in both ICSI and SCNT group,and some of them were stage-specific expressed.Functional analysis of differentially expressed lincRNAs revealed that lincRNAs specifically expressed at different stages were involved in various biological processes.Further analysis revealed that hub lincRNAs in the red module(four cell)were transcription initiation realted and hub lincRNAs in the salmon module(blastocyst)were material metabolism related.(6)Maternal genes were more conserved than zygotic genes during embryogenesis of four species.There were more maternal genes conserved between pig and human than that between mouse and human,but fewer than that between monkey and human.Simultaneously,there were more zygotic genes conserved between pig and human than that between mouse and human as well as between monkey and human.Transcriptome correlation analysis revealed that transcriptome of pig was more similiar with that of human than that of monkey and mouse.(7)In the SCNT group,the biggest change of transcriptome occurred at the stage of morula,which was later than that in the ICSI group.The number of transcripts,alternative splicing genes and total genes expressed at the stage of four cells and eight cells were fewer than that at the same stages in the ICSI group.6080 DEGs were identified at the same stages between SCNT and ICSI group.Most DEGs were identified at the stage of eight cells and blastocyst,and they mainly played roles in RNA processing and transport,lipid metabolism and singnal transduction.(8)In the SCNT group,transcriptomes at the stage from zygote to four cells were similar with that at the same stages in the ICSI group.However,transcriptomes at the stage of egiht cells and morula were similar with that at the stages from zygote to four cells in the ICSI group.157 of 375(41.9%)key genes related to embryo development were differentially expressed between SCNT and ICSI group at the same stage.