| The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved,regulated,and predictable cell divisions.It mainly consists of sperm-egg binding to the formation of zygote,embryonic cell division(cleavage),zygotic genome activation(ZGA),compaction of morula,formation of blastocyst with implantability.This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages.Pigs are important meat animals and are also potential biomedical model animals and organ donors in xenotransplantation for human.In the 21st century,the basic research of life sciences has developed rapidly,and the theoretical and technological developments of stem cells and gene editing continue to make breakthroughs.How to apply these cutting-edge theories and technical achievements to pig breeding,construction of medical model pigs,and development of xenotransplantation donors is an important challenge for scientists in this field.Given the dominance of pigs in the disease model,understanding the mechanism of early embryonic development in pigs and breaking through the bottleneck of establishing porcine embryonic stem cells are key tasks for all researchers.Looking at the process of mouse ESC establishment,it will take a long time to successfully establish ESCs on other large mammals.Only the preimplantation embryos can provide us with reference information.We hope to provide a strong theoretical support for the establishment of ESC by analyzing the developmental mechanisms of early embryos in order to better promote the exploration of pigs as a disease model.So far,with the help of emerging technologies,it has become urgent to fully analyze the mechanism of early embryonic development in pigs.Compared with mouse embryonic development,pig embryos have long developmental stages prior to implantation,and embryo development patterns vary greatly.There are few related basic researches,and the available reference literature is very limited.In the past,people have always believed that non-coding RNAs(ncRNAs)are the by-products of transcription,that is,“transcription trash”.Recent studies have shown that these ncRNAs are not trash,but are actually functional molecules.In addition,the number of ncRNAs is related to the complexity of the organism,which means that it may have a more important role in more advanced animals.Moreover,ncRNAs have been shown to be less conserved than coding genes in various species,and we have reason to suspect that ncRNAs contribute to pig-specific gene expression regulatory networks.In this study,advanced molecular techniques such as high-throughput transcriptome sequencing were used to systematically identify pig embryonic development-related regulatory genes and ncRNAs(endogenous small interfering RNAs:endo-siRNAs;intergenic long non-coding RNAs:lincRNAs),and reveals a unique molecular regulatory network of early embryonic development represented by swine as a large mammal.The specific findings are as follows:(1)Libraries from sncRNAs were prepared from 5×10~9 sperms,7845 oocytes and 6800zygotes of pig and sequenced by high-throughput deep sequencing(llumina).We obtained 90431,244480 and 219971 sncRNA sequences completely matched the pig genome from the sperms,oocytes and zygotes.In addition,we used the bioinformatics methods that we established to classify ncRNAs in pig gametes and zygotes,and identified three sources of endo-siRNAs:transposon elements-associated endogenous small RNAs(TE-siRNAs),Endogenous small RNAs from long hairpin(Lhp-siRNAs)and endosiRNAs derived from inverted-complement mRNA(IC-siRNAs).We performed expression analysis of differential endo-siRNAs between zygote and sperm or oocyte,and focused on the high-expressed endo-siRNAs in zygotes.Deep analysis showed many high-expressed TEs-siRNAs mapped to the ORF2 and 3?UTR regions of porcine L1 retrotransposon(Figure 2B).We refer to these L1-specific endo-siRNAs as L1-siRNAs.Using multiple sequence alignments and RACE methods,we found the L1 accociated antisense transcript:an unannotated long non-coding RNA(asL1).365bp dsRNA structure could be formed between L1 ORF2 and asL1,and 9 L1-siRNAs with high reads mapped to the region.Finally,by RNA interferencing and other experimental methods,we show the L1-siRNAs regulate early embryonic development by inhibiting the activity of L1 retrotransposition.This work can contribute to understanding the functional role of abundant endo-siRNAs in embryonic development.(2)Early embryonic single-cell transcriptome maps of large mammals other than human or mice are currently not reported.Therefore,we aimed at the early embryonic single blastomere transcriptome sequencing in pigs and pioneered a complete isolation system for pig inner cell mass and trophoblast and single cell isolation system for preimplantation porcine embryo.Finally,transcriptome sequencing was performed on 106 samples of single blastomeres at various stages of the porcine early embryos.The bioinformatics analysis revealed that the global transcriptome profiles of porcine early embryos were dynamically changed with embryonic development;It was determined that genomic activation of porcine zygote occurred in the four-to eight-cell stage;Identification of 73 key candidate genes involved in the regulation of blastomere heterogeneity in morula;Through weighted gene co-expression network analysis(WGCNA),specific gene expression regulatory networks corresponding to specific developmental stages of early embryos were identified;Finally,by comparison gene expression regulatory networks of human,mouse,and bovine,the regulatory networks of early embryonic development in large animals such as pigs may be very different from those of small animals.Many new genes of pigs that have not been functionally researched have been excavated.These new genes have contributed to specific gene expression regulatory networks for large mammals such as pigs,enriching our knowledge.(3)Due to incomplete genome annotation of pigs and insufficient depth of previous sequencing data,functional lncRNAs with low expression levels cannot be excavated,leading to stagnation of pig lncRNAs.In this study,we first systematically predict lncRNAs in individual pig blastomeres by the 10G sequencing data.The candidate transcripts were scored using a non-dependent sequence annotation coding algorithm(CPAT,CNCI,PLEK),yielding 43303 transcripts representing the final lincRNAs transcript,corresponding to 18515 lincRNAs loci.The number of lincRNAs predicted by this study is very large,and it can provide a huge database for the study of porcine early embryonic lincRNAs.In addition,for lincRNAs,the apparent heterogeneity of each blastomere at the four-cell stage suggests that the presence of heterogeneous of lincRNAs prior to the expression of the protein-coding gene,it may indicate that lincRNAs can be involved in the regulation of coding genes,resulting in the heterogeneity of subsequent coding genes expressed in individual blastomeres.By combining the lincRNAs with the coding genes for WGCNA analysis,we have obtained a number of developmental regulatory networks for the expression of lincRNAs and mRNA during developmental stages... |
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