| The cleavage rate of in vitro cultured sheep embryos is faster than that of embryos in vivo,mainly due to the absence of negative regulatory factors in the in vitro culture environment.The inhibitory factors in the fallopian tubes could affect the embryo gene network through paracrine signaling,which regulates the cleavage rate by maintaining the balance between positive and negative regulations.When cultured in vitro,the positive regulatory gene is up-regulated,and/or the negative regulatory gene is down-regulated in the embryos,which is manifested as the high cleavage rate.The signaling pathways regulating pluripotency of stem cells,MAPK,PI3K-Akt,and Wnt,as well as NANOG,OCT4,SOX2,Klf4,and c-Myc genes that facilitate the proliferation of in vitro cultured embryos.The expressions of Y chromosome genes,such as SRY and TSPY,are also involved in the regulation of cell proliferation,but the underlying mechanisms remain unclear.The single-cell RNA-seq technology provides an effective tool to comprehensively observe the gene expression in embryos,accurately describe the spatiotemporal specificity of differentially expressed genes(DEGs),and precisely analyze the influences on key regulatory pathways.Therefore,this technique could help address the challenges in the transcriptome sequencing of single embryo of sheep.The transcriptome analysis of single embryo of sheep could clarify the genome transcription differences of embryos in the cleavage stage in vivo versus in vitro,exploring the transcriptome characteristics in different developmental stages,and revealing the mechanisms involved in the rapid proliferation of embryos cultured in vitro.In this study,the sheep embryos of 8-cell,16-cell,and morula stages cultured in vitro or obtained in vivo were used as the samples,for which the Smart-Seq2 amplification technology and Illumina Hi Seq Xten high-throughput sequencing were performed for the investigation,and gene overexpression and si RNA were also performed in mouse embryos for the verification of the functions of the DEGs.The findings are as follows:1.The in vitro and in vivo sheep embryos of 8-cell,16-cell,and morula stages were subjected to Smart-Seq2 amplification to construct the c DNA library,and the quality of the library was assessed using the Agilent-2100 bioanalyzer.The results showed that the c DNA fragments of the 18 samples were in the range of 1-2 kb,and the total amount of products was 60.8-376 ng.Illumina Hi Seq Xten high-throughput sequencing was performed for the transcriptome sequencing of the c DNA library,which showed that the Q30 of the 18 samples ranged from 92.22% to 94.02%,the contents of A-T and C-G were similar,and93.17-94.26% of the clean reads in different stages of the cleavage stage could be aligned to the reference genome.2.Bioinformatics analysis of the transcriptome sequencing data of the in vivo and in vitro sheep embryos at different cleavage stages was performed.The results showed that for the IVF embryos,170 DEGs were identified from the 8-cell stage to 16-cell stage,and 417 DEGs were identified from the 16-cell stage to morula stage.For the in vivo embryos,840 DEGs were identified from the 8-cell stage to 16-cell stage,and6631 DEGs were identified from the 16-cell stage to the morula stage.For the comparisons between the in vivo versus in vitro embryos,2226,2389,and 5965 DEGs were identified in the 8-cell stage,16-cell stage,and morula stage,respectively.The sequencing findings were verified by q RT-PCR,which showed consistent data on the expression levels.GO analysis was performed for the DEGs of different stages,which showed that the DEGs of IVF embryos were mainly clustered into several events including material synthesis,function regulation,and cell proliferation.while in vivo embryos were mainly clustered into aspects such as energy metabolism and material synthesis.The positive regulation of cell proliferation(GO:0008284),regulation of cell proliferation(GO:0042127),and signaling pathways regulating pluripotency of stem cells(map04550)were clustered in all stages of IVF embryos,in which the expressions of NANOG genes were significantly different in the above-mentioned pathways.3.Immunofluorescent labeling was performed for the NANOG,POU5f1,and c-MYC proteins in the cleavage stage of sheep embryos.NANOG and POU5f1 proteins can be detected from 8-cell to blastocyst;The c-MYC protein can only be detected during the blastocyst stage and exhibits cytoplasmic expression.The mouse embryos of cleavage stage were used for microinjection of overexpression and interference vectors of NANOG gene in the fertilized egg,respectively,and q RT-PCR was used to detect the expressions of KLF4,STAT3,SOX2,and OCT4 genes,and the embryonic development efficiency was assessed.After overexpression,the respective percentages of zygotes developed into the 2-cell,4-cell,8-cell,morula,and blastocyst stages were 100%,77.78%,70.37%,62.96%,and 44.44%,which were higher than in the control group.The expressions of KLF4,STAT3,SOX2,and OCT4 genes were up-regulated in all the stages of embryonic development.While after interference,the respective percentages of zygotes developed into the2-cell,4-cell,and 8-cell stages were 80.00%,72.00%,and 48.00%,which were lower than in the control group;no embryo developed into the morula stage.The expressions of KLF4 and STAT3 genes were detected in the 4-cell and 8-cell stages,but the expressions were down-regulated.OCT4 gene was only detected in the 8-cell stage,which was also down-regulated.SOX2 gene expression was not detected.4.The reference-free transcriptome analysis of the sequencing data was performed,which screened 51Y-chromosome expression genes.The SRY gene was expressed in 8-cell,16-cell,and morula stages of sheep embryos in vitro,with FPKM values of 34.15,50.47,and 54.68,while in vivo embryos with 8-cell,16-cell,and morula stages had FPKM values of 5.12,13.85,and 22.38.The mouse embryos of cleavage stage were used as the subjects,and the SRY gene overexpression and interference vectors were microinjected into the fertilized eggs.Then q RT-PCR was used to detect the expressions of SOX9,NANOG,OCT4,and CTNNB1 genes,and the embryonic development efficiency was assessed.After overexpression,the respective percentages of zygotes developed into the 2-cell,4-cell,8-cell,and morula stages were89.13%,71.74%,43.48%,and 4.35%;the percentages of zygotes developed into 2-cell,4-cell,and 8-cell stages were higher than in the control group,while the percentages of zygotes developed into morula stage were lower than in the control group;no zygote developed into the blastocyst stage.The expressions of four genes,SOX9,NANOG,OCT4,and CTNNB1,were up-regulated in all the stages of embryonic development.While after interference,the respective percentages of zygotes developed into the 2-cell,4-cell,and 8-cell stage were 84.85%,27.27%,and 18.18%,which were lower than in the control group;no embryo developed into the morula stage.The expressions of SOX9,NANOG,and OCT4 genes was not detected in other stages of embryonic development,and the expression of CTNNB1 gene was lower than in the control group.In summary,we first constructed the c DNA library of single sheep cleavage embryos based on the Smart-Seq2 amplification technique,and then Illumina Hi Seq Xten high-throughput sequencing technology was used for the transcriptome sequencing of the embryos of cleavage stage based on the library.The functions of DEGs were mainly clustered in function regulation and cell proliferation in IVF embryos,and mainly clustered in energy metabolism in the in vivo embryos.In the absence of paracrine signals,the expressions of positive and negative regulatory genes in IVF embryos were both up-regulated to achieve a new balance,in which NANOG genes promoted the rapid proliferation of IVF embryos through the stem cell maintenance signaling pathways.After gene overexpression and interference,NANOG acted as a positive regulatory gene for embryonic proliferation and promoted the proliferation of in vitro embryos of cleavage stage through the signaling pathways regulating pluripotency of stem cells.The SRY gene is expressed in sheep embryos from the 8-cell to morula stages,which promoted the proliferation of in vitro embryos of cleavage stage through upregulating the genes including NANOG and POU5f1.These findings have great significance for investigating the mammalian embryonic transcriptome,sheep embryo regulation,in vitro production of sheep embryos,and improving the efficiencies of relevant biotechnology techniques. |
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