| Buffalo(Bubalus bubalis)is an important economic livestock species irn South Asia.And buffalo has many good characteristics:resistant to rough feeding,strong adaptability;high quality of dairy products,strong aroma;long service life;Buffalo meat is less fat;and buffalo is easy to manage.However,its economic value is limited by its low reproductive efficiency.There are many factors that cause its low reproductive efficiency,for example,the suitable age of buffalo breeding is older than yellow cattle and cows;the progesterone levels of female buffalo are much lower than those of yellow cattle during pregnancy;and the estrus characteristics of female buffalo are not obvious,and its calving interval is longer than yellow cattle and cows,also the female buffalo is not sensitive to the treatment of reproductive hormones.Under laboratory conditions,we found that the number of vesicles and oocytes that suitable for IVM(in vitro maturation)collected from buffalo ovaries was less than cattle and cows.At the same time,during the development of pre-implantation embryos,the rate of cleavage and blastocyst for buffalo are lower than yellow cattle,which indicate that there may exist a unique regulatory mechanism during the development of buffalo pre-implantation embryos.Before the zygote genome is activated,the early development of the embryo is mainly regulated by the maternal genome.Parthenogenetic activation of mammalian oocytes is an important research topic in reproductive biology.In addition,parthenogenesis is an important research model because it does not require any paternal factors,making it an ideal model for studying maternal genomes.Maternal genes play a role in regulatory networks,which requires the participation of many other genes.Therefore,a panoramic understanding of the role of these maternal genes in early embryonic development is necessary.However,so far,there is no research using buffalo parthenogenesis as model,to exploring dynamic changes in protein levels and transcriptome levels during pre-implantation embryo that are regulated by maternal genomes(without paternal genomic interference).Therefore,this study used maternal genomics function as an entry point to research the reproductive mechanism of buffalo by combining the techniques of quantitative mass spectrometry(TMT)-labeled quantitative proteomics and single-cell/embryo transcriptome sequencing,which could study the dynamic function of the maternal genome during the early embryonic development,and lay a foundation for improving our understanding of the regulation mechanism of maternal genome,also it will improve the technology of somatic cell nuclear transfer and parthenogenetic embryonic stem cell.The main findings of this paper are as follows:(?)Quantitative proteomics studies of buffalo oocytes before and after parthenogenetic activation.We discovered that these important differentially expressed proteins are mainly involved in signaling pathways of tight junctions and gap junctions.Then we used the real-time polymerase chain reaction(RT-PCR)and Western blotting to validate the expression for the four differentially expressed proteins(TUBB3,CTNNA1,CDH3,MAP2K1)at the transcriptional and translational levels.Furthermore,we used cellular immunofluorescence to analyze the migration and expression patterns of these proteins,which revealing that the maternal genome may firstly play a role in the formation of cell junctions after parthenogenetic activation.(?)The identificaiton of single-cell/embryo transcriptome files in buffalo parthenogenesis.Combining single-cell transcriptome sequencing technology,the oocytes and embryos from eight stages(GV,MII,2-cell,4-cell,8-cell,16-cell,morula,blastocyst)in embryonic development was obtained and analyzed.A total of 3567 genes were identified as differentially expressed in all successive stages during the development of pre-implantation embryo.At the same time,we also found 10,442 new genes,and the GO analysis of these new genes indicated that they have extensive cellular localization and participate in many molecular functions and biological processes.In addition,we identified eight clusters that were only highly expressed at specific developmental stages and were enriched in many GO clusters and KEGG pathways associated with specific phases.Also,we identified 1,530 hub genes from the gene network that regulated by the maternal genome,which are involved in 11 stage-specific modules.After the visualization using Cytoscape 3.2.1 software,we obtained a complex interaction network of these hub genes,revealing the efficient collaboration between the genes during the development of buffalo embryo.(?)Based on a combination of proteomics and transcriptomics data,we initially screened and identified buffalo maternal expression proteins or genes.In this study,we performed expression clustering and correlation analysis of the transcriptome files from buffalo parthenogenetic embryos.At the same time,the expression of NPM2(nuclear protein 2)and NLRP5(maternal antigen required for embryos;also known as Mater)that identified in proteomic data were analyzed in mRNA level,and we found that their expression in the GV stage were highest compared to all other stages,and after the 8-cell stage,the expression of NPM2 and NLRP5 sharply decreased,and almost not expressed in the blastocyst stage,which revealed that the process of "maternal to zygote transformation"(MZT)may exist in buffalo parthenogenesis,which may occur between the 8-cell and 16-cell stages,which is similar to normal fertilized embryos.(?)To analyze whether the key genes and proteins identified in this study were conserved between different species(human,mouse,and buffalo),we used Blast software to make homology alignment,and the IDs of the genes and proteins that differentially expressed in each stage of buffalo were converted into corresponding IDs of human and mouse,and their bioinformatics analysis(including GO and KEGG)was conducted,which made our results extend to mouse and human.We found that these proteins or genes between different species were showed the higher similarity of sequence,suggesting that these findings in the buffalo maternal genome will have a good reference value for the research of the maternal genome in human and mouse.In summary,our experiment used high-throughput proteomics and transcriptomics techniques to conducte the research of buffalo maternal genome using the buffalo parthenogenesis as the research model.This study provided a rich data resources for further research,and we screened a number of potential maternal effector genes or proteins,which increased our understanding of the regulatory mechanisms of maternal genome during the pre-implantation development in mammals,which also lay a theoretical foundation for further improvement of somatic cell nuclear transfer and embryonic stem cell technology.The strategy of this study also provide a valuable reference for other species to explore the function of the maternal genome. |
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