Effects Of CCND1 And CCND2 In Cumulus-Granulosa Cell On The Development Of Yak Oocytes By Single-cell Sequencing Analysis

To obtain a large number of matured oocytes by in vitro maturation(IVM)is one of the key factorsfor modern reproductive technology including in vitro fertilization(IVF),embryo transfer and nuclear transfer.The quality of oocyte has a significant influence on the successful of fertilization and embryonic development.Compared with in vivo development,the efficiency of IVF and pregnancy was significant low after yaks’ oocytes IVM.Granulosa cells play an important role in the maturation and development of oocytes,which communicate with oocytes through paracrine pathways and gap functions.Granulosa cells can transport nutrients,metabolites and cell second messenger to oocytes,and affect the block and recovery of oocyte meiosis,cytoplasmic maturation,fertilization,and subsequent embryonic developmental potential.Therefore,in this study,we successfully determined the development process of GⅤ stage,MⅠ stage and MⅡ stage by orcein staining,and then RNA was extracted from the granulosa cells of GⅤ stage,MⅠ stage and MⅡ stage,and amplified by Smart-seq to construct cDNA library.The libraries were sequenced using Illumina HiSeqTM2500 platform,and then we successfully obtained the differentially expressed genes of the three stages.CCND1 and CCND2 were analyzed by immunohistochemical,immunofluorescence and RT-qPCR in yak,and the relation between CCND and the developmentalcompete-nceof oocyte.The results as below:(1)According to chromosome morphological characteristics of oocytes during IVM,the results of orcein staining showed that the development process of GⅤ stage,MⅠ stage and MⅡ stagein vitro is 0 h,12 h and 22 h,respectively.(2)We obtained 7540,8491,7520 transcripts in GⅤ stage,MⅠ stage,MⅡ stage respectively when mapped to yak genome.There were 318 up-regulated genes and 304 down-regulated between GⅤ and MⅠ,141 up-regulated genes and 334 down-regulated between MⅠ and MⅡ,191 up-regulated genes and 88 down-regulated between GⅤ and MⅡ.In order to examine the reliability of single-cell-seq data,the RT-qPCR was used to analyze the expression of 6 randomly selected genes,and showed that the results were consistent with the data of single-cell sequencing.(3)After the location by immunohistochemical,qualitative by immunoflunoresce-nce and quantitative analysis by real-time flunorescence quantification and western blotof CCND1 and CCND2 genes,the results showed that CCND1 and CCND2 were both expression in granulosa cells and significant higher in GⅤ stage than in MⅠ and MⅡ stages.(4)The relation between CCND and the developmental competence showed that CCND1 and CCND2 are positiverelated to the mature rate and blastocyst rate of yak oocytes.In summary,we obtained the transcriptome of yak granulosa cells at GⅤ stage,MⅠ stage and MⅡ stage by single-cell sequencing.And thedata showed that thegenes in granulosa cells areactive during meiosis maturation in vitro of yak oocytes,and play key role for meiosismaturations.In addition,the expression of CCND1 and CCND2 werepositiverelevance with the maturation and development of yak oocytes,and suggested that CCND1 and CCND2 could be used as biomarkers for assessing the quality and developmental potential of yak oocytes,which is helpful for providing the basis for the selection of high quality oocytes and improving IVM system,to improve the ability of maturation,embryo development and pregnancy outcome.Moreover,this study first explored the molecular mechanism of granulosa cells associated with yak oocyte maturation,laid the foundation for the analysis of the principle of lower in vitro blastocysts.