Screening Of Differential Gene Expression In Mature Oocytes Of Young And Old Hu Sheep Ewes Based On Single-cell RNA-Seq

As one of China’s unique high reproductive varieties,Hu sheep has high economic value.Reproductive capacity is one of the important factors that affect the efficiency of sheep breeding and reproduction.With increasing age,the reproductive capacity of sheep continues to decrease,and aging of oocytes is the main factor that reduces the reproductive capacity of old sheep.The oocytes from young ewes and older ewes were used in this study to compare the ultrastructure and gene expression by single cell transcriptome sequencing.In order to study the regulation mechanism of aging of oocytes in Hu sheep.the results of this study as follows:i)The zona pellucida of oocytes of Yong Hu sheep has a loose fibrous structure with clear and obvious edges,and the mitochondria were evenly distributed.and the microvilli were short and lie on the surface of the plasma membrane of the oocyte,The perivitelline space of the oocyte was narrow,and the cortical particles were evenly distributed in the cortical area;the edge of the zona pellucida of the Old Hu sheep oocytes was blurred and arranged closely.The number of mitochondria in the cytoplasm became smaller.The distribution of villi is uneven,the number becomes smaller,the perivitelline space becomes wider,and the cortical particles were migrated and gathered,which can be seen in the cytoplasmic area.These results indicate that aging of oocytes has ultrastructure changes.ii)According to sc RNA-Seq sequencing,the original data Q20> 95%,Q30> 85%,the filtered data accounted for more than 80%.There were 614 differentially expressed genes in the young and aging sheep,of which 229 genes were up-regulated and 385 genes were down-regulated.Among them,SEMA3D、Nr CAM、PKD1、PLA2G4D、PLA2G6、PYCR1 involved Mitochondrial membrane potential regulation,antioxidant stress,lipid antioxidant etal.were down-regulated in the old group.These may be key genes involved in oocyte aging.iii)GO enrichment analysis found that differentially expressed genes mainly play roles in membrane,nuclear chromatin,vesicle,signal receptor activity,ATPase activity,cell-cell adhesion,cellular calcium homology,and regulation of phosphatidylcholine catabolic process.the KEGG pathway revealed that the differentially expressed genes were significantly enriched into pathways such as cell growth and death,development,aging,signal transduction,Cholesterol metabolism,ABC transporter,linoleic acid metabolism,metabolic pathway,retinol metabolism,etc.These significantly enriched pathways may be involved in the aging process of Hu sheep oocytes.iiii)In summary,this study analyzed the differences between the oocytes of the Yong Hu sheep and the Old Hu sheep from the morphological and genetic level,In addition,we combined with bioinformatics analysis DEGs and signaling pathways that might be related to the aging of oocytes in Old Hu sheep,Our results provided a research basis for further analysis of the molecular mechanism of aging oocytes in Old Hu sheep.