Study On Screening Polytocous Candidate Genes In Sheep Based On Transcriptome Sequencing And Proteomics Analysis

Litter size is an extremely complex trait,which is regulated by genetics,epigenetic modification,and hormones.However,the molecular genetic mechanism of the polytocous trait is still unclear,and it is urgent to further explore it.In this study,we selected Small Tail Han ewes without FecB mutation but with different reproductive performance as the research object.We tracked and determined the reproductive phenotype.Single cell transcriptome sequencing?scRNA-Seq?,ovarian RNA-Seq and ovarian proteomics analysis were used to screen the differential genes and proteins related to polytocous trait of sheep.Ewes were divided into monotocous group and polytocous group according to the litter size and the number of ovulation based on the simultaneous estrus and intraperitoneal endoscopy.Compared with the monotocous group,the number of mature follicles in the polytocous ewes was higher?P>0.05?,but the average diameter was reduced by 1.62 mm?P<0.05?.The estrus status of two groups of ewes was observed by ram estrus test.Blood samples were collected and serum isolated for the determination of the concentrations of follicle-stimulating hormone?FSH?,luteinizing hormone?LH?,pregnenolone?P₄?,testosterone?T?and estradiol?E₂?during the whole estrus cycle.Compared with the monotocous ewes,the estrus initiation and the estrus termination in the polytocous ewes were 3.75 h earlier?P>0.05?and7.2 h earlier?P>0.05?respectively,but the estrus duration was shortened by 15.61%?P<0.05?.There was no significant difference in the concentrations of five hormones in the serum of the monotocous and polytocous ewes.Nonetheless,the concentration of E₂ in luteal phase increased by 11.09%the?P>0.05?.The results of scRNA-Seq sequencing showed that 415,395 and 507 differentially expressed genes?DEGs?were screened from oocyte?POo/MOo?,cumulus granulosa cell?PCC/MCC?and follicular membrane?PFT/MFT?comparisons,respectively.KEGG pathway enrichment analysis indicated oxidative phosphorylation?OXPHOS?and ribosome pathways play important roles in oocyte maturation and theca cells proliferation,and Valine,leucine and isoleucine pathways are related to the proliferation and function of granulosa cells.Nine DEGs,such as ATP5H,COX7A1,QCR10,GDF9,CD81and HSD17B1,etc.which may be related to the follicular development and the ovulation rate.The interaction analysis between lncRNAs and mRNAs indicated many differential lncRNAs such as MSTRG.230597,MSTRG.291734,MSTRG.1541,and ENSOARG00000026777 etc.,might exert important regulatory roles through their target genes.458 and 506 DEGs were screened by ovarian RNA-Seq from motocotous and polytocous ewes in the follicular phase?PFO/MFO?and motocotous and polytocous ewes in the luteal?PLO/MLO?,respectively.KEGG pathway enrichment analyses indicated OXPHOS,ribosome,ovarian steroid hormone synthesis,etc mainly related to ovarian physiological function and follicular development.Ten DEGs,such as HYAL2,STAR,GDF5,and CYP19A1,etc.were related to the ovarian function of sheep and affected ovulation number and litter size.The interaction between lncRNAs and mRNAs indicated many differential lncRNAs,such as MSTRG.41242 and MSTRG.99308,etc.may play important regulatory roles in their respective pathways through target genes.A total of 5,074 proteins were identified in the ovarian proteomic analysis in the luteal and follicular phases,with 101 and 57 differential abundance proteins in PFO/MFO and PLO/MLO,respectively.KEGG pathway enrichment analysis revealed that OXPHOS,ovarian steroid hormone synthesis,taurine and hypotaurine metabolism and apoptosis pathways were significantly enriched and involved in ovarian function regulation,among which 9 differentially abundance proteins such as COX7A,HYAL2 and StAR,etc.may regulate the polytocous trait of the ewes.The combined analysis of transcription and proteome showed that OXPHOS,ribosomal pathway and ovarian steroid hormone synthesis pathway might be related to polytocous trait of Small Tail Han sheep without FecB mutation.These pathways are related to ovarian function and follicular development,and can be used as key candidate pathways for the formation of polytocous trait in sheep.The STAR,HYAL2,COX7A1,QCR10,GDF9,CD81 and AIFM1 genes involved in these pathways can be used as key candidate genes related to polytocous trait.The above results need further functional verification.The results can provide a theoretical basis for revealing the molecular genetic mechanism and molecular design breeding for the formation of polytocous traits in sheep.