Differential Expression And Functional Prediction Of RNA In Ovaries Of Sheep With Low And High Litter Sizes

Litter size is an important trait that determines the production efficiency of sheep bred for meat.Its detailed investigation can reveal the molecular mechanisms that control the fecundity of sheep and possibly accelerate the breeding process of new varieties of sheep that have high prolificacy.Based on the lambing records of 1851 sheep,6 Monotocous and 6 Polytocous ewes with lambing numbers of 3 or more consecutive parities were selected.After three months of adaptive feeding,identification of the estrus cycle in natural estrus ewes was conducted.The estrus time of the ewes was subject to the climbing span of the ram receiving the test.The bilateral ovaries of sheep that had either multiple（P）or single births（M）in the follicular phase（F）were collected 36 hours after the third estrus（termed MF or PF,respectively,3 in each group,6 in total）,and from monotocous and polytocous sheep in the luteal phase（Luteal phase,L）216 h（9th day）after the third estrus（termed ML and PL,3 in each group,6 in total）.The follicles in follicular phase ovary and luteal phase ovary tissues were collected.Based on RNA-seq and Small RNA-seq techniques,the whole transcriptome expression profiles of sheep ovary tissues with different lamb sizes were obtained to mine the key genes regulating fertility.In this study,mRNA expression profiles of high and low lambing size sheep were established.A total of 1488 differentially expressed mRNAs were detected in MF vs.PF,and 885 mRNAs were detected in ML vs.PL.Through GO,KEGG,WGCNA,STEM and GSEA analysis,it was found that Ferroptosis,lysosome,MAPK signaling pathway and JAK-STAT signaling pathway play an important role in MF vs.PF,and Steroid Biosynthiesis regulate the follicle development process of sheep in ML vs.PL.RNA editing site analysis showed that the main RNA editing sites were A->G editing type is dominant,a total of 39932 were located in the coding region,of which 4898 lead to non-synonymous substitution of amino acids.3’UTR editing loci mainly bind with miR-23a and miR-24 to regulate follicular development.In addition,the variable splicing genes found in this study mainly regulate follicle development through the MAPK and PI3K-Akt signaling pathway.Eleven key regulatory genes,TGFBR3,CTSS,CASP8,RPS6KA1,POSTN,RASL10b,CTSC,JAM3,MAPK3,SIRT1 and STAR were obtained.In this study,LncRNA expression profiles of high and low lambing size sheep were established.The results indicate that the number of up and down-regulated LncRNAs in MF vs.PF was 95 and 111 and 109 and 49,respectively in ML vs.PL.The functional enrichment of the different LncRNAs co-expressed with mRNA was analyzed.The results demonstrated that the KISS1-GnRH-LH/PFH-E2 and EGF-EGFR-RAS-PI3K signaling pathways promoted the initiation of the primordial period,follicular development,and ovulation in the follicular phase（MF vs.PF）.During the luteal phase（ML vs.PL）,the production and development of the corpus luteum in ewes was influenced by the KITLG-KIT/FGF-FGFR/HGF-MET-RAS-ERK signaling pathway.STEM clustering functional enrichment analysis of the differentially expressed LncRNAs indicated that profile11 was principally enriched in the Cytokine-JAK-STAT,PDGF-PDGFR-PI3K,and KITLG-KIT-RAS-ERK signaling pathways.By analysis of the differential expression of the LncRNAs and their expression in each group,LncRNAs XIST（loc101112291）and GTL2（loc101123329）were found to be highly expressed,suggesting that regulation of follicular development was mediated through methylation processes.In this study,miRNA expression profiles were established in sheep with high and low lambing size.A total of 165 miRNAs were detected in MF vs.PF,among which 43 miRNAs were differentially expressed,and 42 miRNAs were differentially expressed in ML vs.PL.Through GO,KEGG,WGCNA,STEM,GSEA analysis,EGF-EGFR-RAS-ERK signaling pathway,EGF-EGFR-RAS-JNK signaling pathway,IL1-IL1R-JNK signaling pathway,IL1-IL1R-P38 signaling pathway were found.The miRNA-related target genes in the RAC/CDC42-PAK-ERK signaling pathway regulate follicular development.In addition,it is inferred that Novel₅41,oar_miR₅41₃p,oar_miR₄32,oar_miR₅41₃p,Novel₅61 and oar_miR₅41₅p play a key regulatory role in the process of follicle development to affect the level of feculity of sheep.The miRNA editing analysis found that the number of base edited reads at the oar-miR-143,oar-miR-148,oar-miR-21,oar-miR-26a and oar-let-7a sites was large,indicating that the number of mutant reads at each of these miRNA sites was large,thus the target genes were further affected and changed.The differentially expressed circRNAs were screened by transcriptome sequencing and their functions were predicted.We found that there were 146 up-and 37 down-regulated circRNAs in MF vs.PF while 14 up and 20 down-regulated circRNA were identified in ML vs.PL.Through DE-circRNA function enrichment annotation analysis by STEM and WGCNA,17 key circRNAs involved in the development of ovarian follicles were identified,which may function through the MAPK,WNT,mTOR,PI3K-AKT,TGF-β,RAP1,Ovarian steroidogenesis and EGF-EGFR-RAS-JNK signaling pathways to affect the litter size of sheep.Moreover,competitive endogenous RNA network analysis revealed the target binding sites for miRNA,such as oar-miR-27a,oar-miR-16b,oar-miR-200a/b/c,oar-miR-181a,oar-miR-10a/b,and oar-miR-432 in the identified DE-cirRNAs.The finally obtained host genes related to lambing traits of sheep include Novel_circ₀008231（PTGDR）,Novel_circ₀014289（CYP11A1）,Novel_circ₀0013554（INSR）,Novel_circ₀013595（SMAD1）,Novel_circ₀002663（ACVR2A）,Novel_circ₀003144（RPS6KA1）,Novel_circ₀005884（SNX73）,Novel_circ₀013561（ARHGAP10）,Novel_circ₀008937（CO12A1）,Novel_circ₀013116（PGR）and Novel_circ₀005497（LTBP1）。In this study,the selected 12 functional genes closely related to reproductive traits,10 LncRNAs,9 miRNAs and 10 circRNAs related to reproductive trait regulation genes were verified by real-time quantitative PCR.The accuracy of sequencing results was verified.To sum up,this research through to the high and low fertility sheep ovarian tissue are analyzed in full transcription form spectrum and screening to a large number of affect the sheep fertility genes and non-coding candidate functions,and build the non-coding genes of key coding gene regulation relationships and interactions network,through the relevant data mining,finally found,EGF and EGFR-RAS-ERK,EGF and EGFR-RAS-JNK signaling pathway,The IL1-IL1R-J-NK,IL1-IL1R-P38 signaling pathway was a key regulatory pathway.