Regulation Of MiR-205 On The Proliferation And Apoptosis Of Leydig Cells

Leydig cells(LCs)are located in the interstitial space between the seminiferous tubules of the testis.The main function of LCs is to synthesize testosterone,which plays a key role in maintaining spermatogenesis and male secondary sexual characteristics.At present,the study on the regulation mechanism of proliferation and differentiation of LCs is mainly focused on rodents,rarely in livestock.Micro RNAs(mi RNAs)are an important post-transcriptional regulation mechanism that participates in almost all biological processes.However,even in the rat LCs,few studies report the function of mi RNAs.In 2017,our group successfully isolated pig stem leydig cells(SLCs)from 7-day-old piglets by enzyme digestion and established a short-term culture system.In this study the primary pig LCs(SLCs and differentiated LCs)and pig LCs with EDS treated for 24 h(the main cell type is SLCs)were used for RNA-seq,expression profiles of the m RNAs and mi RNAs were constructed,mi R-205 was screened for further study.Moreover,the regulatory of mi R-205 on the proliferation and apoptosis of LCs was explored using mimics and inhibitor.The important findings are as follows:1.Identification,isolation and culture of pig leydig cellsThrough PDGFRα immunohistochemical staining,the PDGFRα positive cells were located in the testicular interstitium.The isolated 7-day-old pig primary LCs was treated with 1.0 mg/m L EDS for 24 h,the expression of PDGFRα gene was not significantly changed,while the expression of CYP17A1 gene was significantly decreased(P < 0.05),suggesting that EDS can ablate differentiated pig LCs.In combination with EDS treatment and culture system with pig testicular fluid,SLCs clones were formed after 12 days of culture,confirming that the isolated cells contained SLCs.2.RNA-seq results of the primary group and the EDS-treated groupHigh-throughput sequencing results showed that 4904 genes were significantly differentially expressed between the primary group and the EDS-treated group.The GO annotation and KEGG analysis revealed that genes significantly down-regulated by the EDS-treated group were enriched for steroid hormone synthesis-related pathways.By comparing the mi RNAs sequencing data,15 known mi RNAs were found to be significantly differentially expressed between the primary and EDS-treated groups.The mi R-205 was the highest up-regulation mi RNAs in the EDS-treated group.The q RT-PCR results were consistent with the results of RNA-seq.In addition,the expression of mi R-205 was verified in pig SLCs clones and differentiated LCs.The results revealed that mi R-205 was highly expressed in pig SLCs clones(P < 0.05),suggesting that mi R-205 may be involved in the regulation of pig LCs differentiation.Thus,mi R-205 was screened for further study.3.Spatio-temporal expression profile of mi R-205Expression profiles showed that mi R-205 was widely expressed in various tissues of pigs,and its expression in epididymis was significantly higher than that in other tissues(P <0.05).With the development of pig testes,the expression of mi R-205 was significantly increased(P < 0.05).Furthermore,mi R-205 was significantly higher expressed in mouse Leydig cell line TM3 than mouse sertoli cell line TM4 and B-type spermatogonia cell line GC-1spg(P < 0.05).Bioinformatics analysis showed that mi R-205 is highly conserved among different species.Considering the transfection efficiency of primary cells and the absence of pig Leydig cell lines,subsequent investigations were performed on mouse Leydig cell line TM3.4.Functional study of mi R-205 on leydig cell proliferation and apoptosisThis study was performed on mouse leydig cell line TM3.In vitro overexpression/interference with mi R-205,no expression difference of LCs differentiation marker genes(HSD3B1 and St AR)was detected,speculating that mi R-205 may not regulate LCs differentiation.Results from CCK8,q RT-PCR,WB,TUNEL,EDU and flow cytometry,indicated that mi R-205 induced cell apoptosis but did not affect cell proliferation.Meanwhile,the analysis results was similar in mouse B-type spermatogonia cell line GC-1spg.5.miR-205 target gene was TP53BP2 geneTP53BP2 was identified as a target gene of mi R-205 by a dual fluorescent reporter vector assay.In summary,this study uncovered the m RNAs and mi RNAs expression profiles of pig SLCs and differentiated LCs,revealed that mi R-205 promotes apoptosis in TM3 and GC-1spg cells,demonstrated that TP53BP2 was a target gene of mi R-205.These findings will provide a theoretical basis for improving the reproductive performance of boars.