犏牛睾丸中血睾屏障ZO-1蛋白定位及差异表达调控分析OA

This study aimed to investigate the relationship between abnormal blood-testis barrier(BTB)function and male sterility in interspecific hybrid offspring dzo(cattle-yak)from three perspectives:the expression differences of BTB key genes,the localization differences of tight junction protein ZO-1 in spermatogenic cells,and the targeted regulatory relationships between miRNAs and BTB key genes.Based on the previous testicular transcriptome data,gene set enrichment analysis(GSEA)and differential expression analysis of key BTB genes were performed.Testicular tissues of healthy and sexually mature cattle(n=3,2.5 years),yaks(n=3,4.5 years)and dzo(n=3,4.5 years)were collected.The expression and localization of tight junction protein ZO-1 in testis were detected by Western blot and immunofluorescence staining.Target miRNAs of BTB genes were predicted,and differential expression analysis of these miRNAs was performed based on previous miRNA-seq data.The GSEA enrichment analysis showed that GO functions and KEGG pathway genes related to tissue remodeling,establishment of endothelial barriers,and gap junctions were significantly enriched in the dzo testicular tissue.The relative mRNA expression levels of BTB formation-related genes,including TJP1,TJP2,GJA1,F11R,TJAP1,and CDH2,were significantly higher in dzo than in yaks and cattle(P＜0.05).However,the relative protein expression level of ZO-1 exhibited no significant differences in testicular tissues among dzo,yak,cattle(P＞0.05).Immunofluorescence results indicated that ZO-1 was mainly localized in the intercellular spaces between spermatogonia and primary spermatocytes in the seminiferous tubules of yaks and cattle;whereas in dzo,it was localized on both sides of the primary spermatocytes.The localization of ZO-1 showed obviously significant differences among the three bovine species.Bioinformatics analysis indicated that bta-miR-191 potentially targets TJP1,bta-miR-93 and bta-miR-218 potentially target GJA1,bta-miR-200a and bta-miR-194 potentially target TJAP1,and bta-miR-218 potentially targets CDH2.Moreover,these miRNAs were downregulated in the testes of dzo.The cellular localization of the key BTB protein ZO-1 exhibits abnormalities in dzo testicular tissues,and the expression of BTB-related genes may be regulated by miRNAs.