| Under the model of large-scale dairy farming where artificial insemination technology is widely used,hundreds of millions of doses of frozen semen are circulated around the world every year,and the semen of breeding bulls plays a crucial role in the genetic improvement of the population.In recent years,with the rapid development and application of high-throughput sequencing technology,many researchers were focusing on the study of non-codingRNA regulation of testicular development and spermatogenesis in dairy cows.However,little has been reported on the regulatory mechanism of non-codingRNAs in testicular development of Chinese Holstein bulls before and after sexual maturity.However,little has been reported on the regulatory mechanism of non-codingRNAs in testicular development of Chinese Holstein bulls before and after sexual maturity.As an important dairy breed in China,it is of great significance to investigate the genetic mechanism and molecular regulation of testicular development in Chinese Holstein bulls.Based on this,the testes of Holstein bulls at two developmental stages(8 weeks of age,young bulls and 80 weeks of age,adult bulls)were used in this experiment,and the testosterone(T)levels in the peripheral blood of Holstein bulls at different developmental stages were detected by radioimmunoassay,and changes in the development of the epithelial cells of the varicose vasculature and spermatogenesis were observed by the help of hematoxyline eosine(HE)staining.The expression profiles of mRNAs and non-codingRNAs(miRNAs,circRNAs and lncRNAs)were further analysed based on the whole transcriptome sequencing data of testis tissues,and the competitive endogenousRNA(ceRNA)regulatory network was constructed,and the genes that were differentially expressed were screened and obtained through the functional enrichment,modularity,Hub,and co-expression analyses,which are related to testis development and spermatogenesis.We screened the differentially expressed genes by functional enrichment,modular analysis,Hub analysis and co-expression analysis to obtain the candidate target genes related to testis development and spermatogenesis:(1)The results showed that there were significant differences in testicular morphology among Holstein bulls at different developmental stages.The diameter of the seminiferous tubules increased with age from 50 μm in the pre-sexually mature testis to 200 μm in the post-sexually mature testis,and the interstitial connective tissue was larger in post-sexually mature testis than pre-sexually mature testis,allowing spermatozoa to migrate to the basal part of the convoluted seminiferous tubules.Meanwhile,testosterone levels in the testes of Holstein bulls increased significantly(P < 0.05)from 0.003 ± 0.001 ng/m L before sexual maturity to 0.167 ± 0.0078ng/m L.(2)A total of 16,956 differentially expressed(P <0.05)lncRNAs(DEL)and 7,970 differentially expressed mRNAs(DEG)were identified.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses indicated that target genes of DEL and DEG were mostly enriched in processes related with spermatogenesis.Co-expression analyses screened a total of 3642 gene pairs with significant relationships,and further Hub analyses identified six lncRNAs(LNC.11740.1,LNC.13308.1,LNC.17541.1,LNC.32897.1,NONBTAT026018.2,and NON-BTAT028534.1)and enriched to 71 mRNAs regulated by it.Functional analyses of differentially expressed Hub LncRNA-mRNA pairs showed that the cell cycle checkpoint,spindle assembly and sister chromatid segregation functions were more active in sexually mature testes,suggesting that spermatogenesis was higher in sexually mature testes than in pre-sexually mature testes.(3)A total of 683 known miRNAs and 431 novel miRNAs were identified as significantly expressed(P <0.05).1641 up-regulated and 1506 down-regulated known miRNA target genes and 1100 up-regulated and 2254down-regulated novel miRNA target genes were obtained.6501 up-regulated and down-regulated miRNA target genes were obtained using miRanda target gene prediction software.A total of 6501 up-and down-regulated miRNA target genes were obtained using miRanda target gene prediction software.The results of GO and KEGG functional enrichment analyses investigated the potential functions of differentially expressed genes during testicular development,and miRNAs associated with reproductive system development were identified.The miRNAs(17＿16057-5p,X＿45311-3p,bta-miR-34 c,bta-miR-375,17＿15431-5p,10＿2547-3p,and bta-miR-449b)and mRNAs(BCL2L11,ADAMTSI1,MED1,and PRDM1)were associated with reproductive system development and others with the process of spermatogenesis such as mRNAs(STRB and BCL2L11)and miRNAs(bta-miR-134,bta-let-7e,bta-miR-181 b,and bta-miR-214).(4)A total of 3032 circRNAs and 683 miRNAs were identified as significantly differentially expressed(P<0.05).Based on the above results,a circRNA-miRNA-mRNA ceRNA(competing endogenousRNA)regulatory network containing 3298 targeted regulatory axes was constructed by DEG.Functional enrichment analysis showed that most of the gene functions in the ceRNA regulatory network in the sexually mature testis were related to amino acid metabolism,glucose metabolism and signal transduction.Module analysis showed that the ceRNA regulatory network in the sexually mature testis exhibited a more positive regulatory role in spermatogenesis cycle checkpoints,chromosome and cytoplasmic segregation,sperm tail formation,and sperm viability.In this study,we used whole transcriptome sequencing technology to comprehensively analyze gene expression in testicular tissues of Chinese Holstein bulls before and after sexual maturity,and revealed the possible regulatory roles of lncRNAs,miRNAs,and circRNAs in testicular development and spermatogenesis of Holstein cows,which further broadened our understanding of the regulatory roles of non-codingRNAs and their regulatory networks in spermatogenesis.It also helps to further investigate the molecular mechanisms by which mRNAs as well as non-codingRNAs regulate mammalian testicular spermatogenesis,and helps to identify specific marker genes and potential therapeutic targets for testicular development. |
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