| Cattleyak forms the first generation in the cross-breeding of cattle(Bos taurus)and yak(Bos grunniens),the purpose of which is to increase the yak's performance in meat and milk production.The female cattleyak is fertile,while the male remains sterile due to spermatogenic arrest.In the past several years,much work has been done to investigate the mechanism of the spermatogenic arrest of cattleyak.The significant divergences between the transcriptomes and proteomics sequencing between yak and cattleyak testis have suggested the critical role of microRNAs as post-transcriptional regulators in the control of genes expression during spermatogenesis.The spermatogenic cells,including spermatogonia and spermatocytes of cattle,yak,and cattleyak have not been successfully isolated so far and notably,their morphological and phenotypic characteristics and differentially expressed miRNAs profiling remain unknown.In this work,spermatogenic cells were isolated from these bovid species with the STA-PUT method that has been previously used for germ cell sorting in human and mouse,and the isolated cells have been utilized to investigate the mechanisms involved in male sterility observed in cattleyak.The characteristics and size of the isolated cells were investigated through microscopic examination,and the cell types were further identified with Reverse Transcription Polymerase Chain Reaction(RT-PCR)amplification of marker genes.Further,differentially expressed microRNAs profiling of isolated cells from cattleyak compared with yak/cattle were analyzed by using(Affymetrix(?)GeneChip(?)miRNA Arrays).The target genes prediction for differentially expressed miRNAs and their Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed by using different bioinformatics tools.The morphological characteristics revealed that the diameter of yak spermatogonium(14.90 ± 2.30 ?m)and cattle(10.10 ± 1.04 ?m)were significantly(P<0.01)larger than those obtained from cattleyak(8.60 ± 0.92?m).Meanwhile,the diameter of yak spermatocyte(20.50 ± 2.42 ?m)and cattle(19.40 ± 1.50 ?m)were also significantly(P<0.01)larger than those obtained from cattleyak(17.70 ± 2.05 ?m).Whereas,phenotypic characteristics were shown the purity of spermatogonia and spermatocytes isolated from each bovidae species was found to be greater than 85%.The total 66 differentially miRNAs were expressed with the>1.5 fold change among the isolated spermatogonia and spermatocytes of cattleyak,cattle and yak.Interestingly,39 differentially expressed miRNAs were obtained in cattleyak compared with yak in which 9 miRNAs were significantly up-regulated and remaining 30 were down-regulated.27 differentially expressed miRNAs were obtained in cattleyak compared with cattle in which 16 miRNAs were significantly down-regulated whereas 11 were up-regulated.Remarkably,48 differentially expressed microRNA were obtained from cattleyak when compared with yak/cattle.STA-PUT presents a convenient,economical and efficient method to isolate spermatogenic cells as it yields more cells within a short time frame.Detected differentially expressed miRNAs in cattleyak compared with cattle/yak were suggesting that,these miRNAs much more likely to play an important role in the spermatogenic arrest of cattleyak via targeting the candidate genes.Whereas,GO revealed the biological functions of targeted genes for differentially expressed miRNAs,and KEGG enrichment analyses showed that MAPK and BREAST CANCER pathways were involved in the top-listed significantly enriched pathway those are very essential for cell differentiation,proliferation and apoptosis etc.The identifications of these miRNAs have provided the valuable information for a better understanding of spermatogenic arrest in cattleyak. |
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