Rna-seq Profiling Of Long Non-coding RNAs And Their Potential Functions In Spermatogenic Arrest Of Cattleyak

Male sterility in cattleyak is caused by spermatogenic arrest,which hinders the effective fixation of fine genes of hybrid cattleyak in yak breeding.For many years,the mechanism of spermatogenic arrest in cattleyak has been studied in many aspects,such as histomorphology,cytogenetics,endocrinology and molecular biology.However,the comparative study of long non-coding RNA?lncRNA?in testis between cattleyak and their parents has not been reported.With the discovery of the important biological functions of lncRNA in mammals,including spermatogenesis,through spatiotemporal dynamic expression,preliminary identification of lncRNA in cattleyak and yak testicular tissues and preliminary exploration of its functions may bring new insights into the molecular mechanism of cattleyak male sterility.In this study,transcriptome sequencing and bioinformatics analysis were performed in testicular tissues of yak and cattleyak aged 12±1months from the same growth condition using the second generation high-throughput sequencer Hiseq 2500,followed by cloning and expression analysis of lncRNA_NONBT-258.STA-PUT method was used to isolate spermatogenic cells from yak and cattleyak,and the function of lncRNA_NONBT-258ONBT-258 was preliminarily studied after siRNA transfection of yak spermatogenic cells.The main results are as follows:1.604 differentially expressed lncRNAs?|Fold Change|>2&P-value<0.05?were screened by transcriptome sequencing.Most of them?469?were low expressed in the testis of cattleyak,and most of the lncRNAs with large differential expression multiples and high significance were also downregulated.The average length and expression level of these lncRNA transcripts were significantly shorter or lower than those of the mRNA transcripts identified together.2.Enrichment analysis of target genes of differentially expressed lncRNAs identified 83and 32 significantly enriched GO terms?P<0.01?and KEGG pathways?P<0.05?,respectively.These target genes are involved in the initiation of cell division,negative regulation of cell cycle and phase change,detection of cell cycle process,damage detection and repair of DNA replication,homologous recombination,endocytosis,programmed cell death,growth,differentiation,apoptosis and metabolism of cell substances,and other important signaling pathways and biological processes during spermatogenesis of cattleyak.3.Quantitative expression analysis of differentially expressed lncRNAs and their target genes in testicular tissues of yak and cattleyak,and qRT-PCR analysis of spermatogenic marker genes and cell cycle-related genes in isolated spermatogenic cells from yak and cattleyak showed that the expression of these molecules in cattleyak was consistent with the sequencing data,which confirmed the reliability of transcriptome sequencing data.4.The higher homology?98.29%?of lncRNA_NONBT-258ONBT-258 between yak and cattleyak indicated that its higher conservativeness under homologous and heterogeneous species might reflect its important function in the testicular tissue of cattleyak to some extent.Bioinformatics analysis further predicted the low coding ability of lncRNA_NONBT-258ONBT-258 and its strong interaction with target gene BRCA1.5.Spermatogenic cells?including spermatogonia and spermatocytes?can be isolated from testicular tissues of yak and cattleyak using STA-PUT device.Specific siRNA transfection into yak spermatogenic cells failed to reduce the expression level of lncRNA_NONBT-258successfully,so it is necessary to optimize the conditions of cell transfection.This experiment showed that there were a large number of differentially expressed lncRNAs in testicular tissues of yak and cattleyak.These target genes of lncRNAs have significant differences in the enrichment of important biological processes and signaling pathways related to cell cycle progression,DNA replication,homologous recombination,correction of abnormal cell proliferation,cell adhesion,migration,immunity,metabolism,programmed death and apoptosis,suggesting that lncRNAs may participate in spermatogenic cell growth,proliferation and differentiation process by regulating the expression of target genes,and then affect the process of spermatogenesis of cattleyak.