Comparative Analysis Of Testis Transcriptome For Yak And Cattleyak Based On RNA-Seq

Cattleyak is the hybrid offspring of yak and cattle,compared with parents of high altitude and harsh natural environment showed stronger adaptability and higher production of causative performance.Because of interspecific hybridization,reproductive isolation in cattle on the performance of F1 generation of male sterility,can not be good parentstraits of inheritance,which greatly restricts the cultivation of genetically modified varieties and the yak.The domestic and foreign scholars have conducted a lot of studies have confirmed that the main reason about male sterility is spermatogenic arrest,but the mechanism remains unclear.This study analysis of yak and cattleyak testis transcriptome spectrum by high-throughput sequencing,to explore the regulation of spermatogenesis cattleyak in the differences in the process,in order to comprehensively study on cattleyak spermatogenic arrest mechanism at the molecular level,and provide a theoretical basis for the study of male sterility in cattleyak.Thus,in this experiment,yak and cattleyak testis histology as the basis,combined with the second generation high-throughput transcriptome sequencing technology(RNA-seq)on testicular parenchyma of yak and cattleyak to construct transcriptome library,through bioinformatics analysis of yak and cattleyak in the differential expression of variable gene and splicing difference,real-time fluorescence quantitative PCR(qRT-PCR)gene expression in the testis validation of differential expression,the main results are as follows:1.Histology showed in cattleyak and yak testis:cattleyak testicular structural integrity,the shape and size of abnormal;and compared with yak,cattleyak seminiferous tube shapes,wall collapse,interstitial;germ cells in the testis was mainly for post spermatogonial cells within the seminiferous tubules of the membrane,monolayer arrangement,and spermatocytes and sperm cells are very rare,instead of yak testis seminiferous tubules from the basement membrane into the lumen are distributed in different developmental stages of germ cells,arranged in a multilayer.2.RNA-Seq successfully constructed the transcriptome of yak and cattleyak testis spectrum,high quality data were obtained from 29.5Gb,after a rigorous screening differential genes,a total of 2960 differentially expressed genes,including 679 genes up-regulated compared with yak,2281 genes expression regulation.3.GO enrichment analysis of differentially expressed genes:GO significant enrichment of a large number of genes involved in spermatogenesis.The up regulation of STRA8 and NLRP14 may lead to the accumulation of primordial germ cells or undifferentiated spermatogonia,and the increase of germ cell apoptosis.SPP1,SPIN2B,PIWIL1 and cell cycle progression and the integrity of the original cell genome,suggesting that spermatogenic arrest may begin in spermatogonia differentiation stage;CDKN2C,CYP26A1,OVOL1,GGN,MAK,INSL6,RNF212,TSSK1B,TSSK2 and TSSK6 and meiosis related to spermatogenesis block intensified these genes down that in the process of meiosis;PRM2,ODF3 and sperm components related to gene expression,resulting in abnormal sperm morphology,structure defects and lack of fertilization ability.4.PATHWAY enrichment analysis of differentially expressed genes:Wnt/beta-catenin signaling pathway is the first of the most significant enrichment pathway,which is a key signaling pathway to regulate cell growth and differentiation.In the Wnt3a pathway,the differentiation of PP2A and TCF/LEF-1 genes by block cattleyak spermatogonial stem cells.5.Alternative splicing:With the yak genome as the reference background,the use of TOPHAT comparison software to compare the transcript sequence found,the variable alternative splicing events in the cattleyak were significantly lower than those in the yak.Presumably due to low variable shear,so that the protein diversity is low,resulting in cattleyak spermatogenic arrest.6.RNA-Seq reliability test:randomly selected 10 different transcriptome sequencing the gene fluorescence quantitative test(Q-PCR)test,both in high-throughput transcriptome sequencing,or low flux Q-PCR levels are "consistent with the results of high and low".Indicating that the RNA-seq method is true and reliable in this experiment.Based on the comparison of histological and transcriptome related analysis,spermatogenic arrest began in spermatogonia differentiation stage,strengthen at meiosis stage,the performance of the sperm morphology and structural abnormalities,resulting in a lack of cattleyak fertilization ability.From the analysis that the variable shear pianniu due to protein diversity caused by low cattle spermatogenesis the mechanism of block.The whole article from histology and transcriptomics to clarify about male infertility,and to provide reference for the research about infertility.