The Identification And Functional Analysis Of MicroRNAs In The Spermatogenic Arrest Of Cattleyak

Cattleyak are the F1 hybrids of cattle and yak,exhibiting much higher performances than yak and the same prominent adaptability to the harsh climate environment of Qinghai-Tibet plateau as yak.However,male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted their effective utilization in yak breeding.MiRNAs are a class of non-coding small RNAs,regulating gene expression at post-transcriptional level by affecting the stability of target mRNAs or inhibiting the process of protein translation.In order to understand the possible regulatory roles of miRNAs involved in the spermatogenic arrest of cattleyak,illumina HISeq was used for the identification of differentially expressed miRNAs between the testis of yak and cattleyak;and the molecular mechanism of bta-miR-34 c regulating the differentiation of spermatogonia in yak was preliminarily analyzed.The main results are as follows:(1)TUNEL-POD staining analysis of the testicular tissues of yak and cattleyaks showed that there were only a single layer of spermatogonia attached to basement membrane of seminiferous tubules and a few spermatocytes,but no round or extended spermatid.Spermatogonia began to apoptosis at the earliest stage and its apoptosis aggravated at the spermatocyte stage,resulting in subsequent spermatogenic arrest;(2)Illumina HISeq and bioinformatics analysis of miRNAs in the testicular tissues of yak and cattleyaks identified 61 differentially expressed miRNAs,in which miR-19b-3p,miR-592,miRNA135 a,miR-378 and miR-184 involved in self-renewal and differentiation of SSCs;miR-34 c and miR-449 a involved in the initiation of meiosis;(3)Spermtogonia and spermatocytes with higher purity were isolated from yak by STA-PUT apparatus via the velocity sedimentation;RT-PCR analysis showed that CD9 and UCHL1 were expressed in the isolated spermatogonia,while Tesmin and SYCP3 were expressed in the isolated spermatocytes;(4)By transfecting bta-miR-34 c mimic and inhibitor into yak spermatogonia,respectively,it was found that bta-miR-34 c might downregulate the expression of E2 F transcription factor by inhibiting the activity of CD4 and CDC25 A,thus promoting the differentiation process of germ cells from mitosis to meiosis;at the same time,bta-miR-34 c might activate p53 pathway to induce germ cell apoptosis by inhibiting the expression of HDAC1 and SIRT1,thus maintaining the steady number of spermatogenic cells in the seminiferous tubules.