Study On Signaling Pathways Related To Meiosis In Male Germline Stem Cells Of Dairy Goat

Male germline stem cells (mGSCs) is one of the most active and attractive biologicalresearch areas in recent years. The studys on mGSCs had broad application prospects in thefield of medicine and biology. mGSCs located near basement membrane in the testis, whichwere the basis of spermatogenesis. The meiosis of mGSCs was regulated by many factors,currently, many genes had been discovered that they were involved in the initiation of meiosis.The current understanding of meiosis regulation is derived primarily from studies in mice, butthe regulation of meiosis in dairy goat is relatively not clear.The results first revealed the expression level of Boule, Stra8, P53and miR-34c in30dpp,90dpp and adult testes of dairy goats. Meanwhile, we detected the maeker genesexpression of germ cell and pluripotent stem cell in the mGSCs isolated. We over-expressedthe genes involved in meiosis in mGSCs and then detected whether some important genesexpression were changed. We discovered some pathways in mGSCs:(1)Boule could make adirect activation to Stra8promoting its transcription and translation;(2)miR-34c was P53dependent in male germline stem cells(mGSCs);(3)Over-expression of Boule and Stra8promoted the expression of miR-34c in dairy goat mGSCs.1. Expression pattern of Boule in dairy goat testis and its function in promoting themeiosis in male germline stem cells (mGSCs)Boule is a conserved gene in meiosis, which encodes RNA binding protein required forspermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesiswhich contributes to infertility. Up to date, the expression and function of Boule in the goattestis are not known. The results first revealed that the expression level of Boule in adulttestes was significantly higher than younger and immature goats, and azoospermia and maleintersex testis. Over-expression of Boule promoted the expression of meiosis-related genes indairy goat mGSCs. The expression of Stra8was up-regulated with over-expression of Bouleanalysed by western-blot and luciferase reporter assay. While, Cdc25A, a downstreamregulator of Boule, was found not to affect the expression of Stra8, and our data illustratedthat Cdc25A did not regulate meiosis via Stra8. The expression of Stra8and Boule was up-regulated by RA induction. Taken together, these results suggested that Boule played animportant role in dairy goat spermatogenesis and that over-expression of Boule may promotespermatogenesis and meiosis in dairy goat.2. Induction of miR-34c by p53leads dairy goat male germline stem cells (mGSCs)to apoptosisMicroRNA (miRNA) is a class of non-coding RNAs that negatively regulate geneexpression. Recent evidences have indicated that miRNAs may play an important role in aseries of biological process. However, the role of miR-34c in male germ cell has littleinformation. The purpose of this experiment is to investigate the effects of miR-34c onapoptosis and proliferation of dairy goatsmale germline stem cells (mGSCs), as well as therelationship between p53and miR-34c. Morphological observation, Brdu staining, flowcytometry, Q-RT-PCR and Western-blot were utilized to ascertain the apoptosis andproliferation of dairy goat mGSCs, through transfection of miR-34c mimics (miR-34c),miR-34c inhibitor(Anti-miR-34c) mimics and inhibitors co-transfected (Mix) compared withcontrol group. The results manifested that overexpressed miR-34c promoted dairy goatmGSCs apoptosis, and suppressed proliferation. Simultaneously, some apoptosis-relatedgenes’ expression level were increased, on the contrary, some proliferation-related geneswere decreased. Accordingly, miR-34c promoted mGSCs to apoptosis and descended theirproliferation, moreover, expression of miR-34c was p53-dependent.3. Expression of miR-34c not through p53in response to over-expression of Bouleand Stra8in male germline stem cells (mGSCs)Reproduction is required for the survival of all animals. Spermatogenesis is an essentialand complex developmental process that ultimately results in production of haploidspermatozoa. Recent studies demonstrate that Boule and Stra8gene play important roles inmeiosis in male germ cells. Role of miR-34c microRNA in the late steps of spermatogenesisis indispensable, remarkably, the main function of miR-34c is to reduce cell proliferationability and promote cell apoptosis. The results first revealed the expression level of Boule,Stra8, P53and miR-34c in30dpp,90dpp and adult testes of dairy goats. The expressionlevel of Boule, Stra8, P53and miR-34c in adult testes of dairy goats was significantly higherthan that of30dpp. Over-expression of Boule and Stra8promoted the expression of miR-34cin dairy goat mGSCs. In previous work, we had demonstrate that miR-34c was P53dependent in male germline stem cells (mGSCs). Now we confirmed that the up-regulation ofmiR-34c was not due to P53protein activated which might be caused by the up-regulation of Boule and Stra8promoting the advance of meiosis. In addition, we found RA (Retinoic Acid)would decrease the expression of p53and miR-34c, did not change the expression of c-Mycgreatly. It suggested that the function of driving differentiation by RA might not be caused byp53.