The Role Of MiR-34c In Goat Male Germline Stem Cells Proliferation And Differentiation

Germ cells were unique in the way that they have the capacity to transmit geneticmaterials to offspring and the study of transfer characteristic in phylad is hot in bioscienceresearch recently. The research on germ cells' differentiation and development was criticalfor animal germplasm optimization and transgenic animal breeding. But its molecularbiology mechanisms had not made any significant progress. Recent study shows that miRNAplayed an essential role in the regulation of mammalian germ cell development, especially formiR-34c, which enhance germ cell characteristics and ensure spermatogenesis.Immunohistochemical and immunofluorescence staining were used to study thedevelopment of male germ cell in Guanzhong dairy goat. By using real-time PCR, theexpression of stage peculiar molecular were detected, and miRNA that associated withspermatogenesis of Guanzhong dairy goat were selected. Target genes of this specificmiRNA were predicted by bioinformatics and dual-luciferase reporter vector of which wasconstructed. The predicted targeted gene was confirmed by vector co-transfectionexperiment. Lastly, to study the effect of miRNA on male goat germ cells proliferation anddifferentiation, miRNA mimics and its inhibitor were used to transfected into the male germstem cells of Guanzhong dairy goat.(1)The study of development of different stages of goat germ cells. The gonads in variedstages were detected by HE staining, immunohistochemical, immunofluorescence stainingand real-time PCR methods, respectively. The results showed that after sex differentiation,the processes of germ cells development can be divided into three stages:(a)gonocyte stage,located in51～59dpc;(b) perspermatogonia,51～59dpc to3month after birth;(c)spermatogonia,which germ cells begin their first meiotic division, located in3month afterbirth. The expression of gene Stra8was abundant when germ cells arrive the meiotic stage,and this gene only expressed in spermatogonia.(2) The screen of spermatogenesis closely related miRNA. The expression level ofmiRNAs were detected in dairy goat testis of different developmental stages by real-timePCR and therefore the germ cells development related miRNA were determined. Thepeculiar miRNA in our study was miR-34c, and our data showed that, the expressioncharacterization of miR-34c was similar to Stra8, which suggests miR-34c hold the potential of meiosis regulation.(3) Targeted genes searching of miR-34c and its function in germ cells differentiation andproliferation of dairy goat. In our study, genes targeted by miR-34c were predicted bybioinformatics. The dual luciferase reporter vector of miR-34c that containing the3'UTRand the gene mutants of miR-34c was constructed. The luciferase activity were determinedand the bioinformatics prediction were verified by dual luciferase reporter system. Ourresearch predicted that Stra8was the targeted gene of miR-34c, which were verified by thedual luciferase reporter system that the seed zone (1361-1367bp)of3'UTR in Stra8genewere tageted by miR-34c. In our study, techniques of transfection, real-time-PCR,westernblotting and BrdU immunofluorescence staining were used to study the the function ofmiR-34c in germ cells differentiation and proliferation of dairy goat. Our data showed that,comparing with control, the expression of Rarg, Stra8and c-Myc was down-regulated aftertransfected with the mimics of miR-34c, and the proliferation of male germ stem cells in dairygoat could be inhibited by miR-34c.