Study On Roles Of MicroRNA-34c In The Differentiation Of Mouse Spermatogonial Stem Cells In Vitro

It is critical to maintaining the functions of tissues and organs that stem cells continue toproduce differentiated cells in animals. The process of spermatogonial stem cells (SSCs)continuingly differentiating into spermatogonia, spermatid and sperm in testis runs throughoutthe entire life of the mammal males. Furthermore, SSCs can be used in regenerative therapyand are also good models for adult stem cell research. microRNAs, usually18-25nt, playingessential roles in gene regulation among a wide range of organizations, including theregulation of self-renewal, cell differentiation, proliferation and apoptosis, it also maintains animportant function in stem cell regulation. microRNA-34c (miR-34c), belonging to themiR-34c family, participating among cellular life processes, having important roles inspermatogenesis as well. Nanos is a conserved RNA-binding protein family, and is necessaryin germ cells development. In mice, Nanos2express specifically in male germ cells. Inspermatogonial stem cells, Nanos2maintain stem cell population during spermatogenesis. Toclarify which roles miR-34c plays when over-express in the progress of spermatogenesis, wepredicted target genes of miR-34c, and constructed dual-luciferase reporter gene vector andmutant vector of predicted target gene, transfected miR-34c mimics and inhibitors intospermatogonial stem cells, cultured with medium containing miR-34c lentiviral venom withadult mouse seminiferous tubules to study the regulatory roles of miR-34c in thedifferentiation of spermatogenesis.1.Isolation, cultivation of mouse spermatogonial stem cellsTestes of6to12day postnatal mice were harvested and the tunica albuginea were peeled.After digested in collagenase IV and trypsin, the cells were differentially plated to removeSertoli and Myoid cells. Different groups combined various medium with factors wereutilized to screen the best system.DMEM+15%KSR plus0.25μmol L-1A83-01were chosento culture the SSCs in vitro without feeders. The cells were passaged and tested bymorphological observation, PCR, immunofluorescence staining, alkaline phosphatase stainingtechniques. It was proved that this system could maintain spermatogonial stem cellsself-renewal state and characteristics. 2.Screening the target gene closely related to mouse spermatogenesismiRWalk and miRDB databases were used to identify and find the precise interactionsites. Nanos2was chosen because of its roles in regulating SSCs’ self-renewal andproliferation. The dual-luciferase reporter gene vectors were constructed using thepmiR-RB-REPORTTMvector and the3’UTR, and mutated3’UTR of Nanos2(Mut-Nanos2)sequences. The activities were measured as the validation of direct-interaction betweenmiR-34c and Nanos2.3.Over-expression miR-34c promotes mouse SSCs to enter spermatogenesisAfter transfected miR-34c mimics into mouse SSCs, miR-34c lentiviral vector in vitrotransfected in seminiferous tubules, combining with qRT-PCR, immunofluorescence staining,western blot and whole-mount staining, our results showed that miR-34c may promotemeiosis progress by interacting with Nanos2leading up regulation of Scp3、Stra8in mousespermatogonial stem cells.