Eif2s3y Promotes Spermatogenic Cell Differentiation From Embryonic Stem Cells In Vitro

The formation of zygote indicates the production of a new individual.In multicellular animals,as the carrier of genetic materials,the generation of germ cells not only ensures the stability and maintains the genetic diversity,but also promotes the evolution of species.However,it is difficult to monitor and study the molecular events of mammalian germ cell generation in vivo.Clinical and epidemiological studies have shown that approximately 13-18% couples suffer from reproductive disorders,and the rate of infertility caused by abnormal male germ cell formation increases dramatically.Therefore,the generation of germ cells in vitro has become one of the most important subjects in life science research.The establishment of an in vitro induction system for germ cells could provide a research model to study germ cell pathogenesis,thus providing a theoretical basis and potential value for the treatment of human reproductive disorders.In mice,eukaryotic translation initiation factor 2,subunit 3,structural gene Y-linked(Eif2s3y),as a gene on the short arm of Y chromosome,has been proven to play a key role in spermatogenesis.In the absence of the short arm of Y chromosome,the overexpression of Eif2s3 y not only rescued the normal proliferation of spermatogonia,but also promoted it to differentiate into a meiosis stage.Furthermore,Eif2s3 y and Sry can interact with each other to guarantee normal spermatogenesis and this even ensured the production of functional round sperms in the absence of the Y chromosome.In male mice,the mutation of Eif2s3 y will result in a significantly smaller testicular volume and azoospermia.Although the function of this gene has been confirmed by the aforementioned studies,the underlying mechanisms needs to be further explored.In this study,we used cytokines to induce the differentiation of mouseembryonic stem cells(mESC)from primordial germ cell-like cells(PGCLCs)to the stage of spermatogonial stem cell-like cells(SSCLCs)and finally obtained spermatid-like cells(SLCs).Our further studies showed that Eif2s3 y not only determined the self-renewal of mESC,but also improved the induction efficiency of SSCLCs and SLCs.Moreover,when the induced SSCLCs were transplanted into the seminiferous tubule of infertile male mice,normal spermatogenesis and healthy offspring could be obtained.The main contents of this study are as follows:1.Establishment of the in vitro induction system for mESC to differentiate into SLC.We successfully created the in vitro induction system for mESC to differentiate into SLC based on the simulation of the in vivo developmental process of germ cells and data of our previous works.First,ActivinA and bFGF were used to induce mESCs into epiblast-like cells(EpiLCs).These Epi LCs were further treated by feeder cells combined with cytokines including BMP4,BMP8 A,SCF and Insulin to be induced to differentiate into PGCLCs.During the induction process of PGCLCs to SSCLCs,feeder cells and cytokines including GDNF,b FGF,BMP4,LIF,Insulin and high concentration of KSR were used.In order to verify the biological function of the induced cells,SSCLCs were transplanted into the seminiferous tubules of azoospermia mice.Eight weeks later,male germ cells at various developmental stages were observed in the seminiferous tubules of the recipient mice,and mature sperms were observed to fill in the epididymis of those mice.In order to guarantee successful meiosis in vitro,RA and low temperature were used in the system,and the induced haploid cells were proven to express acrosome proteins in vitro.Moreover,flow cytometry analysis showed that the percentage of haploid cells was approximately 9.8%.2.Eif2s3 y suppresses the pluripotency state and promotes the proliferation rate of mESCs.Considering the critical function of Eif2s3 y in spermatogenesis,we constructed a monoclonal cell line overexpressing Eif2s3 y in our induction system and investigated the effect of this gene on the biological features of these mESCs.The results showed that Eif2s3 y inhibited the pluripotency and promoted the proliferation rate of mESCs.Further tests proved that Eif2s3 y affected the pluripotency mainly through regulating TET1 and histone methylation levels of these cells,and regulated proliferation rate mainly through regulating expression levels of Cyclin A and Cyclin E.3.Eif2s3 y promotes differentiation of SSCs.We compared the expression levels of Eif2s3 y in SSCs,Sertoli cells and sperms and found the highest level of Eif2s3 y was in SSCs.Using a SSC cell line GC-1,we found that overexpression of Eif2s3 y inhibited the expression level of self-renewal-related genes Plzf and Gfra1,meanwhile promoting the expression level of meiosis-initiating genes Stra8 and Sycp3.In accordance with this,Eif2s3 y knockdown decreased the expression level of Stra8 and Sycp3.These results showed that Eif2s3 y can efficiently promote the differentiation of SSCs.This has provided a good foundation for our further investigation.4.Eif2s3 y enhances the efficiency of mESC to SLC transition.Eif2s3y plays a very important role in the process of mESCs to SLCs transition.Our data have shown that Eif2s3 y can promote the expression of DAZL,NGN3 and VASA in SSCLCs,indicatingan enhancement in SSCLC differentiation.By flow cytometry analysis,the percentage of VASA-positive cells in SSCLC overexpressing Eif2s3 y was approximately 13.3%,while this percentage was significantly lower in the control group(7.95%),suggesting that Eif2s3 y promoted the mESCs to SSCLCs transition.Furthermore,Eif2s3 y increased the expression levels of VASA,SYCP3,Acrosin and PRM1 in the induced SLC.Cell cycle analysis results showed that the percentage of haploid cells was approximately 11% in the Eif2s3y-overexpressing cells,compared with 8.6% of the control group.These results indicated that Eif2s3 ypromoted the induction of SSCLCs from PGCLCs and also increased the induction efficiency of mESCs to SLCs.In summary,this study established a novel induction system for the differentiation of mESCs to SLCs using all defined factors in vitro,and highlighted the critical role of Eif2s3 y in this process.Our work may provide help for the investigation of male germ cell development and may also provide a potential theoretical basis for clinical treatment of male infertility.