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Reconstitution of male germline cell specification from mouse embryonic stem cells using defined factors in vitro

Cell Death & Differentiation (2019) | Download Citation


In vitro induction of functional haploid cells from embryonic stem cells (ESCs) has been reported by several groups. However, these reports either involve complex induction process with undefined induction factors or show low-induction efficiency. Here, we report complete meiosis in vitro from ESCs with defined induction factors. ESCs were first induced into primordial germ cell-like cells, which were further induced into male germline cells, including spermatogonial stem cell-like cells (SSCLCs) and spermatid-like cells. Importantly, the obtained SSCLCs were functional as infertile male mice sired healthy offspring via SSCLC transplantation. Further, we found that eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) was essential for spermatogenesis. Eif2s3y-overexpressing ESCs showed enhanced spermatogenesis in vitro, as demonstrated by higher expression levels of SSC-specific markers during SSCLC induction process, improved reproductive ability recovery of infertile male mice, and increased efficiency of haploid cell induction. Our work provides a convenient and efficient approach to obtain functional male germline cells.

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  1. 1.

    Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell. 2011;146:519–32.

  2. 2.

    Ishikura Y, Yabuta Y, Ohta H, Hayashi K, Nakamura T, Okamoto I, et al. In vitro derivation and propagation of spermatogonial stem cell activity from mouse pluripotent stem cells. Cell Rep. 2016;17:2789–804.

  3. 3.

    Zhou Q, Wang M, Yuan Y, Wang X, Fu R, Wan H, et al. Complete meiosis from embryonic stem cell-derived germ cells in vitro. Cell Stem Cell. 2016;18:330–40.

  4. 4.

    Zhao Y, Ye S, Liang D, Wang P, Fu J, Ma Q, et al. In Vitro modeling of human germ cell development using pluripotent stem cells. Stem Cell Rep. 2018;10:509–23.

  5. 5.

    Li N, Mu H, Zheng L, Li B, Wu C, Niu B, et al. EIF2S3Y suppresses the pluripotency state and promotes the proliferation of mouse embryonic stem cells. Oncotarget. 2016;7:11321–31.

  6. 6.

    Yamauchi Y, Riel JM, Ruthig VA, Ortega EA, Mitchell MJ, Ward MA. Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction. Science. 2016;351:514–6.

  7. 7.

    Murata C, Sawaya H, Nakata K, Yamada F, Imoto I, Kuroiwa A. The cryptic Y-autosome translocation in the small Indian mongoose, Herpestes auropunctatus, revealed by molecular cytogenetic approaches. Chromosoma. 2016;125:807–15.

  8. 8.

    Yamauchi Y, Riel JM, Ruthig V, Ward MA. Mouse Y-encoded transcription factor Zfy2 is essential for sperm formation and function in assisted fertilization. PLoS Genet. 2015;11:e1005476.

  9. 9.

    Yamauchi Y, Riel JM, Stoytcheva Z, Ward MA. Two Y genes can replace the entire Y chromosome for assisted reproduction in the mouse. Science. 2014;343:69–72.

  10. 10.

    Goodyear S, Brinster R. Spermatogonial stem cell transplantation to the testis. Cold Spring Harb Protoc. 2017;2017:pdbprot094235.

  11. 11.

    Ganguli N, Wadhwa N, Usmani A, Kunj N, Ganguli N, Sarkar RK, et al. An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation. Stem Cell Res Ther. 2016;7:142.

  12. 12.

    Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod. 2003;69:612–6.

  13. 13.

    Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating meiosis: the case for retinoic acid. Biol Reprod. 2012;86:35.

  14. 14.

    Jan SZ, Hamer G, Repping S, de Rooij DG, van Pelt AM, Vormer TL. Molecular control of rodent spermatogenesis. Biochim Biophys Acta. 2012;1822:1838–50.

  15. 15.

    Mazeyrat S, Saut N, Grigoriev V, Mahadevaiah SK, Ojarikre OA, Rattigan A, et al. A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis. Nat Genet. 2001;29:49–53.

  16. 16.

    Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature. 2004;427:148–54.

  17. 17.

    Saitou M, Miyauchi H. Gametogenesis from pluripotent stem cells. Cell Stem Cell. 2016;18:721–35.

  18. 18.

    Eguizabal C, Montserrat N, Vassena R, Barragan M, Garreta E, Garcia-Quevedo L, et al. Complete meiosis from human induced pluripotent stem cells. Stem Cells. 2011;29:1186–95.

  19. 19.

    Irie N, Weinberger L, Tang WWC, Kobayashi T, Viukov S, Manor YS, et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell. 2015;160:253–68.

  20. 20.

    Magnusdottir E, Dietmann S, Murakami K, Gunesdogan U, Tang F, Bao S, et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat Cell Biol. 2013;15:905–15.

  21. 21.

    Nakaki F, Hayashi K, Ohta H, Kurimoto K, Yabuta Y, Saitou M. Induction of mouse germ-cell fate by transcription factors in vitro. Nature. 2013;501:222–6.

  22. 22.

    Yokonishi T, Sato T, Katagiri K, Komeya M, Kubota Y, Ogawa T. In vitro reconstruction of mouse seminiferous tubules supporting germ cell differentiation. Biol Reprod. 2013;89:15.

  23. 23.

    Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 2004;101:16489–94.

  24. 24.

    Wang S, Wang X, Ma L, Lin X, Zhang D, Li Z, et al. Retinoic acid is sufficient for the in vitro induction of mouse spermatocytes. Stem Cell Rep. 2016;7:80–94.

  25. 25.

    Yang S, Yuan Q, Niu M, Hou J, Zhu Z, Sun M, et al. BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2. Reproduction. 2017;153:211–20.

  26. 26.

    Endo T, Romer KA, Anderson EL, Baltus AE, de Rooij DG, Page DC. Periodic retinoic acid-STRA8 signaling intersects with periodic germ-cell competencies to regulate spermatogenesis. Proc Natl Acad Sci USA. 2015;112:E2347–56.

  27. 27.

    Matsubara Y, Kato T, Kashimada K, Tanaka H, Zhi Z, Ichinose S, et al. TALEN-mediated gene disruption on Y chromosome reveals critical role of EIF2S3Y in mouse spermatogenesis. Stem Cells Dev. 2015;24:1164–70.

  28. 28.

    Li M, Liu C, Zhu H, Sun J, Yu M, Niu Z, et al. Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs). J Cell Biochem. 2013;114:294–302.

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We appreciate Dr. Christian Dani of University of Nice Sophia Antipolis for excellent comments and revisions. This work was supported by grants from the China National Basic Research Program (JFYS 2016YFA0100203), Doctoral Scientific Research Foundation of 2017 (Z109021715), the Program of National Natural Science Foundation of China (31572399 and 31272518) and China Postdoctoral Science Foundation (K3080217064).

Author contributions

N.L., W.T.M., Q.Y.S., M.F.Z., and W.Q.L. performed the experiments. N.L., W.T.M., Z.Y.D., C.Y.W., and B.W.N. analyzed the data. J.L.H. designed the project. N.L. and W.T.M. wrote the manuscript, and J.L.H. revised the manuscript.

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  1. College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China

    • Na Li
    • , Wentao Ma
    • , Qiaoyan Shen
    • , Mengfei Zhang
    • , Zhaoyu Du
    • , Chongyang Wu
    • , Bowen Niu
    • , Wenqing Liu
    •  & Jinlian Hua


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The authors declare that they have no conflict of interest.

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Correspondence to Jinlian Hua.

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