, Volume 71, Issue 2, pp 563–572 | Cite as

Induction of goat bone marrow mesenchymal stem cells into putative male germ cells using mRNA for STRA8, BOULE and DAZL

  • Yan-Li Zhang
  • Pei-Zhen Li
  • Jing Pang
  • Yong-Jie Wan
  • Guo-Min Zhang
  • Yi-Xuan Fan
  • Zi-Yu Wang
  • Nie-Hai Tao
  • Feng WangEmail author
Original Article


Bone mesenchymal stem cells (BMSCs) have the capacity to differentiate into germ cells (GCs). This study was conducted to develop a non-integrated method of using RNA transfection to derive putative male GCs from goat BMSCs (gBMSCs) in vitro by overexpressing STRA8, BOULE and DAZL. The gBMSCs were induced by co-transfection these three mRNAs together (mi-SBD group) or sequential transfection according to their expression time order in vivo (mi-S + BD group). After transfection, a small population of gBMSCs transdifferentiated into early germ cell-like cells and had the potential to enter meiosis. These cells expressed primordial germ cell specific genes STELLA, C-KIT and MVH, as well as premeiotic genes DAZL, BOULE, STRA8, PIWIL2 and RNF17. Importantly, the expression level of meiotic marker synaptonemal complex protein 3 significantly increased in these transfected two groups compared with control cells by qRT-PCR, immunofluorescence and western blot analysis (P < 0.05). Moreover, the protein expression of MVH was significantly higher in mi-S + BD group than that in mi-SBD group (P < 0.05). In addition, compared with control group, the methylation rate of imprinted gene H19 decreased in these two transfected group (P < 0.05), and the rate was significantly lower in mi-S + BD group compared with mi-SBD group (P < 0.05). This study helps to understand the mechanisms of action of key genes in GCs differentiation and also provides a novel system for in vitro induction of male GCs from stem cells.


BMSCs Germ cells STRA8 DAZL BOULE Differentiation 



Goat bone mesenchymal stem cells


Embryonic stem cells


Germ cells


Induced pluripotent stem cells


Primordial germ cells


Quantitative real-time PCR


Retinoic acid


Bone morphogenetic protein 4


Differentially methylated regions



We thank all the participants of stem cell group to conduct the experiment and revise the manuscript.


This work was supported by the Natural Science Foundation of China (No. 31201802) and Natural Science Foundation of Jiangsu Province (No. BK20161444).

Author’s contribution

YLZ and PZL conceived and conducted the study and wrote the manuscript, JP, WYJ, WZY, FYX and ZGM helped to performe the experiments, NHT revised the manuscript, FW supervised the project. All authors contributed to discuss the results.

Compliance with ethical standards

Competing interests

The authors declare that there are no competing interests associated with the manuscript.


  1. Anderson EL, Baltus AE, Roepersgajadien HL, Hassold TJ, de Rooij DG, van Pelt AM, Page DC (2008) Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice. Proc Natl Acad Sci USA 105:14976–14980CrossRefGoogle Scholar
  2. Clark AT (2015) DNA methylation remodeling in vitro and in vivo. Curr Opin Genet Dev 34:82–87CrossRefGoogle Scholar
  3. Deng W, Lin H (2002) Miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev Cell 2:819CrossRefGoogle Scholar
  4. Drusenheimer N, Wulf G, Nolte J, Lee JH, Dev A, Dressel R, Gromoll J, Schmidtke J, Engel W, Nayernia K (2007) Putative human male germ cells from bone marrow stem cells. Soc Reprod Fertil Suppl 63:69–76Google Scholar
  5. Easley CA 4th, Phillips BT, McGuire MM, Barringer JM, Valli H, Hermann BP, Simerly CR, Rajkovic A, Miki T, Orwig KE, Schatten GP (2012) Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells. Cell Rep 2:440–446CrossRefGoogle Scholar
  6. Fraune J, Brochierarmanet C, Alsheimer M, Volff JN, Schücker K, Benavente R (2016) Evolutionary history of the mammalian synaptonemal complex. Chromosoma 125:355–360CrossRefGoogle Scholar
  7. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ (2004) Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427:148–154CrossRefGoogle Scholar
  8. Ghasemzadeh-Hasankolaei M, Sedighi-Gilani MA, Eslaminejad MB (2014) Induction of ram bone marrow mesenchymal stem cells into germ cell lineage using transforming growth factor-beta superfamily growth factors. Reprod Domest Anim 49:588–598CrossRefGoogle Scholar
  9. Ghasemzadeh-Hasankolaei M, Eslaminejad MB, Sedighi-Gilani M (2016) Derivation of male germ cells from ram bone marrow mesenchymal stem cells by three different methods and evaluation of their fate after transplantation into the testis. Vitro Cell Dev Biol Anim 52:49–61CrossRefGoogle Scholar
  10. Høyer PE, Byskov AG, Møllgård K (2005) Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries. Mol Cell Endocrinol 234:1CrossRefGoogle Scholar
  11. Hua J, Pan S, Yang C, Dong W, Dou Z, Sidhu KS (2009) Derivation of male germ cell-like lineage from human fetal bone marrow stem cells. Reprod Biomed Online 19:99–105CrossRefGoogle Scholar
  12. Kee K, Angeles VT, Flores M, Nguyen HN, Pera RAR (2009a) Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation. Nature 462:222–225CrossRefGoogle Scholar
  13. Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA (2009b) Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462:222–225CrossRefGoogle Scholar
  14. Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomelí H, Nagy A, McLaughlin KJ, Schöler HR, Tomilin A (2004) Oct4 is required for primordial germ cell survival. EMBO Rep 5:1078CrossRefGoogle Scholar
  15. Kerkis A, Fonseca SA, Serafim RC, Lavagnolli TM, Abdelmassih S, Abdelmassih R, Kerkis I (2007) In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes. Cloning Stem Cells 9:535CrossRefGoogle Scholar
  16. Li Y, Wang X, Feng X, Liao S, Zhang D, Cui X, Gao F, Han C (2014) Generation of male germ cells from mouse induced pluripotent stem cells in vitro. Stem Cell Res 12:517CrossRefGoogle Scholar
  17. Li PZ, Yan GY, Han L, Pang J, Zhong BS, Zhang GM, Wang F, Zhang YL (2017) Overexpression of STRA8, BOULE, and DAZL genes promotes goat bone marrow-derived mesenchymal stem cells in vitro transdifferentiation toward putative male germ cells. Reprod Sci 24:300–312CrossRefGoogle Scholar
  18. Mark M, Jacobs H, Oulad-Abdelghani M, Dennefeld C, Féret B, Vernet N, Codreanu CA, Chambon P, Ghyselinck NB (2008) STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation. J Cell Sci 121:3233–3242CrossRefGoogle Scholar
  19. Mclenachan S, Zhang D, Palomo AB, Edel MJ, Chen FK (2013) mRNA transfection of mouse and human neural stem cell cultures. PLoS ONE 8:e83596CrossRefGoogle Scholar
  20. Medrano JV, Ramathal C, Nguyen HN, Simon C, Reijo Pera RA (2012) Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro. Stem Cells 30:441–451CrossRefGoogle Scholar
  21. Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, Gromoll J, Engel W (2006) Derivation of male germ cells from bone marrow stem cells. Lab Invest 86:654–663CrossRefGoogle Scholar
  22. Nolte J, Michelmann HW, Wolf M, Wulf G, Nayernia K, Meinhardt A, Zechner U, Engel W (2010) PSCDGs of mouse multipotent adult germline stem cells can enter and progress through meiosis to form haploid male germ cells in vitro. Differentiation 80:184–194CrossRefGoogle Scholar
  23. Preskey D, Allison T, Jones M, Mamchaoui K, Unger C (2016) Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts. Biochem Biophys Res Commun 473:743–751CrossRefGoogle Scholar
  24. Rosa A, Brivanlou AH (2010) Synthetic mRNAs: powerful tools for reprogramming and differentiation of human cells. Cell Stem Cell 7:549CrossRefGoogle Scholar
  25. Saitou M, Barton SC, Surani MA (2002) A molecular programme for the specification of germ cell fate in mice. Nature 418:293–300CrossRefGoogle Scholar
  26. Shirazi R, Zarnani AH, Soleimani M, Abdolvahabi MA, Nayernia K, Kashani IR (2012) BMP4 can generate primordial germ cells from bone-marrow-derived pluripotent stem cells. Cell Biol Int 36:1185CrossRefGoogle Scholar
  27. Tanaka SS, Toyooka Y, Akasu R, Katoh-Fukui Y, Nakahara Y, Suzuki R, Yokoyama M, Noce T (2000) The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev 14:841Google Scholar
  28. Yakubov E, Rechavi G, Rozenblatt S, Givol D (2010) Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 394:189–193CrossRefGoogle Scholar
  29. Yan G, Fan Y, Li P, Zhang Y, Wang F (2015) Ectopic expression of DAZL gene in goat bone marrow-derived mesenchymal stem cells enhances the trans-differentiation to putative germ cells compared to the exogenous treatment of retinoic acid or bone morphogenetic protein 4 signalling molecules. Cell Biol Int 39:74–83CrossRefGoogle Scholar
  30. Yu Z, Ji P, Cao J, Zhu S, Li Y, Zheng L, Chen X, Feng L (2009) Dazl promotes germ cell differentiation from embryonic stem cells. J Mol Cell Biol 1:93CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Jiangsu Livestock Embryo Engineering LaboratoryNanjing Agricultural UniversityNanjingChina
  2. 2.Jiangsu Provincial Station of Animal HusbandryNanjingChina

Personalised recommendations