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Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway

Abstract

Spermatogenesis is an important cellular differentiation process that produces the male gametes and remains active throughout the individual’s lifespan. Sertoli cell-only syndrome (SCO) refers to the dysfunction of the male reproductive system, including infertility. Accurate self-renewal of spermatogonial stem cells (SSCs) is essential to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice and MAST4 expression shifted to Leydig cells and spermatids throughout puberty. Mast4 knockout (KO) testes were reduced in size compared to wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. In addition, MAST4 phosphorylated the Ets-related molecule (ERM), specifically the serine 367 residue. The phosphorylation of ERM ultimately controls the transcription of ERM target genes related to SSC self-renewal. The expression of spermatogenesis-associated proteins was significantly decreased whereas Sertoli cell markers were increased in Mast4 KO testes, which was well-founded by RNA-sequencing analysis. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway and this association helps us explore the capacity of SSCs to maintain a vertebrate stem cell niche.

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Fig. 1: Analysis of the testicular structure and expression patterns between WT and Mast4 KO.
Fig. 2: Alteration of ERM according to FGF2 and MAST4 manipulation.
Fig. 3: Interaction between MAST4 and ERM regulates transcriptional activity related to SSC self-renewal.
Fig. 4: MAST4-mediated phosphorylation of ERM at serine 367.
Fig. 5: Analysis of expression pattern related to SSC self-renewal and differentiation in WT and Mast4 KO testes.
Fig. 6: FGF2/MAST4/ERM pathway in SSC self-renewal.

References

  1. 1.

    Chen C, Ouyang W, Grigura V, Zhou Q, Carnes K, Lim H, et al. ERM is required for transcriptional control of the spermatogonial stem cell niche. Nature. 2005;436:1030–4.

    CAS  Article  Google Scholar 

  2. 2.

    Chen S-R, Liu Y-X. Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction. 2015;149:R159–67.

    CAS  Article  Google Scholar 

  3. 3.

    Pui HP, Saga Y. Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling. Mech Dev. 2017;144:125–39.

    CAS  Article  Google Scholar 

  4. 4.

    Niedenberger BA, Busada JT, Geyer CB. Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis. Reproduction. 2015;149:329.

    CAS  Article  Google Scholar 

  5. 5.

    Guo J, Nie X, Giebler M, Mlcochova H, Wang Y, Grow EJ, et al. The dynamic transcriptional cell atlas of testis development during human puberty. Cell Stem Cell. 2020;26:262–76. e264.

    CAS  Article  Google Scholar 

  6. 6.

    Sharma M, Braun RE. Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis. Development. 2018;145:dev151555.

    Article  Google Scholar 

  7. 7.

    Lovelace DL, Gao Z, Mutoji K, Song YC, Ruan J, Hermann BP. The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia. Development. 2016;143:1893–906.

    CAS  Article  Google Scholar 

  8. 8.

    Sun F, Xu Q, Zhao D, Chen CD. Id4 marks spermatogonial stem cells in the mouse testis. Sci Rep. 2015;5:17594.

    CAS  Article  Google Scholar 

  9. 9.

    Aloisio GM, Nakada Y, Saatcioglu HD, Peña CG, Baker MD, Tarnawa ED, et al. PAX7 expression defines germline stem cells in the adult testis. J Clin Investig. 2014;124:3929–44.

    CAS  Article  Google Scholar 

  10. 10.

    Spinnler K, Köhn F, Schwarzer U, Mayerhofer A. Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man. Hum Reprod. 2010;25:2181–7.

    CAS  Article  Google Scholar 

  11. 11.

    Chen L-Y, Willis WD, Eddy EM. Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development. Proc Natl Acad Sci. 2016;113:1829–34.

    CAS  Article  Google Scholar 

  12. 12.

    Sada A, Hasegawa K, Pin PH, Saga Y. NANOS2 acts downstream of glial cell line‐derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells. Stem Cells. 2012;30:280–91.

    CAS  Article  Google Scholar 

  13. 13.

    Puli OR, Danysh BP, McBeath E, Sinha DK, Hoang NM, Powell RT, et al. The transcription factor ETV5 mediates BRAFV600E-induced proliferation and TWIST1 expression in papillary thyroid cancer cells. Neoplasia. 2018;20:1121–34.

    CAS  Article  Google Scholar 

  14. 14.

    Fontanet PA, Ríos AS, Alsina FC, Paratcha G, Ledda F. Pea3 transcription factors, Etv4 and Etv5, are required for proper hippocampal dendrite development and plasticity. Cereb Cortex. 2018;28:236–49.

    Article  Google Scholar 

  15. 15.

    Zhang Y, Wang S, Wang X, Liao S, Wu Y, Han C. Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells. Cell Res. 2012;22:773–6.

    CAS  Article  Google Scholar 

  16. 16.

    Gustin SE, Stringer JM, Hogg K, Sinclair AH, Western PS. FGF9, activin and TGFbeta promote testicular characteristics in an XX gonad organ culture model. Reproduction. 2016;152:529–43.

    CAS  Article  Google Scholar 

  17. 17.

    Yu M, Wang J, Liu W, Qin J, Zhou Q, Wang Y, et al. Effects of tamoxifen on the sex determination gene and the activation of sex reversal in the developing gonad of mice. Toxicology. 2014;321:89–95.

    CAS  Article  Google Scholar 

  18. 18.

    Ishii K, Kanatsu-Shinohara M, Toyokuni S, Shinohara T. FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation. Development. 2012;139:1734–43.

    CAS  Article  Google Scholar 

  19. 19.

    Sun L, Gu S, Li X, Sun Y, Zheng D, Yu K, et al. Identification of a novel human MAST4 gene, a new member of human microtubule associated serine/threonine kinase family. Mol Biol. 2006;40:724–31.

    CAS  Article  Google Scholar 

  20. 20.

    Garland P, Quraishe S, French P, O’Connor V. Expression of the MAST family of serine/threonine kinases. Brain Res. 2008;1195:12–19.

    CAS  Article  Google Scholar 

  21. 21.

    Gongol B, Marin TL, Jeppson JD, Mayagoitia K, Shin S, Sanchez N, et al. Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity. Sci Rep. 2017;7:1–11.

    CAS  Article  Google Scholar 

  22. 22.

    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    CAS  Article  Google Scholar 

  23. 23.

    Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25:1105–11.

    CAS  Article  Google Scholar 

  24. 24.

    Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511.

    CAS  Article  Google Scholar 

  25. 25.

    Yoon KA, Chae YM, Cho JY. FGF2 stimulates SDF‐1 expression through the Erm transcription factor in Sertoli cells. J Cell Physiol. 2009;220:245–56.

    CAS  Article  Google Scholar 

  26. 26.

    Baert J-L, Beaudoin C, Coutte L, De Launoit Y. ERM transactivation is up-regulated by the repression of DNA binding after the PKA phosphorylation of a consensus site at the edge of the ETS domain. J Biol Chem. 2002;277:1002–12.

    CAS  Article  Google Scholar 

  27. 27.

    De Rooij DG, Griswold MD. Questions about spermatogonia posed and answered since 2000. J Androl. 2012;33:1085–95.

    Article  Google Scholar 

  28. 28.

    Lavery R, Lardenois A, Ranc-Jianmotamedi F, Pauper E, Gregoire EP, Vigier C, et al. XY Sox9 embryonic loss-of-function mouse mutants show complete sex reversal and produce partially fertile XY oocytes. Dev Biol. 2011;354:111–22.

    CAS  Article  Google Scholar 

  29. 29.

    Rahmoun M, Lavery R, Laurent-Chaballier S, Bellora N, Philip GK, Rossitto M, et al. In mammalian foetal testes, SOX9 regulates expression of its target genes by binding to genomic regions with conserved signatures. Nucleic acids Res. 2017;45:7191–211.

    CAS  Article  Google Scholar 

  30. 30.

    Rey RA, Grinspon RP. Normal male sexual differentiation and aetiology of disorders of sex development. Best Pract Res Clin Endocrinol Metab. 2011;25:221–38.

    CAS  Article  Google Scholar 

  31. 31.

    Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction. 2003;125:769–84.

    CAS  Article  Google Scholar 

  32. 32.

    Kim J, Jung H, Yoon M. VASA (DDX4) is a putative marker for spermatogonia, spermatocytes and round spermatids in stallions. Reprod Domest Anim. 2015;50:1032–8.

    CAS  Article  Google Scholar 

  33. 33.

    Hickford DE, Frankenberg S, Pask AJ, Shaw G, Renfree MB. DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes. Biol Reprod. 2011;85:733–43.

    CAS  Article  Google Scholar 

  34. 34.

    Sharma M, Srivastava A, Fairfield HE, Bergstrom D, Flynn WF, Braun RE. Identification of EOMES-expressing spermatogonial stem cells and their regulation by PLZF. Elife. 2019;8:e43352.

    Article  Google Scholar 

  35. 35.

    Paduch DA, Hilz S, Grimson A, Schlegel PN, Jedlicka AE, Wright WW. Aberrant gene expression by Sertoli cells in infertile men with Sertoli cell-only syndrome. PloS One. 2019;14:e0216586.

    Article  Google Scholar 

  36. 36.

    Lau X, Munusamy P, Ng MJ, Sangrithi M. Single-cell RNA sequencing of the cynomolgus macaque testis reveals conserved transcriptional profiles during mammalian spermatogenesis. Dev Cell. 2020;54:548–66.

    CAS  Article  Google Scholar 

  37. 37.

    Park O-J, Kim H-J, Woo K-M, Baek J-H, Ryoo H-M. FGF2-activated ERK mitogen-activated protein kinase enhances Runx2 acetylation and stabilization. J Biol Chem. 2010;285:3568–74.

    CAS  Article  Google Scholar 

  38. 38.

    Parker N, Falk H, Singh D, Fidaleo A, Smith B, Lopez MS, et al. Responses to glial cell line-derived neurotrophic factor change in mice as spermatogonial stem cells form progenitor spermatogonia which replicate and give rise to more differentiated progeny. Biol Reprod. 2014;91:92. 91–99

    Article  Google Scholar 

  39. 39.

    Hasegawa K, Saga Y. FGF8-FGFR1 signaling acts as a niche factor for maintaining undifferentiated spermatogonia in the mouse. Biol Reprod. 2014;91:145. 141–8

    Article  Google Scholar 

  40. 40.

    Yang Y, Feng Y, Feng X, Liao S, Wang X, Gan H, et al. BMP4 cooperates with retinoic acid to induce the expression of differentiation markers in cultured mouse spermatogonia. Stem Cells Int. 2016;2016:9536192.

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to Prof. G Yamada and C Tickle for critical reading of this manuscript. This research was supported in part by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIP) (NRF-2019R1A2C3005294, NRF-2017M3A9B3061833 and NRF-2016R1A5A2008630).

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Correspondence to Seong-Jin Kim or Han-Sung Jung.

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Lee, SJ., Park, J., Lee, DJ. et al. Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway. Cell Death Differ (2020). https://doi.org/10.1038/s41418-020-00670-2

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