Skip to main content
Download PDF
  • Research article
  • Published:

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

Cellular & Molecular Biology Letters volume 18, pages 149–162 (2013)Cite this article

An Erratum to the article to this article was published on 29 December 2013

Abstract

γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABAA receptor subunits, including α1, α5, β1, β2, β3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABAAR-specific agonist muscimol reduced the proliferation of SSCs. This GABA regulation of SSC proliferation was shown to be independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.

Abbreviations

bFGF:

basic fibroblast growth factor

CSF-1:

colony-stimulating factor 1

dpp:

days postpartum

GABA:

γ-amino butyric acid

GDNF:

lial cell line-derived neurotrophic factor

MEF:

mouse embryonic fibroblasts

pH3:

phosphorylated histone H3

SSCs:

spermatogonial stem cells

TUNEL:

terminal deoxynucleotidyl transferase dUTP nick end labeling These authors contributed equally to this paper

References

  1. Tegelenbosch, R.A. and de Rooij, D.G. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat. Res. 290 (1993) 193–200.

    Article  CAS  PubMed  Google Scholar 

  2. Oatley, J.M. and Brinster, R.L. Spermatogonial stem cells. Methods Enzymol. 419 (2006) 259–282.

    CAS  PubMed  Google Scholar 

  3. Kanatsu-Shinohara, M. and Shinohara, T. Germline modification using mouse spermatogonial stem cells. Methods Enzymol. 477 (2010) 17–36.

    CAS  PubMed  Google Scholar 

  4. Kubota, H., Avarbock, M.R. and Brinster, R.L. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 101 (2004) 16489–16494.

    Article  CAS  PubMed  Google Scholar 

  5. Oatley, J.M., Avarbock, M.R. and Brinster, R.L. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J. Biol. Chem. 282 (2007) 25842–25851.

    Article  CAS  PubMed  Google Scholar 

  6. Lee, J., Kanatsu-Shinohara, M., Inoue, K., Ogonuki, N., Miki, H., Toyokuni, S., Kimura, T., Nakano, T., Ogura, A. and Shinohara, T. Akt mediates self-renewal division of mouse spermatogonial stem cells. Development 134 (2007) 1853–1859.

    Article  CAS  PubMed  Google Scholar 

  7. Oatley, J.M., Oatley, M.J., Avarbock, M.R., Tobias, J.W. and Brinster, R.L. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development 136 (2009) 1191–1199.

    Article  CAS  PubMed  Google Scholar 

  8. He, Z., Jiang, J., Kokkinaki, M. and Dym, M. Nodal signaling via an autocrine pathway promotes proliferation of mouse spermatogonial stem/progenitor cells through Smad2/3 and Oct-4 activation. Stem Cells 27 (2009) 2580–2590.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Yeh, J.R., Zhang, X. and Nagano, M.C. Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells. J. Cell Sci. 124 (2011) 2357–2366.

    Article  CAS  PubMed  Google Scholar 

  10. Liu, H., Wang, Z., Li, S., Zhang, Y., Yan, Y.C. and Li, Y.P. Utilization of an intron located polyadenlyation site resulted in four novel glutamate decarboxylase transcripts. Mol. Biol. Rep. 36 (2009) 1469–1474.

    Article  CAS  PubMed  Google Scholar 

  11. Li, S., Zhang, Y., Liu, H., Yan, Y. and Li, Y. Identification and expression of GABAC receptor in rat testis and spermatozoa. Acta Biochim. Biophys. Sin. (Shanghai) 40 (2008) 761–767.

    CAS  Google Scholar 

  12. Hu, J.H., He, X.B. and Yan, Y.C. Identification of gamma-aminobutyric acid transporter (GAT1) on the rat sperm. Cell Res. 10 (2000) 51–58.

    Article  CAS  PubMed  Google Scholar 

  13. Kanbara, K., Okamoto, K., Nomura, S., Kaneko, T., Watanabe, M. and Otsuki, Y. The cellular expression of GABA(A) preceptor alpha1 subunit during spermatogenesis in the mouse testis. Histol. Histopathol. 25 (2010) 1229–1238.

    CAS  PubMed  Google Scholar 

  14. Kanbara, K., Mori, Y., Kubota, T., Watanabe, M., Yanagawa, Y., and Otsuki, Y. Expression of the GABAA receptor/chloride channel in murine spermatogenic cells. Histol. Histopathol. 26 (2011) 95–106.

    CAS  PubMed  Google Scholar 

  15. LoTurco, J.J., Owens, D.F., Heath, M.J., Davis, M.B., and Kriegstein, A.R. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 15 (1995) 1287–1298.

    Article  CAS  PubMed  Google Scholar 

  16. Ben-Ari, Y. Excitatory actions of gaba during development: the nature of the nurture. Nat. Rev. Neurosci. 3 (2002) 728–739.

    Article  CAS  PubMed  Google Scholar 

  17. Markwardt, S. and Overstreet-Wadiche, L. GABAergic signalling to adultgenerated neurons. J. Physiol. 586 (2008) 3745–3749.

    Article  CAS  PubMed  Google Scholar 

  18. Liu, X., Wang, Q., Haydar, T. F. and Bordey, A. Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAPexpressing progenitors. Nat. Neurosci. 8 (2005) 1179–1187.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Andang, M., Hjerling-Leffler, J., Moliner, A., Lundgren, T.K., Castelo-Branco, G., Nanou, E., Pozas, E., Bryja, V., Halliez, S., Nishimaru, H., Wilbertz, J., Arenas, E., Koltzenburg, M., Charnay, P., El Manira, A., Ibanez, C.F. and Ernfors, P. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature 451 (2008) 460–464.

    Article  PubMed  Google Scholar 

  20. Fernando, R.N., Eleuteri, B., Abdelhady, S., Nussenzweig, A., Andang, M. and Ernfors, P. Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 5837–5842.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Yuan, Z., Hou, R. and Wu, J. Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation. Cell Prolif. 42 (2009) 123–131.

    Article  CAS  PubMed  Google Scholar 

  22. Oatley, J.M., Avarbock, M.R., Telaranta, A.I., Fearon, D.T. and Brinster, R.L. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 9524–9529.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. McLean, D.J., Friel, P.J., Johnston, D.S. and Griswold, M.D. Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol. Reprod. 69 (2003) 2085–2091.

    Article  CAS  PubMed  Google Scholar 

  24. Phillips, B.T., Gassei, K. and Orwig, K.E. Spermatogonial stem cell regulation and spermatogenesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365 (2010) 1663–1678.

    Article  CAS  PubMed  Google Scholar 

  25. Ma, Y.H., Hu, J.H., Zhou, X.G., Mei, Z.T., Fei, J. and Guo, L.H. Gammaaminobutyric acid transporter (GAT1) overexpression in mouse affects the testicular morphology. Cell Res. 10 (2000) 59–69.

    Article  CAS  PubMed  Google Scholar 

  26. Marcon, L., Zhang, X., Hales, B.F., Nagano, M.C., and Robaire, B. Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro. Biol. Reprod. 83 (2010) 228–237.

    Article  CAS  PubMed  Google Scholar 

  27. Faulkner-Jones, B.E., Cram, D.S., Kun, J. and Harrison, L.C. Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic beta-cells and other peripheral tissues of mouse and rat. Endocrinology 133 (1993) 2962–2972.

    Article  CAS  PubMed  Google Scholar 

  28. Geigerseder, C., Doepner, R., Thalhammer, A., Frungieri, M.B., Gamel-Didelon, K., Calandra, R.S., Kohn, F.M. and Mayerhofer, A. Evidence for a GABAergic system in rodent and human testis: local GABA production and GABA receptors. Neuroendocrinology 77 (2003) 314–323.

    Article  CAS  PubMed  Google Scholar 

  29. Sieghart, W., Fuchs, K., Tretter, V., Ebert, V., Jechlinger, M., Hoger, H., and Adamiker, D. Structure and subunit composition of GABA(A) receptors. Neurochem. Int. 34 (1999) 379–385.

    Article  CAS  PubMed  Google Scholar 

  30. Bouche, N., Lacombe, B. and Fromm, H. GABA signaling: a conserved and ubiquitous mechanism. Trends Cell Biol. 13 (2003) 607–610.

    Article  CAS  PubMed  Google Scholar 

  31. Meizel, S. Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol. Reprod. 56 (1997) 569–574.

    Article  CAS  PubMed  Google Scholar 

  32. Hu, J.H., He, X.B., Wu, Q., Yan, Y.C. and Koide, S.S. Biphasic effect of GABA on rat sperm acrosome reaction: involvement of GABA(A) and GABA(B) receptors. Arch. Androl. 48 (2002) 369–378.

    Article  CAS  PubMed  Google Scholar 

  33. de Rooij, D.G. and Russell, L.D. All you wanted to know about spermatogonia but were afraid to ask. J. Androl. 21 (2000) 776–798.

    PubMed  Google Scholar 

  34. Singh, S.R., Burnicka-Turek, O., Chauhan, C. and Hou, S.X. Spermatogonial stem cells, infertility and testicular cancer. J. Cell. Mol. Med. 15 (2011) 468–483.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Meng, X., de Rooij, D.G., Westerdahl, K., Saarma, M. and Sariola, H. Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis. Cancer Res. 61 (2001) 3267–3271.

    CAS  PubMed  Google Scholar 

  36. Lee, J., Kanatsu-Shinohara, M., Morimoto, H., Kazuki, Y., Takashima, S., Oshimura, M., Toyokuni, S. and Shinohara, T. Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. Cell Stem Cell. 5 (2009) 76–86.

    Article  CAS  PubMed  Google Scholar 

  37. Waheeb, R. and Hofmann, M.C. Human spermatogonial stem cells: a possible origin for spermatocytic seminoma. Int. J. Androl. 34 (2011) e296–305; discussion e305.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Young, S.Z. and Bordey, A. GABA’s control of stem and cancer cell proliferation in adult neural and peripheral niches. Physiology (Bethesda). 24 (2009) 171–185.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Shanghai Key Laboratory for Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031, China

    Yong Du, Zhao Du, Hongping Zheng, Dan Wang, Shifeng Li, Yuanchang Yan & Yiping Li

Authors
  1. Yong Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhao Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hongping Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shifeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuanchang Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yiping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yiping Li.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Du, Y., Du, Z., Zheng, H. et al. GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells. Cell Mol Biol Lett 18, 149–162 (2013). https://doi.org/10.2478/s11658-013-0081-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-013-0081-4

Keywords

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us