- Research article
GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells
Cellular & Molecular Biology Letters volume 18, pages 149–162 (2013)
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.
basic fibroblast growth factor
colony-stimulating factor 1
γ-amino butyric acid
lial cell line-derived neurotrophic factor
mouse embryonic fibroblasts
phosphorylated histone H3
spermatogonial stem cells
terminal deoxynucleotidyl transferase dUTP nick end labeling These authors contributed equally to this paper
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.
Oatley, J.M. and Brinster, R.L. Spermatogonial stem cells. Methods Enzymol. 419 (2006) 259–282.
Kanatsu-Shinohara, M. and Shinohara, T. Germline modification using mouse spermatogonial stem cells. Methods Enzymol. 477 (2010) 17–36.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Ben-Ari, Y. Excitatory actions of gaba during development: the nature of the nurture. Nat. Rev. Neurosci. 3 (2002) 728–739.
Markwardt, S. and Overstreet-Wadiche, L. GABAergic signalling to adultgenerated neurons. J. Physiol. 586 (2008) 3745–3749.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Bouche, N., Lacombe, B. and Fromm, H. GABA signaling: a conserved and ubiquitous mechanism. Trends Cell Biol. 13 (2003) 607–610.
Meizel, S. Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol. Reprod. 56 (1997) 569–574.
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.
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.
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.
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.
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.
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.
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.
Electronic supplementary material
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
- Seminiferous Tubule
- Molecular Biology Letter
- Spermatogonial Stem Cell
- Gaba System