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. 2013 Feb 21;18(2):149–162. doi: 10.2478/s11658-013-0081-4

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

Yong Du 1, Zhao Du 1, Hongping Zheng 1, Dan Wang 1, Shifeng Li 1, Yuanchang Yan 1, Yiping Li 1,✉
PMCID: PMC6275874  PMID: 23430456

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.

Electronic Supplementary Material

Supplementary material is available for this article at 10.2478/s11658-013-0081-4 and is accessible for authorized users.

Keywords: Seminiferous Tubule, Muscimol, Molecular Biology Letter, Spermatogonial Stem Cell, Gaba System

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Abbreviations used

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 RA, de Rooij DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat. Res. 1993;290:193–200. doi: 10.1016/0027-5107(93)90159-D. [DOI] [PubMed] [Google Scholar]
  • 2.Oatley JM, Brinster RL. Spermatogonial stem cells. Methods Enzymol. 2006;419:259–282. doi: 10.1016/S0076-6879(06)19011-4. [DOI] [PubMed] [Google Scholar]
  • 3.Kanatsu-Shinohara M, Shinohara T. Germline modification using mouse spermatogonial stem cells. Methods Enzymol. 2010;477:17–36. doi: 10.1016/S0076-6879(10)77002-6. [DOI] [PubMed] [Google Scholar]
  • 4.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;101:16489–16494. doi: 10.1073/pnas.0407063101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Oatley JM, Avarbock MR, Brinster RL. 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. 2007;282:25842–25851. doi: 10.1074/jbc.M703474200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lee J, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Kimura T, Nakano T, Ogura A, Shinohara T. Akt mediates self-renewal division of mouse spermatogonial stem cells. Development. 2007;134:1853–1859. doi: 10.1242/dev.003004. [DOI] [PubMed] [Google Scholar]
  • 7.Oatley JM, Oatley MJ, Avarbock MR, Tobias JW, Brinster RL. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development. 2009;136:1191–1199. doi: 10.1242/dev.032243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.He Z, Jiang J, Kokkinaki M, 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. 2009;27:2580–2590. doi: 10.1002/stem.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Yeh JR, Zhang X, Nagano MC. Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells. J. Cell Sci. 2011;124:2357–2366. doi: 10.1242/jcs.080903. [DOI] [PubMed] [Google Scholar]
  • 10.Liu H, Wang Z, Li S, Zhang Y, Yan YC, Li YP. Utilization of an intron located polyadenlyation site resulted in four novel glutamate decarboxylase transcripts. Mol. Biol. Rep. 2009;36:1469–1474. doi: 10.1007/s11033-008-9337-x. [DOI] [PubMed] [Google Scholar]
  • 11.Li S, Zhang Y, Liu H, Yan Y, Li Y. Identification and expression of GABAC receptor in rat testis and spermatozoa. Acta Biochim. Biophys. Sin. (Shanghai) 2008;40:761–767. [PubMed] [Google Scholar]
  • 12.Hu JH, He XB, Yan YC. Identification of gamma-aminobutyric acid transporter (GAT1) on the rat sperm. Cell Res. 2000;10:51–58. doi: 10.1038/sj.cr.7290035. [DOI] [PubMed] [Google Scholar]
  • 13.Kanbara K, Okamoto K, Nomura S, Kaneko T, Watanabe M, Otsuki Y. The cellular expression of GABA(A) preceptor alpha1 subunit during spermatogenesis in the mouse testis. Histol. Histopathol. 2010;25:1229–1238. doi: 10.14670/HH-25.1229. [DOI] [PubMed] [Google Scholar]
  • 14.Kanbara K, Mori Y, Kubota T, Watanabe M, Yanagawa Y, Otsuki Y. Expression of the GABAA receptor/chloride channel in murine spermatogenic cells. Histol. Histopathol. 2011;26:95–106. doi: 10.14670/HH-26.95. [DOI] [PubMed] [Google Scholar]
  • 15.LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron. 1995;15:1287–1298. doi: 10.1016/0896-6273(95)90008-X. [DOI] [PubMed] [Google Scholar]
  • 16.Ben-Ari Y. Excitatory actions of gaba during development: the nature of the nurture. Nat. Rev. Neurosci. 2002;3:728–739. doi: 10.1038/nrn920. [DOI] [PubMed] [Google Scholar]
  • 17.Markwardt S, Overstreet-Wadiche L. GABAergic signalling to adultgenerated neurons. J. Physiol. 2008;586:3745–3749. doi: 10.1113/jphysiol.2008.155713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Liu X, Wang Q, Haydar T F, Bordey A. Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAPexpressing progenitors. Nat. Neurosci. 2005;8:1179–1187. doi: 10.1038/nn1522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Andang M, Hjerling-Leffler J, Moliner A, Lundgren TK, 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 CF, Ernfors P. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature. 2008;451:460–464. doi: 10.1038/nature06488. [DOI] [PubMed] [Google Scholar]
  • 20.Fernando RN, Eleuteri B, Abdelhady S, Nussenzweig A, Andang M, Ernfors P. Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Proc. Natl. Acad. Sci. U. S. A. 2011;108:5837–5842. doi: 10.1073/pnas.1014993108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yuan Z, Hou R, Wu J. Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation. Cell Prolif. 2009;42:123–131. doi: 10.1111/j.1365-2184.2009.00589.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc. Natl. Acad. Sci. U. S. A. 2006;103:9524–9529. doi: 10.1073/pnas.0603332103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.McLean DJ, Friel PJ, Johnston DS, Griswold MD. Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol. Reprod. 2003;69:2085–2091. doi: 10.1095/biolreprod.103.017020. [DOI] [PubMed] [Google Scholar]
  • 24.Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2010;365:1663–1678. doi: 10.1098/rstb.2010.0026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ma YH, Hu JH, Zhou XG, Mei ZT, Fei J, Guo LH. Gammaaminobutyric acid transporter (GAT1) overexpression in mouse affects the testicular morphology. Cell Res. 2000;10:59–69. doi: 10.1038/sj.cr.7290036. [DOI] [PubMed] [Google Scholar]
  • 26.Marcon L, Zhang X, Hales BF, Nagano MC, 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. 2010;83:228–237. doi: 10.1095/biolreprod.110.083568. [DOI] [PubMed] [Google Scholar]
  • 27.Faulkner-Jones BE, Cram DS, Kun J, Harrison LC. Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic beta-cells and other peripheral tissues of mouse and rat. Endocrinology. 1993;133:2962–2972. doi: 10.1210/en.133.6.2962. [DOI] [PubMed] [Google Scholar]
  • 28.Geigerseder C, Doepner R, Thalhammer A, Frungieri MB, Gamel-Didelon K, Calandra RS, Kohn FM, Mayerhofer A. Evidence for a GABAergic system in rodent and human testis: local GABA production and GABA receptors. Neuroendocrinology. 2003;77:314–323. doi: 10.1159/000070897. [DOI] [PubMed] [Google Scholar]
  • 29.Sieghart W, Fuchs K, Tretter V, Ebert V, Jechlinger M, Hoger H, Adamiker D. Structure and subunit composition of GABA(A) receptors. Neurochem. Int. 1999;34:379–385. doi: 10.1016/S0197-0186(99)00045-5. [DOI] [PubMed] [Google Scholar]
  • 30.Bouche N, Lacombe B, Fromm H. GABA signaling: a conserved and ubiquitous mechanism. Trends Cell Biol. 2003;13:607–610. doi: 10.1016/j.tcb.2003.10.001. [DOI] [PubMed] [Google Scholar]
  • 31.Meizel S. Amino acid neurotransmitter receptor/chloride channels of mammalian sperm and the acrosome reaction. Biol. Reprod. 1997;56:569–574. doi: 10.1095/biolreprod56.3.569. [DOI] [PubMed] [Google Scholar]
  • 32.Hu JH, He XB, Wu Q, Yan YC, Koide SS. Biphasic effect of GABA on rat sperm acrosome reaction: involvement of GABA(A) and GABA(B) receptors. Arch. Androl. 2002;48:369–378. doi: 10.1080/01485010290099246. [DOI] [PubMed] [Google Scholar]
  • 33.de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J. Androl. 2000;21:776–798. [PubMed] [Google Scholar]
  • 34.Singh SR, Burnicka-Turek O, Chauhan C, Hou SX. Spermatogonial stem cells, infertility and testicular cancer. J. Cell. Mol. Med. 2011;15:468–483. doi: 10.1111/j.1582-4934.2010.01242.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Meng X, de Rooij DG, Westerdahl K, Saarma M, Sariola H. Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis. Cancer Res. 2001;61:3267–3271. [PubMed] [Google Scholar]
  • 36.Lee J, Kanatsu-Shinohara M, Morimoto H, Kazuki Y, Takashima S, Oshimura M, Toyokuni S, Shinohara T. Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. Cell Stem Cell. 2009;5:76–86. doi: 10.1016/j.stem.2009.04.020. [DOI] [PubMed] [Google Scholar]
  • 37.Waheeb R, Hofmann MC. Human spermatogonial stem cells: a possible origin for spermatocytic seminoma. Int. J. Androl. 2011;34:e296–305. doi: 10.1111/j.1365-2605.2011.01199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Young SZ, Bordey A. GABA’s control of stem and cancer cell proliferation in adult neural and peripheral niches. Physiology (Bethesda) 2009;24:171–185. doi: 10.1152/physiol.00002.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]

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