Evidence for a peripheral GABAergic system in the testis: Initiation of Leydig cell proliferation via GABAA receptors

Abstract

Recently, the neurotransmitter γ-aminobutyric acid (GABA) has been identified in the endocrine compartment of adult testis. Both rodent and human Leydig cells are capable OF synthesising and storing GABA by glutamate decarboxylase (GAD) and vesicular GABA transporter (VIAAT/VGAT). Furthermore, Leydig cells posses GABAA and GABAB receptors. These results raise the question about a physiological role of testicular GABA. A hint to GABAergic involvement in cell proliferation came from studies in the brain, where GABA activates and regulates cell proliferation of immature neurons. In contrast to adult Leydig cells, which proliferate albeit at a low level to sustain their cell number, developing Leydig cells are fast proliferating. Thus Leydig cells in young rodents expressed not only the proliferation marker PCNA, but also all components of a GABAergic system, namely GAD, VIAAT/VGAT and the GABAA receptor subunit α1, evidenced by immunohistochemistry and RT-PCR. In order to examine whether GABA affects Leydig cell proliferation we investigated TM3 cells, a murine Leydig cell line, bearing resemblance to immature neurons. Like Leydig cells in-vivo TM3 cells expressed active GAD, shown by an enzymatic assay, as well as VIAAT/VGAT, GABAA and GABAB receptor subunits indicated by RT-PCR. These cells responded to GABA and GABAA agonist isoguvacine by significantly increased cell proliferation in a colorimetric assay. The GABAA antagonist bicuculline blocked the GABA or isoguvacine induced proliferation, whereas GABAB agonists or antagonists had no effect. Western blot analysis revealed that stimulation with GABA or isoguvacine also increased levels of PCNA in TM3 cells. In conclusion, our results show that GABA is able to induce proliferation of TM3 cells by GABAA receptor activation. The present data implies that testicular GABA may play a crucial role in the maturation of Leydig cells in the developing endocrine compartment of the testis. Supported by DFG Graduiertenkolleg 333.