Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

Abstract

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.

Insights into GABA Receptor Signalling in TM3 Leydig Cells

“…The detection of binding sites and GABA A receptor subunits alone does also not allow to conclude to typical, functional receptors. Although our previous immunohistochemical studies performed in developing and adult testes showed a GABA A (α1) subunit protein in Leydig cells [8, 10], the full subunit composition in Leydig cells, like in any other peripheral endocrine organs, are to our knowledge not known.…”

“…TM3 cells thus resemble in particular the cells of the Leydig cell lineage found in the developing testis [10]. In some of our studies we also used isolated mouse Leydig cells, which, as our unpublished RT-PCR results showed, possess α1, α2, β3 and γ3 subunit mRNAs.…”

“…TM3 Leydig cells are the main model system used for our studies, and as previously defined, they produce GABA, express the GABA A receptor subunits α1, α2, β1, β3 and γ1 and respond to GABA and GABA A agonist treatment by increased proliferation [10]. TM3 cells thus resemble in particular the cells of the Leydig cell lineage found in the developing testis [10].…”

“…Therefore, we suggested that GABA is involved in development of Leydig cells in postnatal testis. Furthermore, based on actions of GABA agonists/antagonists on TM3 cells, which express GABA A α, β and γ subunits, we concluded that GABA A receptors are responsible for the initial signal transduction [8, 10]. …”

“…However, we recently identified another GABAergic function in the testis. We found that GABA and a GABA A agonist stimulate proliferation of TM3 cells [10], a murine cell line of immature Leydig cells [1, 11]. This action appeared to be mediated via a GABA A receptor, since the effect could be evoked by the GABA A receptor agonist isoguvacine and was blocked by bicuculline, a GABA A receptor antagonist.…”