Background
Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the vertebrate central nervous system. In addition to its well established function as neurotransmitter, locally synthesized GABA and GABA receptors are also present in endocrine organs, for example, in somatotrophs (GH-producing cells) of the anterior pituitary lobe [
1‐
3] and in pancreatic islet cells [
4‐
6]. In both endocrine tissues GABA is regulating the synthesis and the release of hormones. The release of glucagon and growth hormone was shown to be controlled by GABA in an auto-/paracrine manner.
In our previous work we recently identified another GABAergic system located in adult Leydig cells in rodent and human testis [
7]. Since Leydig cells possess both isoforms of the GABA synthesizing enzyme glutamate decarboxylase, GAD65 and GAD67, vesicular GABA transporter (VGAT), as well as several GABA
A and GABA
B receptor subunits, GABA may act as an auto-/paracrine molecule regulating Leydig cell function. Some evidence for a role in release of testosterone came from pharmacological studies in rat Leydig cells, which respond to GABAergic stimulation with increased testosterone production [
8,
9]. What other roles GABA may have in endocrine Leydig cells and which GABA receptors are mediating these actions are not known.
In the central nervous system evidence for a non-synaptic, trophic role of GABA in neurogenesis during embryonic development is mounting [
10‐
15]. Thus GABA stimulates progenitor cells to proliferate in different regions of the developing brain [
16‐
20]. Since these neuronal progenitor cells are also capable of synthesizing GABA and possess GABA receptors, GABA executes this trophic function in an auto-/paracrine fashion [
21]. Further non-synaptic actions of GABA in the developing brain that are evolving include regulation of migration and motility of embryonic neurons [
22‐
24]. While in general, cellular responses to GABA are mediated through GABA
A, GABA
B and GABA
C receptors and the intracellular signaling pathways associated with them [
25], in respect to both cell proliferation and migration in the developing brain, contribution of GABA
A receptors was reported [
18,
19,
21,
26,
27]. Thus, although its precise regulation may depend on the region and cell type affected, GABA emerges as an important signal for cell proliferation and migration.
In view of this role of GABA in the brain, the question arises, whether GABA may influence cell proliferation processes in the testis, for example in Leydig cells, which bear GABA receptors [
7]. In the testis of adult mammals, however, Leydig cells have only a marginal turnover rate and show low mitotic activity [
28‐
30]. Due to the fact that Leydig cells in postnatal testis proliferate to a much greater extend than in adult testis [
31‐
35], we sought to study postnatal testes of mice and rats at age of five-six days after birth. At this point of development two distinct types of Leydig cells are found, namely steroidogenic fetal Leydig cells with a typical rounded morphology clustered together in groups and spindle-shaped mesenchymal precursor cells of adult Leydig cells, which are located primarily in peritubular regions. The latter are not able to synthesize steroids, but are strongly proliferating and differentiate to Leydig progenitor cells during the second postnatal week in rodents [
36‐
38]. Fetal Leydig cells may also increase in number during postnatal development, albeit to a smaller degree [
31,
39]. Thus, the endocrine compartment of postnatal testis bears developing and highly proliferating cells of the adult Leydig cell lineage. Therefore, in this study we addressed the questions whether a local GABAergic system is present in postnatal testis and may be involved in proliferation of Leydig cells.
Methods
Animals
Testes and other tissues were obtained from adult, 3–6 months old (n = 12; Sprague-Dawley, Wistar) and five-six days old male rats (n = 14; Sprague-Dawley), as well as from adult (n = 4; BALB/c) male mice, which were bred at the Technische Universität München, Germany. Testes were also obtained from five-six days old male mice (n = 9; BALB/c), which were bred at the Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina. According to the National Institute of Health Guide for the Care and Use of Laboratory Animals, they were painlessly killed under ether anesthesia by exsanguinations and organs were rapidly removed. Testes were either frozen until isolation of mRNA and preparation for GAD activity measurements, or fixed in Bouin's solution overnight at 4°C and then embedded in paraffin.
Antibodies and antisera
For immunohistochemistry, immunocytochemistry and Western blot analyses we employed rabbit polyclonal antiserum against GAD, which recognizes both isoforms GAD65 and GAD67 (DPC Biermann, Bad Nauheim, Germany); rabbit polyclonal antiserum against VGAT (SySy Synaptic Systems GmbH, Göttingen, Germany); rabbit polyclonal antiserum against GABAA-α1 (Alomone Labs Inc., Jerusalem, Israel), sheep polyclonal antiserum against GABAB-R1 and GABAB-R2 (gift from Graham Disney and Fiona Marshall, GlaxoWellcome R&D Inc., Stevenage, UK), mouse monoclonal antiserum against PCNA (Merck Biosciences, Schwalbach, Germany) and mouse monoclonal antiserum against β-Actin (Sigma, Deisenhofen, Germany).
Cell culture
TM3 cells are an established Leydig cell line. They derived from mouse Leydig cells [
40,
41] and were cultured in F12-DME medium (pH 7.2; Sigma, Deisenhofen, Germany) supplemented with 5 × 10
4 IU/l penicilline, 5 × 10
4 μg/l streptomycine, 5% horse serum (all from Biochrom AG, Berlin, Germany) and 2.5% fetal calf serum FCS Gold (PAA GmbH, Cölbe, Germany). AtT20 cells, a mouse adenohypophysial corticotroph tumor cell line [
42,
43], were cultured in F12-DME medium (pH 7.2; Sigma, Deisenhofen, Germany) supplemented with 2 mM L-glutamine (Sigma, Deisenhofen, Germany), 15% horse serum (Biochrom AG, Berlin, Germany) and 2.5% fetal calf serum FCS Gold (PAA GmbH, Cölbe, Germany). Both cell lines were kept at 37°C in a humidified atmosphere containing air and carbon dioxide (95%/5% vol/vol). In order to study proliferation and cellular PCNA content, TM3 cells were cultured for 24 h in serum-reduced medium (1% fetal calf serum, 2.5% horse serum). This treatment yields a synchronization of the cell cycle [
40,
44]. TM3 cells were incubated subsequently in the same serum-reduced medium with 10
-5 M GABA, 10
-5 M GABA
A agonist isoguvacine, 10
-5 M GABA
B agonist baclofen, as well as combinations with 10
-5 M GABA
A antagonist bicuculline and 10
-5 M GABA
B antagonist phaclofen (BIOTREND GmbH, Köln, Germany) for 5, 10, 15, 30 min and for 24 h.
Cell proliferation studies
TM3 cells (5 × 10
3 cells per well) were plated on 96-well plates (Nunc, Wiesbaden, Germany) and incubated for 24 h in the presence or absence of GABA, isoguvacine, baclofen, bicuculline and phaclofen. One experiment included 32 replicate wells per treatment. As previously described [
45,
46], cell proliferation was determined by using the CellTiter 96 AQ
ueous One Solution cell proliferation assay (Promega, Mannheim, Germany). The specificity and sensitivity of this method was previously evaluated in our lab by comparison with a [
3H]thymidine incorporation assay [
45].
RNA preparation and RT-PCR
Isolation of RNA from rodent testes, as well as RT and PCR for GAD65/67, VGAT and GABA receptor subunits were performed as described previously [
47]. Conditions of PCR amplification consisted of 30 or 35 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 60 s, followed by final extension for 5 min at 72°C). Oligonucleotide primers, as specified in Table
1, were synthesized according to published sequences. Verification of cDNAs was achieved by direct sequencing [
47].
Table 1
Sequences of oligonucleotide primers used for PCR amplification
GAD67 | NM_008077 | 393 | 5'-CTTCTTCCGGATGGTCATCTC-3' |
| | | 5'-ACGAGCAACATGCTATGGTCT-3' |
GAD65 | NM_008078 | 372 | 5'-GATATGGTTGGATTAGCAGC-3' |
| | | 5'-CATTTTCCCTCTCTCATCAC-3' |
VGAT | NM_009508 | 300 | 5'-CCTGTACGAGGAGAACGAAG-3' |
| | | 5'-AGCAGACTGAACTTGGACAC-3' |
GABAA α1 | NM_010250 | 231 | 5'-CTACAGCAACCAGCTATACCC-3' |
| | | 5'-GCTCTCTGTTTAAATACGTGG-3 |
GABAA α2 | NM_008066 | 282 | 5'-AAGGCTCCGTCATGATACAG-3' |
| | | 5'-ACTAACCCCTAATACAGGC-3' |
GABAA α3 | NM_008067 | 418 | 5'-GACTTGCTTGGTCATGTTGTTGGG-3' |
| | | 5'-CAGAGGCCCTGGAGATGAAGAAGA-3' |
GABAA β1 | NM_008069 | 540 | 5'-ATGATGCATCTGCAGCCA-3' |
| | | 5'-TGGAGTTCACGTCAGTCA-3' |
GABAA β2 | NM_008070 | 402 | 5'-GCATGTATGTCTGCAGGA-3' |
| | | 5'-CTGACACCTACTTCCTGA-3' |
GABAA β3 | NM_008071 | 224 | 5'-AGCCAAGGCCAAGAATGATCG-3' |
| | | 5'-TGCTTCTGTCTCCCATGTACC-3' |
GABAA γ1 | X55272 | 191 | 5'-TTTCTTACGTGACAGCAATGG-3' |
| | | 5'-CATGGGAATGAGAGTGGATCC-3' |
GABAA γ2 | NM_008073 | 351 | 5'-GCAATGGATCTCTTTGTA-3' |
| | | 5'-GTCCATTTTGGCAATGCG-3' |
GABAA γ3 | NM_008074 | 251 | 5'-TGTCGAAAGCCAACCATCAGG-3' |
| | | 5'-GACTTGCACTCCTCATAGCAG-3' |
GABAB R1 | NM_019439 | 350 | 5'-ATGTGGTAACCATCCAGC-3' |
| | | 5'-AGAAGATCGGCTACTACG-3' |
GABAB R2 | XM_194138 | 354 | 5'-CATCATCTTCTGTAGCAC-3' |
| | | 5'-TCTGTGAAGTTGCCCAAG-3' |
Immunohistochemistry
Testicular distribution of GAD, VGAT, PCNA and GABA
A/B receptor subunits were examined in deparaffinized sections (5 μm) of Bouin's fixed testes of rats and mice using an Avidin-Biotin-Peroxidase (ABC) immunohistochemical method as described previously [
48]. Specific antisera against GAD65/67 (dilution 1:500), VGAT (dilution 1:750), GABA
A-α1 (dilution 1:750), GABA
B-R1 (dilution 1:1.000–1:500), GABA
B-R2 (dilution 1:1000–1:500) and PCNA (dilution 1:1000–1:500) were employed. A biotin coupled polyclonal goat anti-rabbit antiserum (diluted 1:500; Jackson Inc., West Grove, USA), a biotin coupled goat anti-sheep antiserum (diluted 1:200; Dianova, Hamburg, Deutschland) or a biotin coupled goat anti-mouse antiserum (diluted 1:500; Jackson Inc., West Grove, USA) served as secondary antiserum. Diaminobenzidine (DAB) was used as a chromogen. Sections incubated with buffer alone, buffer containing mouse, sheep or rabbit non-immune serum, respectively, served as controls for all samples. The sections were examined with a Axiovert photomicroscope (Zeiss, Oberkochen, Germany).
Immunocytochemistry
TM3 cells were cultivated on glass cover slips (2 × 10
4 cells per cover slip) for 1 day. They were then fixed and handled as previously described [
49]. For immunolocalization an antiserum recognizing GAD65/67 and an antiserum recognizing VGAT was carried out overnight at 4°C (diluted 1:1000 in 0.02 M potassium phosphate buffered saline containing 2% goat non-immune serum, pH 7.4). Immunoreactivity was visualized using a secondary polyclonal goat anti-rabbit antiserum (diluted 1:200; Dianova, Hamburg, Germany) labeled with fluorescein isothiocyanate (FITC). For control purposes either the specific antiserum was omitted or incubations with rabbit non-immune serum (dilution 1:10.000) were carried out instead. Sections were examined with a Axiovert microscope (Zeiss, Oberkochen, Germany), equipped with a FITC filter set.
Western blotting
Western blot analyses were performed with minor modifications as described previously [
50]. In brief, TM3 cells and for control purposes tissue of mouse brain were lysated and homogenized in 62.5 mM Tris-HCL buffer (pH 6.8) containing 10% sucrose and 2% SDS by sonication, mercaptoethanol was added (10%), and the samples were heated (95°C for 5 min). Protein content was recorded [
51] using a folin phenol quantitation method (DC protein assay, Bio-Rad GmbH, München, Germany). Then, 15 μg protein per lane was loaded on Tricine-SDS-polyacrylamide gels (12.5%), electrophoretically separated, and blotted onto nitrocellulose. Samples were probed with antiserum directed specifically against GAD65/67, PCNA and β-Actin (incubation overnight at 4°C, dilution 1:500). Immunoreactivity was detected using peroxidase labeled goat anti-rabbit antiserum (diluted: 1:5000; Jackson Inc., West Grove, USA) or peroxidase coupled goat anti-mouse antiserum (diluted: 1:5000; Jackson Inc., West Grove, USA) and enhanced chemiluminescence (Amersham Buchler, Braunschweig, Germany). Integrated optical density of Western blot reaction with antiserum directed against PCNA and β-Actin in TM3 cells was measured using Scion Image 4.0.2 (Scion Corporation, Frederick, USA) as previously described in detail [
52].
GAD activity measurements
Determination of GAD activity by measuring the production of radiolabeled carbon dioxide (CO
2) from
14C-glutamate was performed as described previously [
7,
53]. In brief, TM3 cells, AtT20 cells and rat tissue samples were homogenized in 60 mM potassium phosphate buffer (pH = 7.1), containing 0.5% Triton X-100, 1 mM 2-aminoethyl-isothiouronium bromide and 1 mM phenylmethanesulphonyl fluoride (Sigma, Deisenhofen, Germany), centrifuged, and the supernatants were used in the assay. The assay was performed in a total reaction volume of 60 μl, containing 20 μl of sample and 0.1 mM EDTA, 0.5% Triton X-100, 0.1 mM DTT, 0.05 mM pyridoxal phosphate, 9 mM L-glutamate, 3.3 μCi/ml
14C-glutamate (Biotrend, Köln, Germany, specific activity: 50–60 mCi/mmol) and 60 mM potassium phosphate buffer. The reaction mix was incubated for 1 h at 37°C and then stopped by adding 100 μl of 10% trichloracetic acid per vial. The released CO
2 was absorbed on benzethonium hydroxide-drenched filter disks, and bound radioactivity was determined using a Tri-Carb 2100 liquid scintillation counter (Packard, Meriden, USA). The values obtained were normalized to protein content measured by DC protein assay (Bio-Rad GmbH, München, Germany) described above. Rat tissue samples that were heated to 95°C for 5 min served as negative controls.
Statistical analyses
Statistic analyses were performed using GraphPad Prism 3.02 (GraphPad Software, San Diego, USA). The results obtained in cell proliferation and GAD assay experiments were compared using one-way-analysis of variance ANOVA followed by Newmann-Keuls test. The results received in Western blot experiments were compared using one-way-analysis of variance ANOVA followed by Dunnett's test. Data shown are expressed as means+SEMs (standard error of the mean).
Discussion
The present study shows that crucial components of a GABAergic system are present in the endocrine compartment of postnatal rodent testis and that GABA stimulates proliferation of TM3 Leydig cells via GABAA receptors. These results suggest that GABA may regulate cell proliferation of fetal Leydig cells and/or mesenchymal precursors of the adult Leydig cell lineage in an auto-/paracrine manner. We therefore suggest that GABA may contribute to the morphogenesis of the testis.
Previously we and others demonstrated first details of a local GABAergic system in the endocrine compartment of adult rodent and human testis [
7,
54]. Adult Leydig cells possess enzymatically active GAD, VGAT and several GABA
A and GABA
B receptor subunits. Both isoforms GAD65 and GAD67 were present in rats and mice [
7]. The functional significance of a testicular GABAergic system is not well known, but auto-/paracrine modulation of testosterone production in Leydig cells is a possibility suggested by studies describing stimulating effects of GABA on testosterone production in rats [
8,
9]. Since hormonal influences clearly govern steroid production of the adult testis, the modulatory effect of GABA on testosterone may however not be the main effect of GABA.
We rather speculated that GABA may exert trophic effects in the testis. This assumption was based on the trophic action of GABA via GABAA receptors in the developing brain. We focused in this study therefore on the postnatal testis, which bears proliferating cells including fetal Leydig cells and cells of the adult Leydig cell lineage.
It is widely accepted that two distinct Leydig cell populations are present during the first postnatal week in mouse and rat testis [
33,
34,
36‐
39], namely steroidogenic fetal Leydig cells and mesenchymal precursors of adult Leydig cells. The first mentioned form conspicuous clusters in the interstitium [
31,
34,
55]. Although fetal Leydig cells represent a differentiated cell population, there is evidence for a moderate increase in the number of these cells during the first two weeks of postnatal development [
31,
39]. In contrast, non-steroidogenic mesenchymal precursor cells of the adult Leydig cell lineage are located primarily in peritubular regions and proliferate strongly. They differentiate during the second postnatal week into progenitor cells and from the end of the third week on into newly formed, immature and then into fully functional mature adult Leydig cells [
33,
34,
36‐
38].
Our immunohistochemical findings indeed evidenced dramatic proliferative events in the postnatal testis. Interstitial and peritubular cells in the testis of five days old rats expressed the proliferation marker PCNA, which was used in testicular tissues before [
56‐
58]. Among these cells are likely connective tissue cells and endothelial cells [
59‐
61], but also fetal Leydig cells and mesenchymal precursors of adult Leydig cells, as judged by their typical location and morphology. Since the latter are undifferentiated in nature [
34,
36‐
38], we could not use specific markers to distinguish them from other cell types.
Our study links Leydig cells proliferation and local testicular GABA synthesis. This is based first on the fact that we identified GABA synthesis and GABAA receptors in the postnatal testis of rodents, and second on the proliferative action of GABA and GABAA agonists in TM3 Leydig cells.
We identified only fetal Leydig cells, characterized by their rounded morphology and clustered appearance in the testicular interstitium, to possess GAD67 and VGAT. In contrast to adult testis, GAD65 was not detected. GAD67 was, however, found to be enzymatically active in rat testicular tissue of the same developmental stage. These two results together allow the conclusion that only fetal Leydig cells possess the pivotal molecules to synthesize and store GABA.
The present investigation provides insights into the possible targets of testicular GABA. As evidenced by RT-PCR studies, several GABA
A receptor subunits are expressed in the postnatal testis. GABA
B receptors were not found in postnatal testis, a result in contrast to our previous study in the adult rodent testis [
7]. Immunolocalization of GABA
A receptor subunits was hampered, due to availability of suitable antisera, but localization of GABA
A-α1 revealed presence on rat fetal Leydig cells, but also on spindle-shaped interstitial cells. At least some of the last mentioned cells are very likely to represent mesenchymal precursor cells of the adult Leydig cell lineage. Thus according to the immunolocalization of GABA
A-α1, both fetal Leydig cells and precursors of adult Leydig cells, are possible targets for GABA in the postnatal testis.
The number of fetal Leydig cells increases moderately in rodents during the first two weeks of postnatal development [
31,
39] and it is possible that GABA may mediate this effect. Another possibility is that GABA may modulate cell proliferation of mesenchymal precursor cells of adult Leydig cells or other GABA
A receptor bearing testicular cell types. Based on our results in the present study, it is possible that GABA might even be a start signal leading to proliferation and differentiation of mesenchymal precursors of adult Leydig cells. Interestingly, this signal is as yet unknown [
34,
37,
38]. Thyroid hormone may be involved, but participation of LH and androgens in the initiation of adult Leydig cell development was ruled out [
37,
39,
62‐
66].
Clearly, in-vivo evidence for such a crucial role of testicular GABA is as yet missing, but unequivocal evidence for a proliferative action of GABA via GABA
A receptors was provided by cell culture experiments using TM3 Leydig cells, which possess GABA
A receptor subunits. Involvement of GABA
A receptors was suggested by the use of the pharmacologically well defined GABA
A agonist isoguvacine and by the use of the GABA
A antagonist bicuculline. Interestingly, this signaling pathway is in analogy to studies in the developing brain, where GABA also induces cell proliferation of neuronal progenitors and other neuronal cell types via activation of GABA
A receptors [
10‐
13].
In summary, a GABAergic system exists already in postnatal rodent testis and differs from the one in adult testis, since one of the two GAD isoforms as well as GABAB receptor subunits are missing. Nevertheless, it appears functional and our results suggest that GABA has similar roles in the developing brain and in the developing testis, namely to act as a trophic factor affecting the morphogenesis of crucial cells in these two organs.
Authors' contributions
CG carried out most of the experiments, participated in the study design, performed statistical analyses and drafted the manuscript. RFGD carried out part of RT-PCR, Western blotting and immunocytochemistry. AT and AK provided technical assistance. AM conceived of the study, and participated in its design, coordination and writing of the manuscript. All authors read and approved the final manuscript.