The major neurotransmitter of the central nervous system, gamma-aminobutyric acid (GABA), exerts its actions through GABA_A, GABA_B and GABA_C receptors. GABA and GABA receptors are, however, also present in several non-neural tissues, including the endocrine organs pituitary, pancreas and testis. In the case of the rat testis, GABA appears to be linked to the regulation of steroid synthesis by Leydig cells via GABA_A receptors, but neither testicular sources of GABA, nor the precise nature of testicular GABA receptors are fully known. We examined these points in rat, mouse, hamster and human testicular samples. RT-PCR followed by sequencing showed that the GABA-synthesizing enzymes glutamate decarboxylase (GAD) 65 and/or GAD67, as well as the vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT) are expressed. Testicular GAD in the rat was shown to be functionally active by using a GAD assay, and Western blot analysis confirmed the presence of GAD65 and GAD67. Interstitial cells, most of which are Leydig cells according to their location and morphological characteristics, showed positive immunoreaction for GAD and VIAAT/VGAT proteins. In addition, several GABA_A receptor subunits (α1–3, β1–3, γ1–3), as well as GABA_B receptor subunits R1 and R2, were detected by RT-PCR. Western blot analysis confirmed the results for GABA_A receptor subunits β2/3 in the rat, and immunohistochemistry identified interstitial Leydig cells to possess immunoreactive GABA_A receptor subunits β2/3 and α1. The presence of GABA_A receptor subunit α1 mRNA in interstitial cells of the rat testis was further shown after laser microdissection followed by RT-PCR analysis. In summary, these results describe molecular details of the components of an intratesticular GABAergic system expressed in the endocrine compartment of rodent and human testes. While the physiological significance of this peripheral neuroendocrine system conserved throughout species remains to be elucidated, its mere presence in humans suggests the possibility that clinically used drugs might be able to interfere with testicular function.

Progesterone production by the corpus luteum is a process vital for reproduction. In humans its secretion is stimulated by the placental hormone human chorionic gonadotropin (hCG), and this stimulatory action can also be observed in cultured human luteinized granulosa cells (GCs). We now provide evidence that opening of a Ca(2+)-activated K(+) channel, the BK(Ca), is crucially involved in this process. Immunohistochemistry and RT-PCR revealed the presence of the pore-forming alpha-subunit in human luteinized GCs and in luteal cells of human, macaque, and rat, implying that BK(Ca) channels are important throughout species. Blocking of BK(Ca) channels by iberiotoxin attenuated hCG-induced progesterone secretion. The inhibitory action of iberiotoxin suggests that BK(Ca) channels are activated in the course of hCG-induced steroidogenesis. In search of physiological activators we used an electrophysiological approach and could preclude a direct regulation of channel activity by hCG or GC-derived steroids (progesterone and 17beta-estradiol). Instead, the peptide hormone oxytocin and an acetylcholine (ACh) agonist, carbachol, evoked transient BK(Ca) currents and membrane hyperpolarization. These two molecules are both secreted by GCs and act via raised intracellular Ca(2+) levels. The release of oxytocin is stimulated by hCG, and a similar mechanism is likely in the case of ACh. We conclude that BK(Ca) channel activity in GCs is mediated by components of the intraovarian signaling system, thereby interlinking a systemic hormonal and a local neuroendocrine system in control of steroidogenesis.

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.