The vesicular GABA transporter vGAT, is present in sperm and testis.

Abstract

Objective: The amino acid gamma aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in the CNS. GABA is transported across the plasma membrane into cells by transporter proteins. Four such proteins have been cloned and sequenced (GAT 1-4). A protein different from GAT 1-4, the vesicular GABA transporter (VGAT) has been identified, mediating GABA transport from cytoplasm into storage vesicles called small synaptic vesicles in neurons. Recently, a role for GABA has been proposed in reproductive medicine. Specific GABAergic proteins have been identified in human sperm and oocytes and a possible role for GABA in the initiation of the acrosome reaction has been proposed. We have previously reported the presence of specific GABA transport proteins in human and rat spermatozoa and testis. In this study we have identified VGAT in rat testis by immunoblotting immunohistochemistry and RT-PCR. Design: Experimental design in laboratory setting Materials/Methods: Immunohistochemistry: Male Sprague-Dawley rats were sacrificed and testis were removed and fixed in formalin/picric acid. Fixed testis were rinsed and finally cut for indirect fluorescence. Sections of rat testis and spermatozoa were incubated in rabbit antiserum to VGAT for 18-22 hours. The tissues were then washed and incubated with fluorescin isothiocyanate conjugated donkey anti rabbit secondary antibodies. Sections were examined in a laser scanning confocal imaging system equipped with a krypton/argon mixed gas laser. SDS-PAGE and Immunoblotting: Homogenized samples were denatured in SDS-PAGE sample buffer. Equal amounts of protein were loaded and subjected to analysis on a 7.5% SDS-PAGE gel. Proteins were transferred to nitrocellulose and following blocking, the blots were probed overnight with rabbit anti VGAT. Immunoreactive protein bands were visualized following incubation with peroxidase-conjugated goat-anti-rabbit IgG. Reverse transcriptase PCR: Messenger RNA was prepared from rat testis and the mRNA was transcribed into cDNA using a commercially available kit. Primers used for PCR were designed on basis of sequences for cloned vesicular GABA transport. The validity of the PCR procedure was tested by gel electrophoreses and DNA sequencing. Results: Immunohistochemistry: Confocal images of rat testis sections demonstrated strong immunofluorescence in the cytoplasm of germ cells. Peritubular cells did not exhibit staining. Confocal images of rat sperm cells incubated with antibodies to VGAT demonstrated immunofluorescence in the head region. SDS-PAGE: The testis VGAT protein reacted with the rabbit anti VGAT antibody. The immunoreactivity was observed as a band in the 60 kDa region RT-PCR: With the oligonucleotide primer pair based on the cDNA sequence from the rat VGAT, a PCR product matched the size predicted from the location of the primers. This PCR product was sequenced and found identical to rat VGAT. Conclusions: The results from both immunohistochemistry and immunoblots in this study strongly indicate the presence of the vesicular GABA transporter in rat testis. The identification of mRNA expression of VGAT in rat testis confirms this finding. Several GABAergic proteins have now been identified in testis and spermatozoa, this is suggestive of a role for GABA in male reproductive organs and/or spermatozoa. Supported By: This project was supported by grants from the Swedish Medical Council 14X-07164 and the Karolinska Institutes Research Funds.