Possible expression of functional glutamate transporters in the rat testis

Abstract

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by -glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [ 3 H]Glu was accumulated in testicular crude mitochondrial fractions in a temperatureand sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.

Expression of multiple glutamate transporter splice variants in the rodent testis

“…The study strongly supports the prior studies and conclusions of Takarada et al, 20 who showed that GLAST and GLT1 were detectable but that these proteins were typically smaller than those in brain. Similarly, our study supports the prior finding of functional GLT1 in testis.…”

“…9,14 The current study demonstrates that multiple splice variants of these two transporters are present and that some of these splicings result in smaller proteins of a size commensurate with those detected by Takarada et al 20 A more fine-grained comparison of results is unfortunately difficult because of limited information as to the precise epitopes being detected by commercial antibodies used in prior studies. Thus, the GLT1 antibody from Chemicon (Temecula, CA, USA), used by Takarada et al, 20 is directed against an undisclosed epitope in the last 30 amino acids of the carboxyl-terminal region, rendering it potentially able to detect GLT1a, GLT1b and/or GLT1c. The Chemicon antibody has been shown, in western blotting, to detect brain proteins varying between 42 and 71 kDa, 39,40 a size range that precludes immediate distinction of the forms of GLT1 protein detected.…”

“…The commercial GLAST antibody used by Takarada et al 20 is directed against the sequence QLIAQDNEPEKPVADSETKM, which corresponds to the extreme carboxyl-terminal region of rat GLAST (information kindly communicated to the authors by Millipore, Billerica, MA, USA). Thus, the results would accord with our data, which shows a small pool of C-terminal-immunoreactive GLAST.…”

“…The prior literature in this area is limited; the expression of glutamate transporters in the testis (as opposed to associated structures such as the vas deferens) has been studied by two groups that we are aware of. Thus, Takarada et al 20 have demonstrated the presence of mRNA for the glutamate transporters glutamate aspartate transporter (GLAST; also called EAAT1), glutamate transporter 1 (GLT1; also called EAAT2), EAAC1, EAAT4 and EAAT5 in testis. However, in their study, mRNA species for GLAST and GLT1 were reported (in Northern blots) as being smaller than expected, suggesting that any translated protein would be smaller than the normal fulllength form.…”

“…However, in their study, mRNA species for GLAST and GLT1 were reported (in Northern blots) as being smaller than expected, suggesting that any translated protein would be smaller than the normal fulllength form. Takarada et al 20 also demonstrated, using immunocytochemistry, some labeling for GLAST protein in interstitial cells, but labeling was absent in Western blotting, a disparity attributed to differential methodological sensitivities. Conversely, both immunolabeling and western blotting revealed the presence of GLT1, but again it was at a lower molecular weight than the predicted size for full-length GLT1, and EAAT4 was not detectable.…”