GABA and human spermatozoa : characterization and regulation of GABA transport proteins

Abstract

The present project aimed at investigating the interaction between GABA and human spermatozoa under in vitro conditions. Our initial hypothesis was that human spermatozoa had specific binding proteins for GABA and that the binding of GABA to such binding sites could affect sperm function. Studies on swim-up preparations of human spermatozoa incubated with radiolabelled GABA in the presence of unlabelled GABA, alternatively displacers of GABAA/B receptors and GABA transport proteins, indicated that GABA specific binding sites were present on the surface of human spermatozoa. The potency of the different inhibitors indicated that the binding sites possibly represented GABA transport proteins. No effect on sperm motility or AR was observed following incubation with GABA.

Further studies indicated that human spermatozoa are capable of a carrier mediated GABA uptake. The uptake is dependent on the concentration of chloride and sodium in the external medium, and the kinetic properties of the carrier resembled high affinity GABA transport proteins. Uptake of radiolabelled GABA into human spermatozoa in the presence of steroids was investigated. We examined progesterone, other steroids known to increase calcium influx, and steroids known to be ineffective as stimulators of calcium influx in human sperm. The results demonstrated a twofold increase in GABA uptake following preincubation with P or steroids known to stimulate calcium influx. In contrast, an inhibition of GABA uptake following preincubation with activators of protein kinase C was observed. The results suggest a role for PKC in the regulation of GABA transport in human spermatozoa. The reduced GABA uptake following incubation with phorbol esters and diacyl glycerol (DAG) analogues, as well as the lack of effect observed following addition of inactive DAG analogues, strongly indicate such a regulatory pathway.

The existence of GABA transporter m-RNA and proteins in human testis and sperm was examined using RT-PCR, immunohistochemistry and immunoblotting. Oligonucleotide primer pairs were designed from nucleotide sequences for the three cloned GABA transport proteins identified in human brain: GAT-1, GAT-3 and BGT-1. From homogenized human testis, PCR products of predicted size and with homology to respective GABA transport proteins were identified. This finding indicates the MRNA expression of all three GABA transporters in human testicular tissue. Studies on the protein expression of GABA transporters in sperm cells were performed with SDS-PAGE and immunoblotting techniques. Immunoblots using polyclonal antibodies raised against rat GAT-1, GAT-2 and GAT-3 recognized bands of approximately 65kDa, indicating cross-reactivity to GABA transporters present in human sperm cells.

Pharmacological characterization of GABA uptake with specific inhibitors of GABA transport revealed a unique sequence in the relative potencies of the inhibitors tested. The results may indicate the presence of more than one GABA transporter in the plasma membrane of human spermatozoa. Immunoblots and immunohistochemistry studies indicate the presence of one or several GABA transporters in human spermatozoa and testis, these results are supported by pharmacological studies using specific inhibitors of GABA transport. These studies add new information to the present knowledge concerning GABA and human spermatozoa. They also for the first time describe specific GABA transport proteins in the male reproductive tract. Further studies are however, required to elucidate the functional role for these proteins in terms of human reproduction.