Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

Abstract

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter y-aminobutyric acid, is shown to be expressed in the testis of several different species.Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.Neurotransmitter-synthesizing enzymes are predominantly expressed in the nervous system, where their enzymatic products function in synaptic transmission. A similar restricted pattern of gene expression has also been inferred for neuropeptides, and a large amount of evidence suggests that these peptides also function in neurotransmission. However, in recent years, several neuropeptides have also been shown to be expressed in nonneuronal tissues, suggesting a broader function for these peptides than heretofore anticipated. Thus, oxytocin and vasopressin have been shown to be expressed in the thymus (12). Preprotachykinin A mRNA, the common precursor for substance P and neurokinin A, is also expressed in the thymus (A. Ericsson, V.

Expression of multiple glutamate transporter splice variants in the rodent testis

“…These roles include the production of gamma-aminobutyric acid and glutathione. Since gamma-aminobutyric acid appears to be synthesized in both sperm 57 and interstitial glia-like Leylig cells, 58 this would be compatible with the view that GLAST1a and GLAST1b might supply some glutamate for this synthetic process in Leydig cells, while multiple glutamate transporters might contribute to substrate provision in sperm. Similarly, high levels of glutathione and the synthetic machinery to produce it are present in the testis, 59 particularly in cells such as spermatogonia, spermatocytes and Sertoli cells.…”

Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10.

“…6). The largest numbers of grains were at bands p11.2 and p12 (18 grains); a significant number (7 grains) were at band p13. There was no significant hybridization to other human chromosomes.…”

“…In early work, Escherichia coli GAD was crystallized (2) allowing identification of its pyridoxal 5'-phosphate (PLP, or vitamin B6) cofactor binding site, -XaaHis-Lys(E-PLP)-Xaa- (3,4). Multiple forms of GAD (5,6) with distinct tissue expression (1,7), subcellular localization (8)(9)(10), and developmental expression (11,12) have since been reported. Biological functions of GAD and GABA extend beyond regulation of neurotransmission to include effects on the immune system as well as modulation of cell proliferation, protein synthesis, and metabolism (for reviews see refs.…”

Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region.

“…1). GAD is expressed in neurons, in pancreatic /3 cells, and in cells of the testis and fallopian tube (2)(3)(4)(5). In neurons and pancreatic d cells, GAD immunoreactivity is concentrated around synaptic vesicles and synaptic-like microvesicles, respectively, a localization that may facilitate the uptake of GABA into these vesicles (6).…”

“…CHO cells (CHO-Kl cells) and COS-7 cells were grown as described (24,25). For transient transfections, cells were seeded on coverslips and incubated on day 2 (-40% confluency) with 5 ,ug of plasmid DNA and 15 ,ul of Lipofectin per ml (GIBCO/BRL) for [5][6][7] hr. On day 4, cells were fixed and processed for immunocytochemistry as described (24).…”