Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic

Abstract

Testosterone and oestradiol can modulate GABA synthesis in sexually regressed gold®sh. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD 65 and GAD 67 , EC 4.1.1.15). Full-length GAD clones were isolated from a gold®sh cDNA library and sequenced. Gold®sh GAD 65 encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD 65 . Gold®sh GAD 67 encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD 67 . Gold®sh GAD 65 and GAD 67 are 63% identical. Sexually regressed male and female gold®sh were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol signi®cantly increased GAD 65 mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a signi®cant increase only in telencephalon. GAD 67 mRNA levels were not affected by steroids in females. In contrast, both steroids induced signi®cant decreases of GAD 65 and GAD 67 mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.

Sexually Dimorphic Expression of Glutamate Decarboxylase mRNA in the Hypothalamus of the Deep Sea Armed Grenadier, <i>Coryphaenoides (Nematonurus) armatus</i>

“…These data demonstrate that sex differences in GAD are specific to the GAD65 isoform in the hypothalamus of the armed grenadier. These observations are similar to data in sexually regressed goldfish, where GAD65 mRNA levels are 2-fold higher in the hypothalamus of males compared to females [Bosma et al, 2000]. Sex differences in the GABAergic system have also been observed in both immature and sexually active mammals [see McCarthy, 1995 for review].…”

“…Moreover, in the trout hypothalamus and preoptic region the distribution of neurons expressing both GAD65 mRNA and immunoreactivity for the estrogen receptor strongly overlap , suggesting that GABA neurons are directly sensitive to estradiol. Indeed, in vivo implantation of estradiol upregulates hypothalamic GAD65 mRNA levels in sexually regressed female goldfish [Bosma et al, 2000]. In contrast, estradiol treatment suppressed hypotha- lamic GAD65 mRNA levels in sexually regressed male goldfish.…”

“…Higher levels of GAD65 measured in the hypothalamus of the male grenadier may be a reflection of sex-related differences in sex steroid production by the pre-spawning gonad of this semelparous species. Based on work in sexually inactive goldfish [Bosma et al, 2000] and neonatal rats [McCarthy, 1995;Davis et al, 1996], it is equally plausible that sex differences in GAD65 mRNA levels in the hypothalamus result from a differential response to low levels of circulating sex steroids.…”

“…The expression of GAD mRNA transcripts in grenadier hypothalamus, telencephalon and cerebellum was studied using a modified semiquantitative RT-PCR method described previously in our laboratory [Bosma et al, 2000]. Two µl aliquots (ෂ10 ng) of cDNA were subjected to PCR amplification in the presence of [α-35S]dATP, using specific primers for each GAD form (GenBank accession numbers: GAD65, AF4327; GAD67, AF4328; GAD3, AF43279).…”

“…A PCR for grenadier β-actin (GenBank accession number AF 159716) was carried out as internal standard for each cDNA sample. The relative expression of GAD was expressed in relation to β-actin levels [Bosma et al, 2000]. Levels of expression of β-actin were similar between the tissues examined, allowing the comparison of relative GAD expression in the telencephalon, hypothalamus and cerebellum of males and females.…”