Dopamine D1 receptor blockage potentiates AMPA-stimulated luteinising hormone release in the goldfish.

Previous microarray analyses of the goldfish hypothalamus led us to hypothesise that dopamine could potentially inhibit the excitatory effects of glutamate on luteinising hormone (LH). Post-spawning female goldfish were pre-treated (-4.5 h) with either saline (C; control), SCH 23390 (S; D(1) -receptor antagonist) or sulpiride (L; D(2) -receptor antagonist), followed by an i.p. injection, at -0.5 h, of saline or the glutamate agonist AMPA (A, SA or LA). Blood, hypothalamus and telencephalon tissues were collected. Serum LH was not affected in the S, L, A, or LA groups relative to control as determined by radioimmunoassay. The SA group, however, showed a 289% (P<0.0005) increase in serum LH compared to either treatment alone or control. Real-time reverse transcriptase-polymerase chain reaction identified the mRNAs for ionotropic (Gria2a, Gria4) glutamate receptor subunits, activin ßa, isotocin, and cGnRH-II as being significantly affected by some of the treatments. The same experiment conducted with sexually-regressed female fish showed a very different LH profile, indicating that this mechanism is seasonally-dependent. We also show that i.p. injection of 1 µg/g isotocin was able to increase LH levels by 167% in sexually regressed female fish relative to controls. Taken together, these results demonstrate that blockage of the D(1) receptor primes post-spawning goldfish for AMPA-stimulated LH release, and provides further insights into the central regulation of reproduction.

Growth hormone and insulin-like growth factor of naked carp (Gymnocypris przewalskii) in Lake Qinghai: expression in different water environments.

Here, we report the cloning and characterization of growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-II from naked carp (Gymnocypris przewalskii), a native teleost fish of Lake Qinghai in the Qinghai-Tibet Plateau of China. The GH of naked carp encodes for a predicted amino acid sequence showing identities of 63%, 63%, 91% and 94% with cherry salmon, rainbow trout, zebrafish and grass carp, respectively. Compared to common carp and goldfish, evolutionary analysis showed that genome duplication has had less influence on the relaxation of purifying selection in the evolution of naked carp GH. Sequence analysis of naked carp IGF-I (ncIGF-I) and ncIGF-II showed a high degree of homology with known fish IGF-I and IGF-II. To investigate effects of salinity and ionic composition of the aquatic environment on the GH-IGF axis in naked carp, male fish held in river water were assigned randomly to 4 groups: RW (river-water), RW+Na (NaCl in RW), RW+Mg (MgCl(2) in RW) and LW (lake-water) groups. The concentrations of Na(+) in RW+Na and Mg(2+) in RW+Mg were equal to the concentrations of these ions in lake-water. After 2 days of exposure, the plasma IGF-I levels in the RW+Na and LW groups were significantly higher than the control group (RW), and the plasma GH levels of the LW group were also significantly higher than the RW group. The somatostatin (SS) levels in the hypothalamus significantly increased in the RW+Na group. After 5 days of exposure, these hormone levels did not differ significantly among groups. These results indicate that while the plasma GH and IGF-I levels are osmosensitive, the absence of a change in GH secretion in RW+Na might be partly due to a transiently increased release of hypothalamic SS induced by the stress of neutral-saline water. This is the first report of a salinity-induced increase of GH-IGF-I circulating levels in Cypriniformes.

The effects of GABA agonists on glutamic acid decarboxylase, GABA-transaminase, activin, salmon gonadotrophin-releasing hormone and tyrosine hydroxylase mRNA in the goldfish (Carassius auratus) neuroendocrine brain.

GABA plays a pivotal role in reproduction by regulating luteinising hormone (LH) release from the anterior pituitary. Current evidence indicates that there is a prominent stimulatory effect of GABA on LH release in teleost fish which results from enhanced gonadotrophin-releasing hormone (GnRH) release and decreased dopamine turnover in the brain and pituitary. We hypothesised that there may be additional mechanisms underlying LH release in goldfish and investigated the relative mRNA levels of GABA synthesising enzymes (GAD65 and GAD67), degrading enzyme (GABA-T), activin betaa and betab, salmon GnRH (sGnRH), and tyrosine hydroxylase (TH) with the real-time reverse transcriptase-polymerase chain reaction after GABA agonist treatment. Sexually regressed female goldfish were i.p. injected with either the GABA(A) agonist muscimol (1 microg/g body weight) or the GABA(B) agonist baclofen (10 microg/g body weight). Both agonists significantly increased serum LH after 6 h. Muscimol decreased GAD65 (approximately ten-fold), GABA-T (approximately 15-fold) and TH (approximately three-fold) mRNA in the telencephalon. Baclofen significantly reduced GAD67 (approximately two-fold) and GABA-T (approximately two-fold) mRNA levels in the hypothalamus. Activin betaa, but not activin betab, steady-state mRNA was increased approximately three- to four-fold in both the hypothalamus and telencephalon after baclofen treatment. There was no change in sGnRH mRNA levels in either tissue after GABA agonist treatment. We show that the GABA(A) and GABA(B) receptor agonists have differing and rapid effects on gene transcription in the goldfish neuroendocrine brain and, by affecting specific targets, we identify putative genomic mechanisms underlying GABA-stimulated LH release in fish.

Gamma-aminobutyric acid up-regulates the expression of a novel secretogranin-II messenger ribonucleic acid in the goldfish pituitary.

An RNA-arbitrarily primed PCR differential display strategy was used to identify candidate genes in the pituitary that are up-regulated by endogenously activated gamma-aminobutyric acid (GABA) systems that may also be involved in the control of reproduction. Goldfish were injected with the GABA metabolism inhibitor gamma-vinyl-GABA (GVG), known for its high efficiency to specifically increase endogenous brain and pituitary GABA levels in this species, resulting in higher levels of circulating gonadotropin-II (GTH-II). Several transcripts related to hormone secretion, signal transduction pathways, and messenger RNA (mRNA) editing were shown to be up-regulated after GVG injection. Among these transcripts we characterized an mRNA coding for the secretory vesicle protein secretogranin-II (SgII), a member of the chromogranin family, which is the precursor of a novel 34 amino acid neuropeptide, goldfish secretoneurin (SN). A semiquantitative PCR developed to measure pituitary SgII mRNA levels showed a 5-fold increase in GVG treated fish vs. control fish. Moreover, GVG treatment specifically increased SgII mRNA levels in gonadotrophs, concomitant with a decrease in GTH-II cell content. In addition, i.p. injection of synthetic goldfish SN increased GTH-II release in goldfish pretreated with the dopamine antagonist domperidone. Activation of GABAergic neurons has two effects, enhancing in vivo GTH-II release and up-regulating SgII mRNA specifically in goldfish gonadotrophs. Together with our SN bioactivity data, this suggests the existence in the pituitary of an autocrine or paracrine mechanism linked to the regulated secretory pathway in the gonadotrophs.

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Influence of GABA on gonadotrophin release in the goldfish.

The influence of GABA on pituitary gonadotrophin (GTH) release in the goldfish was studied by means of in vivo and in vitro techniques. It was found that GABA injected intraperitoneally caused an increase of serum GTH levels in regressed or early maturing fish, but not in late maturing animals. Moreover, injection of a GABA transaminase inhibitor caused a significant increase of GABA within the hypothalamus and pituitary, and a dose-dependent increase in serum GTH levels. To determine if this effect could be exerted directly at the level of the pituitary, dispersed pituitary cells in static incubation or in perifusion were exposed to increasing concentrations of GABA or its agonists muscimol and baclofen. None of these drugs was able to modify the spontaneous or GnRH-induced secretion of GTH, indicating that the in vivo effect of GABA was most likely mediated via another hypothalamic factor. Using in vitro incubation of pituitary slices, it was found that GABA caused a dose-related stimulation of GnRH release at the level of the pituitary, providing a possible explanation for the observed in vivo stimulatory effect of GABA on GTH release. Since the seasonal effect of GABA in vivo indicated a possible interaction of GABA with sexual steroids, GABA was given intraperitoneally to female goldfish implanted with either testosterone or estradiol. We found that the stimulatory effect of GABA on GTH release was abolished in estradiol-treated females but was still observed in testosterone-implanted fish. Moreover, estradiol but not testosterone caused a decrease of the GABA concentration within the telencephalon.(ABSTRACT TRUNCATED AT 250 WORDS)

GnRH receptors and actions in the control of reproductive function.

The hypothalamic control of reproductive function is expressed through the receptor-mediated actions of GnRH on the pituitary gonadotroph. GnRH receptors in the pituitary gland exhibit prominent variations in number during the ovarian cycle and after changes in steroid feedback, and are modulated by the rate of GnRH secretion from the hypothalamus. In cultured pituitary cells, GnRH receptors undergo down-regulation during exposure to GnRH agonists, followed by a subsequent elevation of sites that is dependent on protein synthesis. GnRH antagonists do not cause receptor down-regulation, but high-affinity antagonist analogs bind for extended periods to cause receptor occlusion and prolonged inhibition of GnRH action. Analysis of the rat pituitary GnRH receptor by photoaffinity labeling reveals two binding subunits of mol. wt 53,000 and 42,000. The receptor-activated processes leading to gonadotropin secretion are highly calcium-dependent, and are initiated by rapid phospholipid hydrolysis with production of arachidonic acid metabolites, diacylglycerol, and inositol phosphates. The role of protein kinase C in gonadotropin secretion is indicated by the ability of phorbol esters and synthetic diacylglycerols to stimulate LH release, the inhibition of protein kinase C and LH release by retinal, and the redistribution of protein kinase C between cytosol and membrane fractions during stimulation of pituitary gonadotrophs by GnRH. It is likely that the effects of arachidonate metabolites are integrated with those of calcium-calmodulin and calcium, phospholipid-dependent protein kinases during the immediate and sustained phases of GnRH-induced gonadotropin secretion.