Dopamine stimulates growth hormone release from the pituitary of goldfish, Carassius auratus, through the dopamine D1 receptors.

Previously, we have demonstrated that ip injection of apomorphine, a nonselective dopamine (DA) agonist, increases serum GH levels in the goldfish, suggesting a possible role of DA in GH regulation. In the present study, the effects of DA on GH release in the goldfish were further characterized using an in vitro perifusion system for pituitary fragments. DA increased GH release in a dose-dependent manner with an ED50 of 0.26 +/- 0.06 microM. SKF38393, a DA D1 agonist, mimicked the GH-releasing effect of DA with an ED50 of 0.41 +/- 0.12 microM. Stereoselectivity consistent with mammalian DA D1 systems was demonstrated for the GH response to SKF38393; only the (+)- but not (-)-enantiomer of SKF38393 induced a dose-dependent GH release. Two other D1 agonists, SKF77434 and SKF82958, were also found to have GH-releasing activity. In contrast, high doses (up to 1 microM) of the DA D2 agonists, bromocriptine and LY171555, did not affect basal GH levels. The receptor specificity for DA-stimulated GH release was further investigated by using D1 and D2 antagonists; the D1 antagonists SCH23390 and SKF83566 completely abolished the GH response to DA or the D1 agonist SKF38393, whereas the D2-specific antagonists domperidone and (-)-sulpiride were not effective in this respect. Taken together, the present study demonstrates that DA is stimulatory to GH release from the pituitary of goldfish, and its action is mediated through receptors resembling the mammalian DA D1 receptors. The apparent similarities of the DA D1 receptor pharmacology between the goldfish and the mammals also indicate that D1 receptor is highly conserved during vertebrate evolution.     

Influence of catecholamines on gonadotropin secretion in goldfish, Carassius auratus

Serum gonadotropin (GtH) concentrations in female goldfish were measured before and at various times after intraperitoneal injection of drugs altering catecholamine synthesis and neural activities. Reserpine, a depleter of neurotransmitter stores, elevated serum GtH levels compared to controls, suggesting the general involvement of neurotransmitters in altering GtH release. 6-Hydroxydopamine, a catecholaminergic neurotoxin, increased serum GtH concentration, suggesting that catecholaminergic neurons inhibit GtH release. Blocking of L-DOPA and dopamine synthesis by alpha-methyl-para-tyrosine and carbidopa, respectively, but not norepinephrine by diethyldithiocarbamate, raised serum GtH values above those of controls. Injections of an alpha-agonist, clonidine, also increased serum GtH concentrations. These results suggest the existence of an inhibitory dopaminergic and a stimulatory alpha-adrenergic influence on GtH release in goldfish.     

Dopamine inhibition of gonadotropin and alpha-melanocyte-stimulating hormone release in vitro from the pituitary of the goldfish (Carassius auratus)

The purpose of the present investigation was to examine the receptor specificity of dopamine inhibition of gonadotropin (GtH) and alpha-melanocyte-stimulating hormone (alpha-MSH) release from the goldfish (Carassius auratus) pituitary in vitro. Pars distalis (PD) and neurointermediate lobe (NIL) fragments of the goldfish pituitary were superfused in vitro under various experimental paradigms; eluate from PD and NIL fragments was analyzed for (GtH) and (alpha-MSH), respectively. Spontaneous GtH release from PD fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited spontaneous GtH release with an estimated ED50 of 10(-4.4) M. Domperidone, a specific D-2 receptor antagonist, reversed the inhibitory action of dopamine and increased spontaneous GtH release. Acute treatment of PD fragments with salmon GnRH (sGnRH) stimulated GtH release; dopamine inhibited GtH release from similarly treated fragments with an ED50 of 10(-7.5) M. The spontaneous release of alpha-MSH from NIL fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited this release with an ED50 of 10(-7.2) M. Acute treatment of NIL fragments with thyrotropin-releasing hormone (TRH) caused acute dose-related increases in alpha-MSH release with an ED50 of 10(-8.2) M; dopamine reversibly inhibited alpha-MSH release from similarly treated fragments with an ED50 of 10(-7.7) M. Both stereoisomers of apomorphine, a dopamine agonist, inhibited GtH release from PD fragments treated with sGnRH; in contrast, alpha-MSH release from NIL fragments treated with TRH was stereospecifically inhibited by (-)-apomorphine, but not by (+)-apomorphine. Domperidone reversed (ED50 = 10(-6.6) M) dopamine (10(-6.3) M) inhibition of GtH release from PD fragments treated with sGnRH. In NIL fragments, the inhibitory action of dopamine (10(-6.3) M) was reversed by domperidone (ED50 = 10(-5.5) M), which restored the acute alpha-MSH release response to TRH. These results suggest the involvement of a low-affinity dopamine/neuroleptic receptor in dopamine inhibition of GtH and alpha-MSH release from the pituitary of the goldfish.     

Influences of norepinephrine, and adrenergic agonists and antagonists on gonadotropin secretion from dispersed pituitary cells of goldfish, Carassius auratus.

Static incubations of dispersed goldfish pituitary cells with 1-100 nM norepinephrine (NE) stimulated gonadotropin (GTH) release. Additions of the alpha-agonist phenylephrine, and the alpha 1-agonist 6-fluoronorepinephrine, but not the alpha 2-agonist clonidine, nor the beta-agonist isoproterenol, also enhanced GTH secretion. The GTH responses to 1 nM NE was significantly inhibited by coincubations with 1 microM of the alpha-antagonist phentolamine, the alpha 1-antagonists prazosine and benoxathian, but not the alpha 2-antagonist yohimbine nor the beta-antagonist propranolol. The GTH responses to NE and phenylephrine were also additive to salmon GTH-releasing hormone (sGnRH)-induced GTH release. These results suggests that NE directly stimulates GTH secretion independent of sGnRH receptors via alpha 1-like adrenergic receptors.     

Prostaglandin synthesis in goldfish heart, Carassius auratus

Potential precursors for prostaglandin (PG) synthesis were measured in goldfish heart and skeletal muscle by gas chromatography. Heart tissue contained docosahexaenoic, arachidonic, eicosapentaenoic, and eicosatrienoic acids in concentrations of 3223 +/- 128, 1216 +/- 7.8, 260 +/- 72.8, and 250 +/- 14 ng/mg wet wt, respectively. 14C-Labeled substrates were examined for their ability to be converted to prostaglandins. Eicosatrienoic and docosahexaenoic acid were not synthesized into prostaglandins, with 66 and 72% of the substrate remaining as free fatty acids, respectively. In contrast, both arachidonic and eicosapentaenoic acids were converted predominantly to PGFs and PGIs. The conversion was time dependent and complete by 30 min. The conversion patterns with eicosapentaenoic acid and arachidonic acid were essentially the same. The data suggest that goldfish cyclooxygenase can utilize two of the four potential substrates for prostaglandin synthesis. As fatty acid levels in fish vary with environmental temperature, substrate availability rather than cyclooxygenase preference may dictate the types of prostaglandins which are produced.     

Stimulation of gonadotropin secretion by intraventricular injection of hypothalamic extracts in the goldfish,Carassius auratus

Goldfish serum gonadotropin (GTH) concentrations were determined by radioimmunoassay 20 min after injection of goldfish brain extracts or synthetic luteinizing hormone-releasing hormone (LH-RH) into the third ventricle in the ventrobasal hypothalamic region. Extracts of the cerebellum and medulla were ineffective. Synthetic LH-RH, and extract of the lateral hypothalamus each significantly increased plasma GTH levels. The results indicate that the hypothalamus of the sexually mature female goldfish contains gonadotropin releasing hormone activity, and that releasing hormone is effective when administered via the intrahypothalamic route.     

Effects of third ventricle injection of prostaglandins on gonadotropin secretion in goldfish,Carassius auratus

The effects of injection of prostaglandin (PG) E₁, PGE₂, and PGF_2α into the third ventricle on serum gonadotropin (GTH) concentrations in the goldfish were tested. Blood samples were taken at 30 min postinjection for radioimmunoassay of serum GTH. PG dosages of 0.5 and 1.0 μg were ineffective. However, PGE₂ and PGF_2α at the 2.0-μg dosage significantly suppressed serum GTH. PGE₁ at a 2.0-μg dosage had no effect. There were no effects on serum GTH when 2.0 μg of PGE₁, PGE₂, or PGF_2α were injected intraperitoneally. The results indicate that PGE₂ and PGF_2α suppress gonadotropin secretion by some action, presumably on the hypothalamus. However, action of PGE₂ and PGF_2α by diffusion from the site of injection to some other brain site or the pituitary cannot be eliminated in the present study.     

Circadian rhythms of melatonin secretion from superfused goldfish (Carassius auratus) pineal glands in vitro

A flow-through, whole-organ culture (superfusion) system was developed, and goldfish pineal glands were maintained at 25 degrees under light-dark (LD) 12:12 cycles, reversed LD 12:12 cycles, continuous dark (DD), or continuous light (LL) conditions for 48 hr. Under LD 12:12 and reversed LD 12:12 cycles, superfused pineal glands showed a rhythmic melatonin secretion: Scotophase was associated with high titers and photophase with low titers. The melatonin secretion rhythms persisted for two cycles under DD conditions, whereas nocturnal rises were suppressed under LL conditions. After the transition from LL to DD conditions on the third day, melatonin showed a nocturnal increase. These results indicate that melatonin secretion from the superfused goldfish pineal gland is directly photosensitive and that the goldfish pineal gland harbors a circadian oscillator which generates melatonin secretion rhythms.     

Dopaminergic regulation of pituitary gonadotrophin-releasing hormone receptor activity in the goldfish (Carassius auratus)

In goldfish, dopamine acts as an endogenous inhibitor of basal and gonadotrophin-releasing hormone (GnRH)-stimulated gonadotrophin release. The purpose of the present study was to investigate the effects of dopamine on the pituitary GnRH receptors in vivo and in vitro in goldfish. The goldfish pituitary contains two classes of GnRH-binding sites, a high-affinity/low-capacity site and low-affinity/high-capacity site. Injection of domperidone, a dopamine antagonist, resulted in a dose- and time-related increase in capacity of both the high- and low-affinity GnRH-binding sites; apomorphine, a dopamine agonist, completely reversed this effect. The effects on GnRH receptor capacity correlated very closely with changes in serum gonadotrophin concentrations. Domperidone was generally without effect on GnRH-binding affinity; however, a small but significant decrease in affinity was observed for the low-affinity binding site at 18 h after injection of the highest dose of domperidone used (40 mumol/kg body weight). Treatment with apomorphine of goldfish pituitary fragments in a perifusion system caused a decrease in the capacity of both the high- and low-affinity GnRH-binding sites without affecting binding affinity; domperidone reversed this effect. It is concluded that the dopaminergic inhibition of basal and GnRH-stimulated gonadotrophin release in goldfish might, in part, be the result of a down-regulation of the pituitary GnRH receptors; this effect of dopamine can be achieved by a direct action at the pituitary level.     

Ovulatory surge of gonadotropin in the goldfish, Carassius auratus.

The periovulatory pattern of circulating gonadotropin is described for the first time in a poikilothermic vertebrate. In sexually mature female goldfish (Carassius auratus) maintained on a 16L:8D photoperiod, ovulation occurs during the latter part of the dark phase, approximately 20 hr after the fish are warmed from 13 to 21°. Serum gonadotropin levels increase gradually during the first half of the light phase, and by the latter part begin to increase rapidly, reaching the peak of the gonadotropin surge by the onset of the dark phase. Gonadotropin levels remain high through the period of ovulation, and decrease markedly by the onset of the following light phase. The findings indicate that the goldfish provides a valuable model for the study of ovulation in teleosts.     

Gonadotropin release after implantation of anti-estrogens in the pituitary and hypothalamus of goldfish Carassius auratus.

Serum immunoreactive gonadotropin (GTH) levels were measured in sexually mature (gravid) female foldfish at 24 hr before and 24 and 72 hr after brain or pituitary stereotaxic implantation of either a blank pellet (cocoa butter only) or a pellet containing one of the anti-estrogens, clomiphene citrate or ICI 46474. Both pituitary implantation and implantation in the nucleus lateral tuberis (NLT) region of a pellet containing ICI 46474 caused a significant increase in serum GTH levels, with the former site having a more pronounced effect. Implantation of ICI 46474 in the nucleus preopticus (NPO) region, the posterodorsal hypothalamus, or the optic tectum (OT) had no significant effects on serum GTH. Implantation of clomiphene citrate in the pituitary caused a significant increase in serum GHT levels, but implantation in the NPO region or OT had no effects. The results demonstrate that the pituitary gland and, to a lesser extent, the NLT region are sites for negative feedback effects of estrogens in sexually mature (gravid) female goldfish.     

Alterations in pituitary GnRH and dopamine receptors associated with the seasonal variation and regulation of gonadotropin release in the goldfish (Carassius auratus)

Seasonal variations in the serum concentrations of gonadotropin (GtH) and the serum GtH response to intraperitoneal injection of domperidone, a specific dopamine receptor antagonist, were examined in goldfish. In addition, the effects of in vivo treatment of goldfish with a superactive analog of salmon gonadotropin-releasing hormone (sGnRH-A) and domperidone on the binding parameters of pituitary GnRH and dopamine receptors were investigated in goldfish. Serum concentrations of GtH and the maximum GtH response to domperidone increased in correlation with advancing gonadal maturation; values increased from those in sexually regressed fish in January to maximal levels observed in fish in late stages of gonadal recrudescence in March, followed by a decrease with gonadal regression. At all stages, injection of domperidone increased serum concentrations of GtH in a dose-related manner; however, the ED50 of domperidone did not vary significantly over the course of the reproductive cycle. Multiple injections of sGnRH-A caused a progressively increasing and more prolonged serum GtH response; as well, multiple sGnRH-A treatment significantly potentiated the serum GtH response to domperidone without altering the ED50 of domperidone. sGnRH-A treatment caused a significant increase in the number of dopamine/neuroleptic receptors in the goldfish pars distalis, accompanied by a nonsignificant increase in dopamine/neuroleptic receptors in the neurointermediate lobe, and significantly increased the number of high-affinity GnRH receptors in the goldfish pituitary. Treatment with domperidone also significantly increased pituitary high-affinity GnRH receptor numbers. Receptor affinities were not significantly altered by either sGnRH-A or domperidone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of endogenous opioids in the regulation of gonadotropin secretion in male goldfish, Carassius auratus

The in vivo effects of the opioid receptor antagonist naloxone (NAL) alone, and in combination with des-Gly10[D-Ala6]LHRH ethylamide (LHRH-A) and the dopamine receptor antagonist domperidone (DOM) on serum gonadotropic hormone (GtH) levels in male goldfish, Carassius auratus, were investigated. NAL caused a significant decrease in serum GtH 1 hr following treatment, with a return to control levels by 2 hr. NAL treatment attenuated the stimulation of GtH levels in response to DOM; NAL treatment 2 hr prior to or 2 hr following DOM resulted in a significantly reduced response to DOM. During late recrudescence, NAL pretreatment significantly blocked the stimulatory effects of LHRH-A on serum GtH. During early recrudescence, when LHRH-A alone did not significantly elevate GtH levels, NAL treatment simultaneously with or 1 hr following LHRH-A significantly elevated serum GtH. In DOM-pretreated fish, combined LHRH-A and NAL treatment resulted in a nine-fold increase in serum GtH compared to DOM alone. These data indicate the ability of NAL to both suppress and increase GtH levels in male goldfish. Interactions between NAL, DOM, and LHRH-A suggest that opioids modulate both dopamine and GnRH secretion, and possibly the pituitary sensitivity to GnRH and dopamine, thus affecting GtH levels.