High density and food deprivation affect arginine vasotocin, isotocin and melatonin in gilthead sea bream (Sparus auratus)

Arginine vasotocin (AVT) and isotocin (IT) levels in plasma and pituitary, and melatonin (MEL) levels in plasma were determined in gilthead sea bream (Sparus auratus) subjected to two different types of stress: i) high density (HD) and ii) food deprivation (NF: non-fed). Fishes were randomly assigned to one of 4 treatments that lasted for 14 days: 1) fed fish under normal low density (ND, 4 kg m(-3)); 2) non-fed (NF) fish under ND; 3) fed fish under high density (HD, 70 kg m(-3)); and 4) non-fed fish under HD. Ten fish from each tank were anaesthetized, weighed and plasma and pituitary samples were taken. Plasma and pituitary AVT and IT content were determined by HPLC, while plasma MEL was assayed by RIA. Plasma AVT and IT values were enhanced in all fish kept at high density. The response of AVT was much stronger than that of IT. The highest pituitary AVT and IT levels were shown in NF fish kept at normal density. The significantly higher plasma MEL levels were measured in fed fish kept at HD. These results suggest a role of AVT, IT and MEL in response of sea bream to a common stress factor, high density. Although food deprivation does not influence AVT and IT plasma levels, it seems to affect hypothalamic synthesis of nonapeptides. Further studies are required to elucidate the complex role of AVT, IT and MEL in the sea bream's response to different stress stimuli.     

High density and food deprivation affect arginine vasotocin, isotocin and melatonin in gilthead sea bream (Sparus auratus).

Arginine vasotocin (AVT) and isotocin (IT) levels in plasma and pituitary, and melatonin (MEL) levels in plasma were determined in gilthead sea bream (Sparus auratus) subjected to two different types of stress: i) high density (HD) and ii) food deprivation (NF: non-fed). Fishes were randomly assigned to one of 4 treatments that lasted for 14 days: 1) fed fish under normal low density (ND, 4 kg m(-3)); 2) non-fed (NF) fish under ND; 3) fed fish under high density (HD, 70 kg m(-3)); and 4) non-fed fish under HD. Ten fish from each tank were anaesthetized, weighed and plasma and pituitary samples were taken. Plasma and pituitary AVT and IT content were determined by HPLC, while plasma MEL was assayed by RIA. Plasma AVT and IT values were enhanced in all fish kept at high density. The response of AVT was much stronger than that of IT. The highest pituitary AVT and IT levels were shown in NF fish kept at normal density. The significantly higher plasma MEL levels were measured in fed fish kept at HD. These results suggest a role of AVT, IT and MEL in response of sea bream to a common stress factor, high density. Although food deprivation does not influence AVT and IT plasma levels, it seems to affect hypothalamic synthesis of nonapeptides. Further studies are required to elucidate the complex role of AVT, IT and MEL in the sea bream's response to different stress stimuli.     

Brain levels of arginine-vasotocin and isotocin in dominant and subordinate males of a cichlid fish

The nonapeptides arginine-vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine-vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirect measures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptors mRNA expression with in situ hybridization or qPCR) and therefore direct measures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL) method to quantify the levels of both AVT and IT in macro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect, we have also investigated the effect of social status on urine storage. As predicted, dominant males stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes.     

Annual changes in brain concentration of arginine vasotocin and isotocin correspond with phases of reproductive cycle in round goby,Neogobius melanostomus

Reproductive cycle of seasonally breeding fish is synchronized with changes of photoperiod and temperature in environment. We hypothesize that arginine vasotocin (AVT) and isotocin (IT) are involved in timing and synchronization of seasonal reproductive activity in the round goby (Neogobius melanostomus). To verify this hypothesis, we examined the annual profiles of brain AVT and IT in round goby males and females in relation to their reproductive cycle. Wild round gobies were exposed to annual environmental changes in their natural habitats from where they were sampled monthly over a year. AVT and IT were measured using HPLC with fluorescence detection preceded by solid-phase extraction. This study shows seasonal variations in brain AVT and IT levels. Profiles of changes were similar in males and females: the peak of AVT was observed before spawning in March-April, whereas that of IT during spawning in May–June. Furthermore, the lowest AVT level was noted out of breeding season from November to January, while the level of IT decreased immediately at the end of the spawning. The results show that high AVT levels correlate with pre-spawning period whereas the highest IT levels correspond to spawning. A significant decline in AVT and IT in non-spawning season coincided with the quiescent phase of gametogenesis in both sexes.     

Arginine vasotocin, isotocin and melatonin responses following acclimation of gilthead sea bream (Sparus aurata) to different environmental salinities

Gilthead sea bream (Sparus aurata) is a euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-adaptive processes and their hormonal control. Immature sea bream acclimated to different salinities, i.e. SW (38 per thousand), LSW (5 per thousand) and HSW (55 per thousand), were kept at 18 degrees C under natural photoperiod. Arginine vasotocin (AVT) and isotocin (IT) in plasma and pituitary were determined by HPLC. Plasma melatonin (Mel) was assayed by RIA. Plasma osmolality, ion concentrations (Na(+), K(+), Ca(2+), Cl(-)) and Na(+),K(+)-ATPase activity in gill were measured. A steady increase in plasma AVT, along with increasing water salinity was observed. Pituitary IT concentration in HSW-acclimated fish was significantly higher than that in LSW group. AVT/IT secretory system of sea bream does appear to be involved in the mechanism of long-term acclimation to different salinities. The distinct roles and control mechanisms of both nonapeptides are suggested. Plasma Mel was significantly higher in LSW compared with both HSW and SW groups. Data indicate that the changes in Mel level are linked to osmoregulation. Further studies are required to elucidate a complex role of AVT, IT and Mel in sea bream osmoregulation.     

Opposite effects of nonapeptide antagonists on paternal behavior in the teleost fish Amphiprion ocellaris

The nonapeptides isotocin (IT) and arginine vasotocin (AVT), along with their mammalian homologs oxytocin and arginine vasopressin, are well known regulators of social behaviors across vertebrate taxa. However, little is known about their involvement in paternal care. Here, we measured the effect of an IT and an AVT V1a receptor antagonist on paternal behaviors in the primarily paternal teleost Amphiprion ocellaris. We also measured the effect of the IT receptor antagonist on aggression in dyadic contests between two non-reproductive fish to assess specificity of the effect on paternal behaviors. Individual differences in levels of paternal behaviors (nips, fanning the eggs, and proportion of the time in the nest) were consistent across spawning cycles when no treatments were administered. The IT receptor antagonist severely reduced paternal behaviors but had no effect on aggression, whereas the AVT V1a receptor antagonist increased paternal behaviors. These results support the idea that IT signaling is crucial for the expression of paternal behavior in A. ocellaris. Based on a previous study showing that the AVT V1a antagonist decreases aggression in dyadic contests, we hypothesize that the antagonist enhances paternal behavior indirectly by reducing vigilance and aggression, thereby alleviating effort directed towards other competing behaviors and allowing for the increased expression of paternal behaviors.     

Day-night variations in plasma melatonin and arginine vasotocin concentrations in chronically cannulated flounder (Platichthys flesus).

Chronically catheterised, free swimming flounder (Platichthys flesus) have been used in experiments examining the day-night variations in circulating levels of melatonin (Mel) and arginine vasotocin (AVT). Under normal photoperiod (16 h light/8 h dark) serial blood samples taken from individual fish demonstrated a Mel rhythm with daytime levels at 09.00 and 15.00 h (238+/-14 and 179+/-12 fmol x ml(-1), respectively) lower than those at 23.00 h (1920+/-128 fmol x ml(-1)). Maintenance of fish in 24-h light abolished the light/dark Mel rhythm and circulating levels were comparable to those measured during the day in fish under normal photoperiod illumination. In fish maintained under 24 h dark, although a daily rhythm was still apparent, at the time when it would be normally dark, plasma Mel concentration was reduced and at times when it would be normally light, levels were higher than in fish maintained under normal light/dark illumination. Plasma AVT concentrations were higher in fish during the day (4.4+/-0.8 fmol x ml(-1)) than those at night (1.5+/-0.4 fmol x ml(-1)), the opposite to that seen with Mel. During acute study infusion of AVT resulted in reduced levels of plasma Mel, although this did not achieve statistical significance. Infusion of Mel did not alter circulating AVT concentration.     

Determination of arginine-vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography

A new HPLC assay for plasma arginine-vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15-220 pmol ml(-1) for AVT r2=0.998 and 10-220 pmol ml(-1) for IT r2=0.996. The detection limits for AVT and IT were 0.8 and 0.5 pmol ml(-1) (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89-93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.     

Exogenous vasotocin alters aggression during agonistic exchanges in male Amargosa River pupfish (Cyprinodon nevadensis amargosae)

Pupfishes in the Death Valley region have rapidly differentiated in social behaviors since their isolation in a series of desert streams, springs, and marshes less than 20,000 years ago. These habitats can show dramatic fluctuations in ecological conditions, and pupfish must cope with the changes by plastic physiological and behavioral responses. Recently, we showed differences among some Death Valley populations in brain expression of arginine vasotocin (AVT). As AVT regulates both hydromineral balance and social behaviors in other taxa, these population differences may indicate adaptive changes in osmoregulatory and/or behavioral processes. To test whether AVT is relevant for behavioral shifts in these fish, here we examined how manipulations to the AVT system affect agonistic and reproductive behaviors in Amargosa River pupfish (Cyprinodon nevadensis amargosae). We administered exogenous AVT (0.1, 1, and 10 microg/g body weight) and an AVP V1 receptor antagonist (Manning compound, 2.5 microg/g body weight) intraperitoneally to males in mixed-sex groups in the laboratory. We found that AVT reduced the initiation of aggressive social interactions with other pupfish but had no effect on courtship. The effects of AVT were confirmed in males in the wild where AVT (1 microg/g body weight) reduced the aggressive initiation of social interactions and decreased aggressive responses to the behavior of other males. Combined, these results show that AVT can modulate agonistic behaviors in male pupfish and support the idea that variation in AVT activity may underlie differences in aggression among Death Valley populations.     

In vitro effect of cortisol and urotensin I on arginine vasotocin and isotocin secretion from pituitary cells of gilthead sea bream Sparus aurata

This study aimed at determining whether in vitro secretion of two neuropeptides, arginine vasotocin (AVT) and isotocin (IT), from pituitary cells of gilthead sea bream Sparus aurata was affected by cortisol and urotensin (UI). Pituitary cells were exposed to 1·4 × 10^?8, 1·4 × 10^?7 and 0·4 × 10^?6 M cortisol and 10^?12, 10^?10 and 10^?8 M UI for 6, 24 and 48 h, respectively. AVT and IT contents were determined in the culture media by high‐performance liquid chromatography (HPLC). An increase in AVT secretion and a decrease in IT secretion were observed at all cortisol doses. UI increased AVT secretion after 6 h of incubation at all doses. After 24 h, however, only the highest dose of UI still displayed an effect. IT secretion was not influenced by UI. It was thus demonstrated that cortisol does influence AVT and IT secretion from S. aurata pituitary cells, while UI regulates AVT secretion, as a component of hypothalamic–pituitary–interrenal (HPI) axis in this species.     

Agonistic interactions elicit rapid changes in brain nonapeptide levels in zebrafish

The teleost fish nonapeptides, arginine vasotocin (AVT) and isotocin (IT), have been implicated in the regulation of social behavior. These peptides are expected to be involved in acute and transient changes in social context, in order to be efficient in modulating the expression of social behavior according to changes in the social environment. Here we tested the hypothesis that short-term social interactions are related to changes in the level of both nonapeptides across different brain regions. For this purpose we exposed male zebrafish to two types of social interactions: (1) real opponent interactions, from which a Winner and a Loser emerged; and (2) mirror-elicited interactions, that produced individuals that did not experience a change in social status despite expressing similar levels of aggressive behavior to those of participants in real-opponent fights. Non-interacting individuals were used as a reference group. Each social phenotype (i.e. Winners, Losers, Mirror-fighters) presented a specific brain profile of nonapeptides when compared to the reference group. Moreover, the comparison between the different social phenotypes allowed to address the specific aspects of the interaction (e.g. assessment of opponent aggressive behavior vs. self-assessment of expressed aggressive behavior) that are linked with neuropeptide responses. Overall, agonistic interactions seem to be more associated with the changes in brain AVT than IT, which highlights the preferential role of AVT in the regulation of aggressive behavior already described for other species.     

Plasma arginine vasotocin and angiotensin II in the water deprived Kelp gull (Larus dominicanus), Cape gannet (Sula capensis) and Jackass penguin (Spheniscus demersus)

1. The basal levels of the osmoregulatory hormones, arginine vasotocin (AVT) and angiotensin II (AII) were measured (by radioimmunoassay) in the plasma of conscious Kelp gulls, Cape gannets and Jackass penguins. 2. The responses of the hormones to similar degrees of hypertonicity and hypovolemia caused by water deprivation have also been determined. 3. Dehydration elevated plasma AVT and plasma AII in all three species. 4. The AVT concentration was increased by 2-4 fold and although in each case the correlation between plasma osmolality and plasma AVT was highly significant (2P less than 0.01), the sensitivity of release was greater in the gull (1.13 pg/ml per mOsm/kg) than in the gannet (0.36 pg/ml per mOsm/kg) or penguin (0.44 pg/ml per mOsm/kg). 5. Dehydration increased plasma AII 3-fold in the three bird types.     

Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness

The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF), arginine vasotocin (AVT), CRF receptor type 1 (CRF-R1), CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1, AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.     

Diurnal variations in plasma arginine vasotocin (AVT) concentrations in the ovine fetus

Previously we have demonstrated the presence of AVT in the blood of fetal sheep. The source is not clear, but AVT has been identified in fetal pineal and pituitary glands. In view of the circadian secretory pattern of the pineal gland, we questioned whether fetal plasma AVT levels might vary diurnally. Plasma samples from five chronically catheterized ovine maternal ewes and fetal lambs 129-135 days' gestation were obtained at 3-19 hourly intervals for 1-2 days (mean +/- S.E.M. = 35 +/- 6 hours. Plasma AVT levels were determined by radioimmunoassay. Results were analyzed by nonlinear curve fitting procedures to relate hormone levels with time of day. Plasma AVT values for maternal ewes did not vary during the day in response to light/dark periods. The curve for mean fetal plasma AVT plotted against time showed oscillations with a period of about 25 hours (p less than 0.05). Peak fetal AVT levels were observed at 1600 hours and minimal levels at 0400 hours. These results indicate that ovine fetal AVT secretion varies diurnally. The site of AVT secretion may be the pineal gland; however, confirmation of this and identification of the physiological stimuli for secretion of fetal plasma AVT require further information.     

Neurohypophysial hormones and renal function in fish and mammals

The two major basic neurohypophysial peptides, arginine vasopressin (AVP) of mammals and arginine vasotocin (AVT) of all non-mammalian vertebrates, share common structure and major roles in regulating renal function. In this review the complexity of AVP actions within the mammalian kidney is discussed and comparisons are made with the emerging picture of AVT's renal effects in fish. It has become apparent that the antidiuretic action of the neurohypophysial hormones is an ancient phylogenetic phenomenon, although this is based upon reduced glomerular filtration in fish by comparison with predominant tubular effects in mammals. Nonetheless, there appears to be retention of AVP effects upon the functional heterogeneity of nephron populations in mammals. Preliminary evidence for the possible existence of V(2)-type (tubular) neurohypophysial hormone receptors in fish, implies possible AVT actions which parallel those in mammals on tubular ion transport. Further insight from recent mammalian tubule microperfusion studies suggests that in teleost fish both apical (tubular lumen) and basolateral (blood borne) AVT have the potential to modulate renal function, though this remains to be examined.     

Effect of indomethacin and vasotocin on oviposition in the hen (Gallus domesticus)

The interrelationship between prostaglandins (PG) and vasotocin (AVT) in the oviposition of the domestic hen was investigated. Single or combined injections of indomethacin (IND), an inhibitor of PG synthesis, and AVT gave delay or induction of oviposition. Injection (i.m.) of IND (5 mg/kg) 5 h before oviposition resulted in 15.1 h (+/- 0.93) delay of oviposition. Injection (i.v.) of AVT (0.1 microgram/kg) 2.5 h before oviposition caused premature oviposition within a few minutes (3.1 +/- 0.2). Combined injection of IND and AVT at 5 h and 2.5 h, respectively, before oviposition caused the delay of oviposition (15.8 h +/- 0.8). The results indicate that IND blocked the induction of oviposition by AVT.     

The role of arginine vasotocin in teleost fish osmoregulation

The AVT system is a key component of the complex mechanism responsible for coordinating the response in teleost fish to hyper-osmotic challenge. The pituitary AVT store falls following transfer to SW from FW and this is accompanied by an increase in circulating AVT levels. This indicates that there has been release of AVT in response to this stimulus and the system is being up-regulated. The effect of an increase in secretion would appear to be predominantly to reduce GFR and urine production as is seen when AVT is injected into FW-adapted fish. This action is consistent with the requirements of hyper-osmotic challenge when water conservation becomes essential.     

Sex and seasonal co-variation of arginine vasotocin (AVT) and gonadotropin-releasing hormone (GnRH) neurons in the brain of the halfspotted goby

Gonadotropin-releasing hormone (GnRH) and arginine vasotocin (AVT) are critical regulators of reproductive behaviors that exhibit tremendous plasticity, but co-variation in discrete GnRH and AVT neuron populations among sex and season are only partially described in fishes. We used immunocytochemistry to examine sexual and temporal variations in neuron number and size in three GnRH and AVT cell groups in relation to reproductive activities in the halfspotted goby (Asterropteryx semipunctata). GnRH-immunoreactive (-ir) somata occur in the terminal nerve, preoptic area, and midbrain tegmentum, and AVT-ir somata within parvocellular, magnocellular, and gigantocellular regions of the preoptic area. Sex differences were found among all GnRH and AVT cell groups, but were time-period dependent. Seasonal variations also occurred in all GnRH and AVT cell groups, with coincident elevations most prominent in females during the peak- and non-spawning periods. Sex and temporal variability in neuropeptide-containing neurons are correlated with the goby's seasonally-transient reproductive physiology, social interactions, territoriality and parental care. Morphological examination of GnRH and AVT neuron subgroups within a single time period provides detailed information on their activities among sexes, whereas seasonal comparisons provide a fine temporal sequence to interpret the proximate control of reproduction and the evolution of social behavior.