The time enzyme in melatonin biosynthesis in fish: Day/night expressions of three aralkylamine N-acetyltransferase genes in three-spined stickleback.

In vertebrates, aralkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is a time-keeping enzyme in melatonin (Mel) biosynthesis. Uniquely in fish, there are several AANAT isozymes belonging to two AANAT subfamilies, AANAT1 and AANAT2, which are encoded by distinct genes. The different substrate preferences, kinetics and spatial expression patterns of isozymes indicate that they may have different functions. In the three-spined stickleback (Gasterosteus aculeatus), there are three genes encoding three AANAT isozymes. In this study, for the first time, the levels of aanat1a, aanat1b and aanat2 mRNAs are measured by absolute RT-qPCR in the brain, eye, skin, stomach, gut, heart and kidney collected at noon and midnight. Melatonin levels are analysed by HPLC with fluorescence detection in homogenates of the brain, eye, skin and kidney. The levels of aanats mRNAs differ significantly within and among organs. In the brain, eye, stomach and gut, there are day/night variations in aanats mRNAs levels. The highest levels of aanat1a and aanat1b mRNAs are in the eye. The extremely high expressions of these genes which are reflected in the highest Mel concentrations at this site at noon and midnight strongly suggest that the eye is an important source of the hormone in the three-spined sticklebacks. A very low level of aanat2 mRNA in all organs may suggest that AANAT1a and/or AANAT1b are principal isozymes in the three-spine sticklebacks. A presence of the isozymes of defined substrate preferences provides opportunity for control of acetylation of amines by modulation of individual aanat expression and permits the fine-tuning of indolethylamines and phenylethylamines metabolism to meet the particular needs of a given organ.

Cortisol stimulates arginine vasotocin and isotocin release from the hypothalamo-pituitary complex of round goby (Neogobius melanostomus): Probable mechanisms of action.

There were two aims of this in vitro perfusion study. Firstly, to determine which class of receptors, glucocorticoid (GRs) or mineralocorticoid (MRs), are involved in cortisol regulation of arginine vasotocin (AVT) and isotocin (IT) release from the hypothalamo-pituitary (H-P) complex of round goby (Neogobius melanostomus). Secondly, to determine which pathways, genomic or non-genomic, are involved in the aformentioned process.The H-P explants were perfused with cortisol (1.4 × 10(-) (7) M, 2.8 × 10(-7) M, 0.4 × 10(-6) M); only the highest dose significantly increased a release of both nonapeptides. In the perfusion of H-P explants, we used cortisol (0.4 × 10(-6) M) in combination with GRs antagonist RU486 (0.3 × 10(-6) M) or MRs antagonist C03DA01 (0.36 × 10(-6) M) or transcription inhibitor Actinomycin D (1 × 10(-7) M). All inhibitors were also tested seperately. The contents of AVT and IT in the perfusion media was determined by high-performance liquid chromatography (HPLC) with UV detection. This study suggested that different mechanisms were involved in the regulation of AVT and IT release from H-P complex in round goby. Apparently it was GRs but not MRs that were involved in cortisol regulation of AVT and IT release. In the case of AVT, our data points to both genomic and non-genomic pathways mediating the effect of cortisol; in the case of IT, it is only the non-genomic pathway. This study presents the first feasible mechanisms of cortisol action on AVT and IT release from the H-P complex in round goby.

Urotensin II inhibits arginine vasotocin and stimulates isotocin release from nerve endings in the pituitary of gilthead sea bream (Sparus aurata).

The aim of this in vitro study was to determine whether arginine vasotocin (AVT) and isotocin (IT) release from nerve endings is affected by urotensin II (UII) in gilthead sea bream pituitary. Primary cultures of pituitary cells were exposed to 10(-12), 10(-10), and 10(-8) M UII for 6, 24, and 48 hr. AVT and IT contents were determined in the culture media by high performance liquid chromatography (HPLC). UII at all doses decreased AVT release after 6, 24, and 48 hr of incubation. IT release was increased by UII only after 24 hr of incubation. This study, for the first time, indicates that UII affects AVT and IT release from nerve endings in the pituitary of Sparus aurata. It is presumed that UII together with AVT and IT may control response to different salinities in fishes.

Brain arginine vasotocin and isotocin in breeding female three-spined sticklebacks (Gasterosteus aculeatus): the presence of male and egg deposition.

Arginine vasotocin (AVT) and isotocin (IT) are fish hypothalamic nonapeptides involved in numerous social and reproductive behaviors. Vasotocinergic and isotocinergic fibers project to different brain areas where peptides act as neurotransmitters and/or neuromodulators. In this study, we measured whole brain levels of bioactive AVT and IT in breeding females of three-spined stickleback (Gasterosteus aculeatus) when they were kept with: (i) courting nest-owners, (ii) courting males that did not build the nest, (iii) non-courting males, and (iv) alone. Only some of the females kept with courting nest-owners deposited eggs. The highest and similar brain AVT levels were in those of females that did not deposit eggs, regardless of whether they were kept with non-courting or courting male, having the nest or not. The highest IT levels were in females that did not deposit eggs but only in those kept with courting male. We suggest that production of AVT in females' brain is stimulated by the presence of male in close proximity, irrespective of whether or not it displays courting behavior, but that of IT is stimulated by male courtship proxies. Moreover, presence of courting or non-courting male that stimulate IT or/and AVT producing neurones may be decisive for final oocyte maturation or egg deposition, because brain levels of both nonapeptides decrease after egg deposition. Similar AVT levels in brains of aggressive and non-aggressive individuals and lack of correlation between brain IT levels and aggressive behavior of females suggest that the nonapeptides are not related to females aggressiveness in three-spined sticklebacks.

Changes in brain arginine vasotocin, isotocin, plasma 11-ketotestosterone and cortisol in round goby, Neogobius melanostomus, males subjected to overcrowding stress during the breeding season.

In natural spawning grounds, breeding round goby, Neogobius melanostomus, males are exposed to various social stimuli, including high density of same-sex competitors and separation from females. We hypothesize that breeding males subjected to overcrowding in the wild experience high stress that affects their socio-sexual behavior and their relationships among conspecifics. We designed an experiment to mimic natural stimulation when highly aggregated breeding males are subjected to same-sex opponents. Males were sampled sequentially from experimental tank stocked at decreasing fish densities of 15 fish/m(2), 9 fish/m(2) and 4 fish/m(2). We studied the effects of overcrowding on male behavior and selected hormones, brain arginine vasotocin (AVT) and isotocin (IT) and plasma 11-ketotestosterone (11-KT) and cortisol as these are known to play roles in reproduction and related social interactions. The highest brain AVT and plasma cortisol levels were measured in non-aggressive males kept in the overcrowded group of 15 fish/m(2). IT level was elevated in fish kept at the lower density of 9 fish/m(2), and at which the males began to display territoriality and aggression. The plasma level of 11-KT was similar in all the males. Brain AVT and IT and plasma cortisol along with behavioral observations can be applied as species-specific indicators of the well-being of round goby males.

Stocking density influences brain arginine vasotocin (AVT) and isotocin (IT) levels in males and females of three-spined stickleback (Gasterosteus aculeatus).

Arginine vasotocin (AVT) and isotocin (IT) are fish neurohormones produced in separate parvocellular and magnocellular preoptic neurons of Teleostei. Apart from well-established peripheral action as hormones they are important neurotransmitters in central nervous system in fish. In the present study, we examined an influence of stocking density on whole brain AVT and IT concentrations in males and females of three-spined stickleback (Gasterosteus aculeatus). In males, the highest AVT levels have been found at stocking densities of 10 and 30 individuals per 30-l tank. On the other hand, in females, AVT concentration was significantly higher in those kept alone. Brain IT concentrations significantly increased along with stocking density only in females and did not change in males. The sex-dependent responses indicate a different stimulation of AVT and IT neurons in males and females. Consequently, roles of the neurohormones in males and females exposed to stress of overcrowding must be different.

Variation in brain arginine vasotocin (AVT) and isotocin (IT) levels with reproductive stage and social status in males of three-spined stickleback (Gasterosteus aculeatus).

Arginine vasotocin (AVT) and isotocin (IT) are fish nonapeptides synthesized in separate hypothalamic neurons from where they are transported to the neurohypophysis for storage and release into circulation. AVT is known to modulate aggression, courtship and parental care or social communication in many species, including fish, amphibians and birds. In this paper we examined a link between the level of AVT and IT in the brain and particular reproductive behavior in males of three-spined stickleback (Gasterosteus aculeatus). AVT and IT levels in whole brain of males of three-spined stickleback vary depending on specific breeding behavior of the individuals and their social status. These studies have shown the highest AVT levels in aggressive males that took care of the eggs. Brain AVT concentrations are also increased in nuptial colored subordinate males that fight to change their social status. On the other hand, IT is significantly higher in aggressive dominant males that defend their territory. IT may be also involved in courtship in three-spined stickleback. These findings highlight the importance of determination of "free", bioavailable neuropeptides' level in behavioral studies.

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.

Determination of the neuropeptides arginine vasotocin and isotocin in brains of three-spined sticklebacks (Gasterosteus aculeatus) by off-line solid phase extraction-liquid chromatography-electrospray tandem mass spectrometry.

A method based on solid phase extraction (SPE) followed by liquid chromatography-electrospray ionisation tandem mass spectrometry for the determination of the nonapeptides arginine vasotocin (AVT) and isotocin (IT) in brains of three-spined sticklebacks (Gasterosteus aculeatus) is described. Separation and detection were optimized using synthetic standards. Limits of detection (LOD) for standard solutions were 160 pg mL(-1) for AVT and 250 pg mL(-1) for IT. The SPE procedure hardly affected the LODs for standard solutions. Mainly because of ion suppression, LODs for AVT and IT in brains were approximately 5 and 25 pg mg(-1), respectively. The concentrations determined in the brain of several fishes ranged from 10 to 500 pg mg(-1) for AVT and from 400 to 4000 pg mg(-1) for IT.

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.

Osmoregulatory and metabolic changes in the gilthead sea bream Sparus auratus after arginine vasotocin (AVT) treatment.

The influence of arginine vasotocin (AVT) on osmoregulation and metabolism in gilthead sea bream Sparus auratus was evaluated by two experimental approaches. In the first, seawater (SW, 36 ppt)-acclimatized fish were injected intraperitoneally with vehicle (vegetable oil) or two doses of AVT (0.5 and 1 microg/g body weight). Twenty-four hours later, eight fish from each group were sampled; the remaining fish were transferred to low saline water (LSW, 6 ppt, hypoosmotic test), SW (transfer control), and hypersaline water (HSW, 55 ppt, hyperosmotic test). After another 24h (48-h post-injection), fish were sampled. The only significant effect observed was the increase of sodium levels in AVT-treated fish transferred to HSW. In the second experiment, fish were injected intraperitoneally with slow-release vegetable oil implants (mixture 1:1 of coconut oil and seeds oil) alone or containing AVT (1 microg/g body weight). After 3 days, eight fish from each group were sampled; the remaining fish were transferred to LSW, SW, and HSW as above, and sampled 3 days later (i.e. 6 days post-injection). In the AVT-treated group transferred from SW to SW, a significant increase vs. control was observed in gill Na(+),K(+)-ATPase activity. Kidney Na(+),K(+)-ATPase activity decreased in the AVT-treated group transferred to LSW and no changes were observed in the other groups. These osmoregulatory changes suggest a role for AVT during hyperosmotic acclimation based on changes displayed by gill Na(+),K(+)-ATPase activity. AVT treatment increased plasma cortisol levels in fish transferred to LSW and HSW. In addition, AVT treatment affected parameters of carbohydrate, lipid, amino acid, and lactate metabolism in plasma and tissues (gills, kidney, liver, and brain). The most relevant effects were the increased potential of liver for glycogen mobilization and glucose release resulting in increased plasma levels of glucose in AVT-treated fish transferred to LSW and HSW. These changes may be related to the energy repartitioning process occurring during osmotic adaptation of S. auratus to extreme environmental salinities and could be mediated by increased levels of cortisol in plasma.