Agnieszka Kleszczyńska

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.

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.

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.

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.

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.

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.