Vasotocin Research Articles

Neurohypophysial Hormones Regulate Amphibious Behaviour in the Mudskipper Goby.

The neurohypophysial hormones, arginine vasotocin and isotocin, regulate both hydromineral balance and social behaviors in fish. In the amphibious mudskipper, Periophthalmus modestus, we previously found arginine-vasotocin-specific regulation of aggressive behavior, including migration of the submissive subordinate into water. This migration also implies the need for adaptation to dehydration. Here, we examined the effects of arginine vasotocin and isotocin administration on the amphibious behavior of individual mudskippers in vivo. The mudskippers remained in the water for an increased period of time after 1–8 h of intracerebroventricular (ICV) injection with 500 pg/g arginine vasotocin or isotocin. The ‘frequency of migration’ was decreased after ICV injection of arginine vasotocin or isotocin, reflecting a tendency to remain in the water. ICV injections of isotocin receptor antagonist with arginine vasotocin or isotocin inhibited all of these hormonal effects. In animals kept out of water, mRNA expression of brain arginine vasotocin and isotocin precursors increased 3- and 1.5-fold, respectively. Given the relatively wide distribution of arginine vasotocin fibres throughout the mudskipper brain, induction of arginine vasotocin and isotocin under terrestrial conditions may be involved also in the preference for an aquatic habitat as ligands for brain isotocin receptors.

Is there convergence in the molecular pathways underlying the repeated evolution of sociality in African cichlids?

Despite wide variation in the complexity of social interactions across taxa, the basic behavioral components of sociality appear to be modulated by conserved hormone pathways. Specifically, the nonapeptide hormones oxytocin and vasopressin and their receptors have been implicated in regulating diverse social behaviors across vertebrates. Here, we took advantage of the repeated evolution of cooperative breeding in African cichlids to investigate whether there are consistent brain gene expression patterns of isotocin and arginine vasotocin (teleost homologues of oxytocin and vasopressin), as well as their receptors, between four closely related pairs of social (cooperative) and non-social (non-cooperative) species. We first found that the coding sequences for the five genes studied were highly conserved across the eight species. This is the first study to examine the expression of both isotocin receptors, and so we performed a phylogenetic analysis that suggests that these two isotocin receptors are paralogues that arose during the teleost genome duplication. When we then examined brain gene expression patterns relative to social system, we found that there were whole-brain gene expression differences between the social and non-social species in many of the species pairs. However, these relationships varied in both the direction and magnitude among the four species pairs. In conclusion, our results suggest high sequence conservation and species-specific gene expression patterns relative to social behavior for these candidate hormone pathways in the cichlid fishes.

The Effects of Melatonin on Brain Arginine Vasotocin: Relationship with Sex and Seasonal Differences in Melatonin Receptor Type 1 in Green Treefrogs (Hyla cinerea).

The neuroendocrine mechanisms by which animals synchronise their physiological state with environmental cues are vital to timing life-history events appropriately. One important endocrine transducer of environmental cues in vertebrates is the pineal hormone melatonin, the secretion of which is directly sensitive to photoperiod and temperature. Melatonin modulates arginine vasotocin (AVT)-immunoreactive (-IR) cell number in the brain of green treefrogs (Hyla cinerea) during the summer breeding season, and this modulation is sexually dimorphic. In the present study, we investigated whether the influence of melatonin on vasotocin varies seasonally. We show that treatment of nonreproductive male green treefrogs with melatonin-filled silastic implants for 4weeks during the winter does not alter vasotocin-IR cell number in any brain region (i.e. nucleus accumbens, amygdala, preoptic area, suprachiasmatic nucleus or ventral hypothalamus). Taken together, these results suggest that the influence of melatonin on AVT is associated with sex and seasonal variation in melatonin receptor expression. We tested this hypothesis by using immunohistochemistry to characterise the distribution of melatonin receptor type 1 (MT1, also known as Mel1a) in the brain of reproductive and nonreproductive male and female frogs. We quantified MT1-IR cell number in regions known to contain AVT cell populations. Reproductive males had significantly more MT1-IR cells than nonreproductive males in all brain regions, including the combined nucleus accumbens, diagonal band of Broca and septum, striatum, amygdala, combined preoptic area and suprachiasmatic nucleus, as well as the ventral hypothalamus. In the accumbens region, where the effect of melatonin on AVT is known to be sexually dimorphic, males had significantly more MT1-IR cells than females during the summer breeding season. Based on these findings, we suggest that MT1 plays a role in mediating the interactions between melatonin and vasotocin that regulate seasonal and sexually dimorphic changes in sociosexual behaviour.

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.

Possible hormonal interaction for eliciting courtship behavior in the male newt, Cynops pyrrhogaster

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7α-hydroxypregnenolone (7α-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7α-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7α-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results.

Arginine vasotocin induces calling behavior with a female social stimulus and interacts with gonadotropins to affect sexual behaviors in male Xenopus tropicalis

Arginine vasotocin (AVT) and the mammalian homologue, arginine vasopressin (AVP), modulate vertebrate social behaviors, including vocalizations in male anurans. To study the impact of AVT and social stimuli on calling in male Xenopus tropicalis, we injected males with vehicle, 1μg, or 10μg AVT and recorded vocalizations under four social contexts (no stimulus, with male call playback, with a female, and with call playback and a female). More males called when injected with 10μg AVT. Furthermore, calling males called only when paired with a female. We identified four call types: long fast trill; short fast trill; slow trill; or click. Next, we injected males with vehicle, 10μg, or 20μg AVT and recorded vocalizations with or without a female. AVT treatment did not affect calling in this experiment, but we confirmed that more males, regardless of AVT treatment, called when a female was present. Then we evaluated the effect of human chorionic gonadotropin (hCG) on male sexual behavior. 20IU hCG elevated behavior compared to controls while the 10IU hCG treatment group was not different from either treatment. Last, we examined the effect of AVT on hCG-induced reproductive behavior. Males were injected with 10IU hCG or with 10IU hCG and 20μg AVT. Males receiving hCG and AVT clasped and called significantly more than males receiving hCG only. Our results suggest that AVT and a female stimulus induce vocalizations in a male pipid anuran, X. tropicalis, and the interaction between gonadotropins and neurohormones influences reproductive behaviors in this anuran amphibian.

Brain levels of nonapeptides in four labrid fish species with different levels of mutualistic behavior

There is strong evidence that brain nonapeptides are implicated as modulators of a wide array of social and reproductive behaviors in fishes. However, the question remains, as to whether there is a link between the distribution of active nonapeptides across brain regions and fishes specific behavioral phenotypes. To explore this link we compared the nonapeptides’ profile across the brains of fishes representing different degrees of mutualistic behavior (here: cleaning behavior). Herein we studied the quantitative distribution of both nonapeptides, arginine vasotocin (AVT) and isotocin (IT), in the brains of four species of fish belonging to the family Labridae: two are obligatory cleaners throughout their entire life (Labroides dimidiatus and Labroides bicolor), one species is a facultative cleaner (Labropsis australis; juveniles are cleaners and adults are corallivorous), and one is a non-cleaner species, corallivorous throughout its entire life (Labrichthys unilineatus). The biologically available AVT and IT concentrations were measured simultaneously in distinct brain macro-areas: forebrain, optic tectum, cerebellum and brain stem, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We showed that the levels of both AVT and IT varied significantly across species, as measured in the whole brain or in the specific macro-areas. Significantly higher AVT concentrations in the cerebellum which were found in the obligate cleaners seemed to be related to expression of mutualistic behavior. On the other hand, the higher levels of brain IT in the non-cleaner L. unilineatus suggested that these might be linked to the development of sexual dimorphism, which occurs only in this non-cleaner species.

Changes in vasotocin levels in relation to ovarian development in the catfish Heteropneustes fossilis exposed to altered photoperiod and temperature.

Photoperiod and temperature are the major proximate factors that activate the brain-pituitary-gonadal-endocrine axis stimulating gonadal recrudescence. Vasotocin (VT), the basic nonapeptide hormone, is secreted by the nucleus preopticus in the hypothalamus and released from the pituitary into circulation as a neurohormone for physiological actions. Additionally, VT is secreted de novo in the ovary of the catfish and has been implicated in ovarian functions. In the present study, we evaluated the changes in VT secretion during altered photoperiod and temperature exposure. The ovarian changes were monitored over gonadosomatic index (GSI) and plasma steroid hormone levels. Exposure of the catfish to long photoperiod (LP, 16L:08D) daily, alone or in combination with high temperature (HT, 28±2°C), for 14 or 28days resulted in a decrease in brain-pituitary VT level with a concomitant increase in plasma and ovarian VT levels. The changes were greater in the LP+HT group on day 28. Concurrently, the treatments stimulated the GSI and plasma estradiol-17β (E2), testosterone (T) and progesterone (P4) levels with higher more responses in the LP+HT group. Exposure of the catfish to short photoperiod (SP, 08L:16D) daily or total darkness (TD, 24L:00D) daily, with or without changing the ambient temperature, for 14 or 28days produced a depressing effect on VT, GSI and steroid hormone levels, the range of the response varied with the temperature. The brain VT level was low except in the TD+NT group. Plasma and ovarian VT levels decreased more in the SP and TD groups under ambient temperature than in the groups at the raised temperature. The GSI and plasma steroid hormones (E2, T and P4) responded in a similar manner. Plasma cortisol level registered a significant increase in all the groups compared to the initial control groups, and the increase was significantly higher on day 28. The simultaneous activation of VT secretion and ovarian recrudescence by photoperiod and temperature suggests the peptide's involvement in the hormonal control of gametogenesis.

The Need for Speed: Neuroendocrine Regulation of Socially-controlled Sex Change.

Socially-controlled functional sex change in fishes is a dramatic example of adaptive reproductive plasticity. Functional gonadal sex change can occur within a week while behavioral sex change can begin within minutes. Significant progress has been made in understanding the neuroendocrine bases of this phenomenon at both the gonadal and the neurobiological levels, but a detailed mechanistic understanding remains elusive. We are working with sex-changing wrasses to identify evolutionarily-conserved neuroendocrine pathways underlying this reproductive adaptation. One key model is the bluehead wrasse (Thalassoma bifasciatum), in which sex change is well studied at the behavioral, ecological, and neuroendocrine levels. Bluehead wrasses show rapid increases in aggressive and courtship behaviors with sex change that do not depend on the presence of gonads. The display of male-typical behavior is correlated with the expression of arginine vasotocin, and experiments support a role for this neuropeptide. Estrogen synthesis is also critical in the process. Female bluehead wrasses have higher abundance of aromatase mRNA in the brain and gonads, and estrogen implants block behavioral sex change. While established methods have advanced our understanding of sex change, a full understanding will require new approaches and perspectives. First, contributions of other neuroendocrine systems should be better characterized, particularly glucocorticoid and thyroid signaling. Second, advances in genomics for non-traditional model species should allow conserved mechanisms to be identified with a key next-step being manipulative tests of these mechanisms. Finally, advances in genomics now also allow study of the role of epigenetic modifications and other regulatory mechanisms in the dramatic alterations across the sex-change process.

Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: phylogeny and evolutionary considerations in teleosts.

Basic and neutral neurohypophyseal (NH) nonapeptides have evolved from vasotocin (VT) by a gene duplication at the base of the gnathostome lineage. In teleosts, VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT precursor genes of Heteropneustes fossilis and Clarias batrachus (Siluriformes, Ostariophysi) were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor sequences were obtained from EST database. The catfish NH sequences were used along with the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH gene loci in various teleost species was done to complement the phylogenetic analysis. In H. fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the deduced precursor proteins showed conserved characteristics of the NH nonapeptide precursors. The genes are expressed in brain and ovary (follicular envelope) of H. fossilis with higher transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance. The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication (3R) and subsequent diploidization in the NH loci. The VT and IT precursor genes are most likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the two superorders. The independent yet consistent retention of VT and IT in the two superorders might be directed by a stringent ligand-receptor selectivity.

Social Status and Arginine Vasotocin Neuronal Phenotypes in a Cichlid Fish

The nonapeptide arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin play a key role in the regulation of social behaviour across vertebrates. In teleost fishes, three AVT neuronal populations have been described in the preoptic area (POA): the parvocellular (pPOA), the magnocellular (mPOA) and the gigantocellular (gPOA). Neurons from each of these areas project both to the pituitary and to other brain regions, where AVT is supposed to regulate neural circuits underlying social behaviour. However, in the fish species studied so far, there is considerable variation in which AVT neuronal populations are involved in behavioural modulation and in the direction of the effect. In this study, the association between AVT neuronal phenotypes and social status was investigated in the Mozambique tilapia (Oreochromis mossambicus). This species is an African female mouth-brooding cichlid fish in which males form breeding aggregations in which dominant males establish territories and subordinate males to act as floaters. With respect to sex differences in AVT neuronal phenotypes, females have a larger number of AVT neurons in the pPOA and mPOA. Within males, AVT appeared associated with social subordination, as indicated by the larger cell body areas of AVT neurons in mPOA and gPOA nuclei of non-territorial males. There were also positive correlations between submissive behaviour and the soma size of AVT cells in all three nuclei and AVT cell number in the mPOA. In summary, the results provide evidence for an involvement of AVT in the modulation of social behaviour in tilapia, but it was not possible to identify specific roles for specific AVT neuronal populations. The results presented here also contrast with those previously published for another cichlid species with a similar mating system, which highlights the species-specific nature of the pattern of association between AVT and social behaviour even within the same taxonomic family.

Arginine vasotocin modulates associative learning in a mutualistic cleaner fish

Social environmental complexity induces structural and biochemical changes in animals’ brains, which are linked to the improvement of animals’ learning abilities. The nonapeptides from the arginine vasopressin (AVP) family (arginine vasotocin, AVT, in non-mammals) play a significant role in the regulation of social behavior, particularly in the formation of social memories and individual recognition. Moreover, the role of AVT in the regulation of interspecific interactions has only recently started to be addressed in the context of cleaner fish mutualisms and learning. Variance in the distribution of AVP receptor expression, which is linked to distinct neural systems (related to the dorsolateral and the dorsomedial telencephalon), is known to be implicated in differences in individual learning processes. Here we asked if the associative learning performance of the Indo-Pacific bluestreak cleaner wrasse (Labroides dimidiatus) is regulated by AVT. We tested the influence of AVT upon the cleaners’ ability to solve two different problems (cue and place discrimination tasks) that in principle differ in ecological relevance and are associated with two different memory systems. We found that AVT affected the learning competence of cleaners differently between tasks, as individual performance showed distinct response selectivity to AVT dosage levels. However, only in the ecologically relevant task was their learning response improved by blocking AVT via treatment with the antagonist Manning compound. Our findings demonstrate that AVT pathways, which are implicated in the regulation of interspecific behavior (i.e., a cleaner’s willingness to seek interactions with clients), are also linked to individual learning ability in the context of mutualistic behavior, and in tune with socio-ecological demands.

Anuran‐like Aquaporins in the Skin and Urinary Bladder of a Urodele, Evidence for Early Gene Duplication in the Lissamphibia

Amphibians are unique among vertebrates in that they obtain water primarily by absorption across their skin. In anurans this is mediated by specific aquaporins (AQPa2 homologs) that become inserted into the apical membrane of the outer most living cell layer in response to dehydration or treatment with arginine vasotocin (AVT). The urinary bladder epithelium also expresses an AQPa2 homolog to regulate the reabsorption of dilute urine when the animals are away from water. Recently, an ortholog of human AQP5 (AQP5a) has been identified in the bladder of the aquatic anurans Xenopus laevis and X. tropicalis that were exposed to dehydration. Here we report the identification of an AQP-a2-like aquaporin (AQP-nv3) in the skin and an AQP5a-like aquaporin in the urinary bladder of the urodele, Notopthalmus viridescens. These results suggest the gene duplication and co-optation of aquaporins to the skin and urinary bladder occurred early in the phylogeny of the lissamphia, prior to the breakup of the Pangea supercontinent. Supported by Grants-in Aid for Scientific Research (26440165) from the Ministry of Education, Culture, Sports, Science and Technology of Japan

Forebrain neuropeptide regulation of pair association and behavior in cooperating cleaner fish

Animals establish privileged relationships with specific partners, which are treated differently from other conspecifics, and contribute to behavioral variation. However, there is limited information on the underlying physiological mechanisms involved in the establishment of these privileged ties and their relationship to individual cooperation levels. The Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus often forages in mixed-sex pairs when cleaning fish clients. Intra-couple conflicts often arise during a joint client inspection, which may alter the overall quality of cleaning service provided. Here we tested two hypotheses: a) whether intra-pair association (i.e. association index), measured with joint interspecific cleaning and intraspecific behavior, is correlated with neuroendocrine mechanisms involving forebrain neuropeptides arginine vasotocin (AVT) and isotocin (IT) and b) whether these neuropeptide level shifts relate to an individual's interspecific service quality. We found that partner support (number of cleaning interactions and tactile stimulation) received by male cleaners increased with association index. When cleaners inspected clients alone, cleaners' cheating decreased with association index for females but not males. AVT levels did not differ according to sex or association level. Forebrain IT levels increased with association index for males, whereas no relationship was found for females. Finally, cleaner cheating varied between sex and forebrain IT levels. Findings indicate that variation in pairs' relationships influences male and female cleaner fish differently and contributes to the variation of brain neuropeptide levels, which is linked to distinct cooperative outcomes.

An essential role of the arginine vasotocin system in mate-guarding behaviors in triadic relationships of medaka fish (Oryzias latipes).

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.

Brain nonapeptide levels are related to social status and affiliative behaviour in a cooperatively breeding cichlid fish.

The mammalian nonapeptide hormones, vasopressin and oxytocin, are known to be potent regulators of social behaviour. Teleost fishes possess vasopressin and oxytocin homologues known as arginine vasotocin (AVT) and isotocin (IT), respectively. The role of these homologous nonapeptides in mediating social behaviour in fishes has received far less attention. The extraordinarily large number of teleost fish species and the impressive diversity of their social systems provide us with a rich test bed for investigating the role of nonapeptides in regulating social behaviour. Existing studies, mostly focused on AVT, have revealed relationships between the nonapeptides, and both social behaviour and dominance status in fishes. To date, much of the work on endogenous nonapeptides in fish brains has measured genomic or neuroanatomical proxies of nonapeptide production rather than the levels of these molecules in the brain. In this study, we measure biologically available AVT and IT levels in the brains of Neolamprologus pulcher, a cooperatively breeding cichlid fish, using high performance liquid chromatography with fluorescence detection. We found that brain AVT levels were higher in the subordinate than in dominant animals, and levels of IT correlated negatively with the expression of affiliative behaviour. We contrast these results with previous studies, and we discuss the role the nonapeptide hormones may play in the regulation of social behaviour in this highly social animal.

Interrelationship between feeding level and the metabolic hormones leptin, ghrelin and obestatin in control of chicken egg laying and release of ovarian hormones

The aim of the present experiment is to examine the role of nutritional status, metabolic hormones and their interrelationships in the control of chicken ovarian ovulatory and secretory activity. For this purpose, we identified the effect of food restriction, administration of leptin, ghrelin 1-18, obestatin and combinations of food restriction with these hormones for 3days on chicken ovulation (egg laying) rate and ovarian hormone release. The release of progesterone (P), testosterone (T), estradiol (E) and arginine-vasotocin (AVT) by isolated and cultured ovarian fragments was determined by EIA. It was observed that food restriction significantly reduced the egg-laying rate, T, E and AVT release and promoted P output by ovarian fragments. Leptin, administrated to ad libitum–fed chickens, did not change these parameters besides promoting E release. Nevertheless, administration of leptin was able to prevent the effect of food restriction on ovulation, T and E (but not P or AVT) release. Ghrelin 1-18 administration to ad libitum–fed birds did not affect the measured parameters besides a reduction in P release. Ghrelin 1-18 administration prevented the food restriction–induced decrease in ovarian T, E and AVT, but it did not change P output or egg laying. Obestatin administrated to control chicken promoted their ovarian P, E and inhibited ovarian AVT release but did not affect egg laying. It was able to promote the effect of food restriction on P, T and AVT, but not E release or egg laying. Our results (1) confirm an inhibitory effect of food restriction on chicken ovulation rate; (2) shows that food restriction–induced reduction in egg laying is associated with a decrease in ovarian T, E and AVT and an increase in ovarian P release; (3) confirm the involvement of metabolic hormones leptin, ghrelin and obestatin in the control of chicken ovarian hormones output; and (4) the ability of metabolic hormones to mimic/antagonize or prevent/promote the effects of food restriction on both egg laying and ovarian hormones demonstrates that nutritional status can influence ovarian ovulatory and endocrine functions via changes in metabolic hormones.

Molecular cloning, sequencing and phylogeny of vasotocin receptor genes in the air-breathing catfish Heteropneustes fossilis with sex dimorphic and seasonal variations in tissue expression.

Vasotocin (VT) is the ortholog of vasopressin (VP) in non-mammalian vertebrates and is known for multiple functions. Teleost fishes have a complete repertoire of known VP/VT receptor subtypes (vasopressin type, VR): two V1A subtypes (V1Aa and V1Ab or V1a1 and V1a2) and five V2 subtypes (V2A1, V1A2, V2B1, V2B2 and V2C). Full-length cDNAs of v1a1, v1a2 and v2 (v2a1) with ORFs of 1,308, 1,137 and 1,527 bp, respectively, were cloned and characterized in the catfish Heteropneustes fossilis (Siluriformes, Ostariophysi). BLAST analysis revealed that the genes encoded three VT receptors, V1a1, V1a2 and V2 of 436, 379 and 509 amino acid residues, respectively. The predicted proteins showed typical features of the seven-transmembrane domain receptor core structure with hallmark triplets Asp-Arg-Tyr/Asp-Arg-His (DRY/DRH) and the variable intracellular loop III of vertebrate neurohypophysial hormone receptors. Phylogenetic analysis of the deduced protein sequences revealed that they clustered with the V1Aa, V1Ab and V2A1, respectively, of other teleosts. The V2R has a sequence identity of 70-76% with V2A1 than with the V2B type (sequence identity 43-49%). Semiquantitative PCR analysis showed that the receptor gene transcripts were expressed ubiquitously in the tissues examined (brain, pituitary, gonads, liver, muscle, kidney and gills) and displayed sex and seasonal fluctuations in a tissue-specific manner. The results form a basis for functional studies on the VT receptors in the catfish.

AVT and IT regulate ion transport across the opercular epithelium of killifish (Fundulus heteroclitus) and gilthead sea bream (Sparus aurata)

The regulatory role of arginine vasotocin (AVT) and isotocin (IT) in Cl− secretion was investigated with the short circuit current (Isc) technique in opercular epithelia of killifish (Fundulus heteroclitus) and gilthead sea bream (Sparus aurata). Sea bream operculum showed ~4-fold lower number of Na/K-ATPase immunoreactive cells and ~12-fold lower secretory current than the killifish. In sea bream opercular membranes, the basolateral addition of AVT (10−6M) significantly stimulated Cl− secretion, while IT (10−6M) was without effect. In killifish, IT produced an immediate dose-dependent stimulation of Cl− secretion with significant effect at doses ≥10−7M and stimulation maxima (∆Isc ~25μA⋅cm−2) at 10−6M. The basolateral addition of bumetanide (200μM) abolished >75% of the effect of IT on Cl− secretion. In turn, AVT had a dual effect on killifish opercular Isc: an immediate response (~3min) with Isc reduction in an inverted bell-shaped dose–response manner with higher current decrease (−22μA⋅cm−2) at 10−8M AVT, and a sustained dose-dependent stimulation of Cl− secretion (stable up to 1h), with a threshold significant effect at 10−8M and maximal stimulation (~20μA⋅cm−2) at 10−6M. Both effects of AVT appear receptor type specific. The V1-receptor antagonist SR 49059 abolished Isc reduction in response to AVT, while the specific V2-receptor antagonist (Tolvaptan, 1μM) abolished the stimulatory action of AVT on Cl− secretion. According to these results, we propose a modulatory role for AVT and IT in Cl− (NaCl) secretion across the opercular epithelium of marine teleost.

Brain nonapeptide and gonadal steroid responses to deprivation of heterosexual contact in the black molly.

ABSTRACTFish may respond to different social situations with changes in both physiology and behaviour. A unique feature of fish is that social interactions between males and females strongly affect the sexual characteristics of individuals. Here we provide the first insight into the endocrine background of two phenomena that occur in mono-sex groups of the black molly (Poecilia sphenops): masculinization in females and same-sex sexual behaviour, manifested by gonopodial displays towards same-sex tank mates and copulation attempts in males. In socially controlled situations, brain neurohormones impact phenotypic sex determination and sexual behaviour. Among these hormones are the nonapeptides arginine vasotocin (AVT) and isotocin (IT), counterparts of the well-known mammalian arginine vasopressin and oxytocin, respectively. To reveal potential hormone interactions, we measured the concentrations of bioactive AVT and IT in the brain, along with those of the sex steroids 17β-estradiol and 11-ketotestosterone in the gonads, of females, masculinized females, males displaying same-sex sexual behaviour and those who did not. These data were supplemented by morphological and histological analyses of the gonads. Correlations between brain nonapeptides and gonadal steroids strongly suggest a cross talk between hormonal systems. In the black molly, the masculinization process was associated with the production of brain AVT and gonadal steroids, whereas same-sex sexual behaviour involves both brain nonapeptides, but neither of the sex steroids. This study extends current knowledge of endocrine control of phenotypic sex and sexual behaviour in fish and for the first time links brain nonapeptides with the occurrence of male-male sexual behaviour in lower vertebrates.