Reproductive roles of the vasopressin/oxytocin neuropeptide family in teleost fishes.

The vertebrate nonapeptide families arginine vasopressin (AVP) and oxytocin (OXT) are considered to have evolved from a single vasopressin-like peptide present in invertebrates and termed arginine vasotocin in early vertebrate evolution. Unprecedented genome sequence availability has more recently allowed new insight into the evolution of nonapeptides and especially their receptor families in the context of whole genome duplications. In bony fish, nonapeptide homologues of AVP termed arginine vasotocin (Avp) and an OXT family peptide (Oxt) originally termed isotocin have been characterized. While reproductive roles of both nonapeptide families have historically been studied in several vertebrates, their roles in teleost reproduction remain much less understood. Taking advantage of novel genome resources and associated technological advances such as genetic modifications in fish models, we here critically review the current state of knowledge regarding the roles of nonapeptide systems in teleost reproduction. We further discuss sources of plasticity of the conserved nonapeptide systems in the context of diverse reproductive phenotypes observed in teleost fishes. Given the dual roles of preoptic area (POA) synthesized Avp and Oxt as neuromodulators and endocrine/paracrine factors, we focus on known roles of both peptides on reproductive behaviour and the regulation of the hypothalamic-pituitary-gonadal axis. Emphasis is placed on the identification of a gonadal nonapeptide system that plays critical roles in both steroidogenesis and gamete maturation. We conclude by highlighting key research gaps including a call for translational studies linking new mechanistic understanding of nonapeptide regulated physiology in the context of aquaculture, conservation biology and ecotoxicology.

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.

Melanins as biomarkers of ovarian follicular atresia in the catfish Heteropneustes fossilis: biochemical and histochemical characterization, seasonal variation and hormone effects.

Follicular atresia is a common feature of the vertebrate ovary that occurs at different stages of folliculogenesis and ovarian regression. It has physiological significance to maintain homeostasis and control fecundity, and ensure removal of post-ovulatory follicular remnants for preparing the ovary for the next cycle. Pigments appear late in the atretic process as indigestible waste formed out of the degradation of the oocytes, follicle wall and granulocytes. In the present study, pigment accumulation was demonstrated by Schmorl's and Perls' staining methods in the atretic ovarian follicles of Heteropneustes fossilis during follicular development and regression. Melanins were characterized spectrophotometrically for the first time in fish ovary. The predominant form is eumelanin, followed by pheomelanin and alkali-soluble melanin. Melanins showed significant seasonal variations with levels low in gonad resting phase, increasing to the peak in the post-spawning phase. The concentration of melanins increased time-dependently in post-ovulated ovary after human chorionic gonadotropin treatment. In the spawning phase, in vitro incubation of ovary slices with estradiol-17ß or dexamethasone for 8 or 16 h decreased both eumelanin and pheomelanin levels time-dependently. The alkali-soluble melanin showed a significant decrease only in the dexamethasone group at 16 h. The results show that melanin assay can be used as a biomarker of follicular atresia in fish ovary, natural or induced by environmental toxicants.

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.

Differential and reproductive stage-dependent regulation of vasotocin secretion by catecholamines in the catfish Heteropneustes fossilis.

Vasotocin (VT) is the basic nonapeptide hormone secreted by the neurohypophysis of non-mammalian vertebrates and is involved in the regulation of osmoregulation, metabolism, cardiovascular function, reproduction and behaviour. Among the reproductive function, VT is specifically implicated in final oocyte maturation, ovulation, oviposition/parturition in teleosts, amphibians, reptiles, and birds. The central catecholaminergic system is involved in the regulation of pituitary hormone secretion including gonadotropin, and mediates also changes in environmental photoperiod and temperature. The close apposition of the VT and catecholaminergic systems in the preoptic area of the hypothalamus signifies a strong possibility of their functional interaction. The aim of the present study was to investigate the effects of exogenously administered catecholamines on VT secretion in two different reproductive phases of female catfish Heteropneustes fossilis. For this, the catecholamine precursor L-dihydroxyphenylalanine (L-DOPA) and catecholamines (dopamine-DA, norepinephrine-NE, and epinephrine-E) were intraperitoneally injected in normal catfish and/or along with α-methylparatyrosine (α-MPT, a tyrosine hydroxylase inhibitor). Brain and plasma VT levels were measured by specific ELISA, 24h post injection. Both L-DOPA and DA inhibited brain and plasma VT levels in a concentration-dependent manner in preparatory and prespawning phases. In contrast, NE elicited dose-dependent effects: the lowest dose (0.5 ng/g body mass, BM) was ineffective, the median dose (1 ng/g BM) stimulated, and the high doses (10 and 100 ng/g BM) inhibited VT levels. E stimulated VT levels dose-dependently. A single injection of α-MPT (250 µg/g BM) strongly inhibited VT when given alone and enhanced the inhibitory effects of L-DOPA and DA in the combination groups. The α-MPT inhibition of VT was significantly reduced by the injection of NE (5 ng/g BM) and was restored or elevated by E. When the adrenergic neurotransmitters were given together with α-MPT, the inhibitory effect of the latter was abolished and VT levels were significantly elevated. Thus, the present data indicate that the physiological changes in VT is differentially regulated by the catecholamines (DA inhibits and NE/E stimulates VT).

Risperidone ameliorates post-traumatic stress disorder-like symptoms in modified stress re-stress model.

The management for post-traumatic stress disorder (PTSD) involves chronic administration of drugs. We have modified the stress re-stress (SRS) model to study the effect of chronic administration of risperidone (RIS) after induction of PTSD in rats. On day-1 (D-1) rats underwent training session for elevated-plus maze (EPM) test. On D-2, rats were subjected to stress protocol of 2 h restraint and 20 min forced-swim test (FST) followed by halothane anesthesia. The rats were exposed to re-stress (FST) on D-8 and at six day intervals on D-14, D-20, D-26 and D-32. The rats were treated with RIS (0.01, 0.1 and 1.0 mg/kg; p.o.) and standard drug, paroxetine (PAX; 10.0 mg/kg; p.o.) from D-8 to D-32. RIS (0.1 mg/kg) and PAX ameliorated SRS-induced immobility. RIS in median dose reversed SRS-induced hypocorticosteronemia both in urine and plasma. RIS in median dose improved SRS-induced behavioral perturbations such as memory impairment and anxiety-like behavior in EPM and Y-maze tests. RIS (0.1 mg/kg) reversed SRS-induced increase in amygdalar serotonin level. RIS (0.1 mg/kg) increased the expression of hippocampal MR thereby reversing the SRS-induced decrease in MR/GR ratio. Pearson's analysis of data on D-32 showed that there was significant correlation of plasma corticosterone, amygdalar serotonin and hippocampal ratio of mineralocorticoid (MR)/glucocorticoid receptor (GR) with SRS-induced behavioral abnormalities. Hence, median dose of RIS shows anti-PTSD-like effect in the modified SRS model. PAX had earlier onset of action in ameliorating behavioral effects of PTSD compared to RIS. However, RIS showed anti-PTSD like effect in sub-therapeutic dose. The mode of anti-PTSD action of RIS seems to involve the HPA-axis and serotonergic system, whereas PAX did not show any significant action on these pathways. The effect of repeated treatment of drugs for PTSD can be evaluated using the modified SRS model.

Estrogen regulation of brain vasotocin secretion in the catfish Heteropneustes fossilis: an interaction with catecholaminergic system.

Vasotocin (VT) is a basic neurohypophysial nonapeptide in non-mammalian vertebrates and is involved in diverse functions like osmoregulation, reproduction, metabolism and behavior. In this study, we report that estradiol-17ß (E(2)) regulates brain and plasma VT secretion through the involvement of the catecholaminergic (CA) system. To demonstrate this, E(2) level was altered through ovariectomy (OVX, 3 weeks) and replacement study with low and high E(2) doses (0.1 and 0.5 µg/g body weight). CA activity was inhibited by treatment with α-methylparatyrosine (α-MPT; 250 µg/g body weight), a competitive inhibitor of tyrosine hydroxylase. VT was assayed by an enzyme immunoassay method. In the sham group, the low E(2) dose produced 82% and 104% increase, respectively, in brain and plasma VT levels. The high E(2) dose decreased the VT levels significantly. The low E(2) dose decreased brain E(2) but elevated plasma E(2). In the high E(2) group, the E(2) level increased further in both brain and plasma. OVX resulted in a significant inhibition (69% and 25%, respectively) of both brain and plasma VT, which was correlated with low E(2) levels. The low E(2) dose not only reversed the inhibition, but increased the VT level in both brain and plasma in comparison to the sham groups. The high E(2) replacement inhibited VT levels further low in both brain and plasma. The α-MPT treatment inhibited VT levels significantly in both sham and OVX groups. The drug treatment abolished partially the restorative effect of the low E(2) dose in the ovariectomized fish. In the high E(2) dose group, α-MPT decreased brain and plasma VT levels further low compared to the sham + 0. 5 µg E(2) group or OVX + 0.5 µg E(2) group except the brain VT level, which increased in the OVX+0.5 µg E(2) group. It is inferred that E(2) may exert biphasic effects on VT through the mediation of the CA system.

Estradiol-17ß modulates dose-dependently hypothalamic tyrosine hydroxylase activity inhibited by α-methylparatyrosine in the catfish Heteropneustes fossilis.

The brain is a target for organizational and activational effects of oestrogens synthesized de novo or transported from the peripheral organs. A neuroprotective role of oestrogens has been documented in a variety of vertebrates. In the present study in the catfish Heteropneustes fossilis, we have demonstrated that estradiol-17ß (E(2)), the major circulating oestrogen at low dosages (0.05 and 0.1 µg/g body weight of fish for 3 days) stimulated hypothalamic tyrosine hydroxylase (TH) activity, and countered the negative effects of ovariectomy (3-week) or α-methylparatyrosine (α-MPT: 250 µg/g body weight, a competitive inhibitor of TH). In contrast, high dosages of E(2) (1 and 2 µg/g body weight of fish for 3 days) were inhibitory and further amplified the inhibitory effects of ovariectomy and α-MPT. The inhibiting role of E(2) was higher in gonad-active (prespawning) phase than gonad-inactive (resting phase) phase. The dual roles of E(2) may ensure a tight regulation of catecholaminergic activity, activating and inhibiting the system against wide fluctuations that are characteristic of seasonally breeding animals.

Risperidone in ultra low dose protects against stress in the rodent cold restraint model by modulating stress pathways.

The present investigation evaluates the anti-stress activity of risperidone (RIS) in the cold restraint stress (CRS) model and related stress pathways. Rats were pretreated with RIS (0.1 and 1.0 mg/kg) for 21 days before subjecting to CRS. Ultra low dose of RIS (ULD; 0.1 mg/kg) in contrast to higher dose (1.0 mg/kg) significantly reduced stress in terms of ulcer index. ULD also reversed stress-induced increase in plasma corticosterone and norepinephrine levels used as markers for the function of hypothalamo-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS) respectively. ULD caused dose and brain region (hippocampus, prefrontal cortex and striatum) specific changes to stress-induced perturbations of serotonin, dopamine and its metabolites indicating modulation of brain monoaminergic system (BMS). ULD did not show any extrapyramidal side effects. Thus, the anti-stress effect ULD is probably mediated through the HPA axis, SNS and BMS. The study indicates a potential use of ULD in stress disorders.

Molecular and functional characterization of catfish (Heteropneustes fossilis) aquaporin-1b: changes in expression during ovarian development and hormone-induced follicular maturation.

The oocytes of the freshwater catfish Heteropneustes fossilis hydrate during hormone-induced meiotic maturation. To investigate if this process may be mediated by aquaporins (AQPs), as it occurs in marine fish producing highly hydrated eggs, the cloning of ovarian AQPs in catfish was carried out. Using degenerate primers for conserved domains of the major intrinsic protein (MIP) family, and 5' and 3'end amplification procedures, a full-length cDNA encoding for an AQP1-like protein was isolated. The predicted protein showed the typical six transmembrane domains and two Asn-Pro-Ala (NPA) motifs conserved among the members of the AQP superfamily. Phylogenetic analysis indicated that the catfish AQP clustered with the teleost-specific aquaporin-1b subfamily, and accordingly it was termed HfAqp1b. Heterologous expression in Xenopus laevis oocytes indicated that HfAqp1b encoded for a functional AQP, water permeability being enhanced by cAMP. Site-directed mutagenesis revealed that cAMP induced the translocation of HfAqp1b into the oocyte plasma membrane most likely through the phosphorylation of HfAqp1b Ser(227). In adult catfish, hfaqp1b transcripts were detected exclusively in ovary and brain and showed significant seasonal variations; in the ovary, hfaqp1b was maximally expressed during the pre-spawning period, whereas in the brain the highest expression was detected during spawning. In vitro stimulation of isolated catfish ovarian follicles with vasotocin (VT) or human chorionic gonadotropin (hCG), which induce oocyte maturation and hydration, elevated the hfaqp1b transcript levels after 6 or 16 h of incubation, respectively. These results suggest that HfAqp1b may play a role during VT- and hCG-induced oocyte hydration in catfish, and that VT may regulate HfAqp1b at the transcriptional and post-translational level in a manner similar to the vasopressin-dependent mammalian AQP2.