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The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHβ, FSHβ, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

In the catfish Heteropneustes fossilis, three nonapeptide hormone genes were identified in the brain preoptic area (POA) and ovary: a pro-vasotocin (pro-vt) and two isotocin gene paralogs viz., a novel pro-ita and conventional pro-itb. In the present study, the regulatory role of catecholamines [CA: dopamine (DA), noradrenaline (NA), adrenaline (AD)] on the expression of these genes were investigated in vitro. DA (1, 10, and 100 ng/mL) inhibited significantly the mRNA expression in both the POA and ovary. NA upregulated the POA mRNA expression in a biphasic manner, the lower concentrations (1 ng and 10 ng) scaled up and the higher concentration (100 ng) scaled down the expression of pro-vt and pro-itb, while only the 1 ng NA scaled up the pro-ita expression. In the ovary, NA upregulated the mRNA expressions at all concentrations; the pro-vt expression was stimulated only at 10 and 100 ng. AD stimulated pro-vt and pro-ita expression in the POA at all concentrations but the pro-itb expression was inhibited at 1 and 10 ng, and stimulated at 100 ng concentrations. In the ovary, AD elicited varied effects; no significant change in pro-vt, a stimulation of pro-ita, and an inhibition of pro-itb at 1 ng, and stimulation of pro-itb at the 10 and 100 ng. The incubation of the POA and ovary with α-methylparatyrosine (MPT, 250 µg/mL, a tyrosine hydroxylase inhibitor) for 8 h downregulated the mRNA expression in the POA but unaltered the expression in the ovary. Pre-incubation with MPT for 4 h, followed by co-incubation with DA, NA or AD for 4 h elicited varied effects. In the POA, the co-incubations with the CAs rescued the inhibition due to MPT. The MPT + DA and MPT + AD treatments reduced the magnitude of the inhibition of pro-vt and pro-itb by MPT. But the pro-ita expression was modestly stimulated in the MPT + AD group. On the other hand, the MPT + NA treatment rescued the MPT effect and elicited 10-folds increase in the expression levels. In the ovary, the changes were: an inhibition in the MPT + DA group, no significant alteration in the MPT + NA group, and a mild stimulation in the MPT + AD group. The results suggest that CAs modulate brain and ovarian nonapeptide gene expression differentially, which is important in the neuroendocrine/endocrine integration of reproduction in the catfish., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mennigen, Ramachandran, Shaw, Chaube, Joy and Trudeau.)

Heteropneustes fossilis is a facultative air-breathing freshwater catfish and inhabits ponds, ditches, swamps, marshes and rivers that dry up in summers. It possesses a pair of unique tubular accessory respiratory organ (air sac), which is a modification of the gill chamber and enables it to live in water-air transition zones. In the catfish, three vasotocin (Vt) receptor gene paralogs viz., v1a1, v1a2 and v2a were identified for Vt actions. In the present study, the receptor gene transcripts were localized in the gill and air sac by in situ hybridization, and their expression levels in relation to water and air deprivation conditions were investigated by quantitative RT-PCR. The catfish were exposed to 1 h and 2 h in gonad inactive (resting) and gonad active (prespawning) phases. The gene paralogs showed overlapping distribution in the respiratory epithelium of primary and secondary lamellae of gills and reduced lamellae of the air sacs. In water deprivation (forced aerial mode of respiration) experiment, v2a expression showed a high fold increase in the air sac, which was unchanged or inhibited in the gill. Both v1a1 and v1a2 expression was significantly upregulated in the air sac but showed varied responses in the gill. The gill v1a1 expression was unchanged in the resting phase and modestly upregulated in the prespawning phase. The gill v1a2 expression was modestly upregulated at 1 h in both phases but unchanged at 2 h. In the air deprivation experiment (forced aquatic respiration), the v2a expression in the air sac was inhibited except for a mild stimulation at 1 h in the prespawning phase. In the gill, the v2a expression was stimulated with a steep upregulation at 2 h in the prespawning phase. Both v1a1 and v1a2 expression was significantly high in the gill but only modestly increased or unchanged in the air sac. The expression patterns point to a functional distinction; the V2 type receptor expression was higher in the air sac during forced aerial respiration, and the V1 type receptor expression was highly prominent in the gill during forced aquatic respiration. Water and air deprivation treatments caused a significant increase in plasma cortisol level, and the stimulation was higher in the water deprivation fish in the resting phase but equally prominent in the water and air deprivation groups in the prespawning phase. The results indicate that the changes in the expression patterns of Vt receptor genes may be a sequel to stress (hypoxic, metabolic and osmotic), and both Vt and cortisol may interact to counter the stress responses. This study shows that Vt has a new role in the control of air sac functions., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Arginine vasotocin (VT) is the basic neurohypophysial nonapeptide hormone in teleosts. VT is also distributed in the ovary of the catfish Heteropneustes fossilis and induces final oocyte maturation (FOM) and ovulation by stimulating the maturation-inducing hormone (MIH). The present study reports the effects of cAMP (0.5 mM), phosphodiesterase inhibitors (IBMX -0.5 mM and theophylline- 0.5 mM), the inositol triphosphate (IP3) receptor inhibitor heparin (10 μg/mL) and the Ca 2+ chelator BAPTA-AM (25 μM) on VT (100 nM) - induced progestin stimulation, FOM and ovulation. Incubation of post-vitellogenic follicles with cAMP, IBMX and theophylline for 0, 8, 16 and 24 h stimulated basal secretion of progesterone (P 4 ), 17-hydroxyprogesterone (17-P) and 17, 20β-dihydroxy-4-pregnen-3-one (MIH) in a duration-dependent manner. The incubation of the follicles with heparin stimulated P 4 modestly, and 17-P and MIH levels in a duration-dependent manner. The incubation of the follicles with BAPTA-AM stimulated P 4 and MIH levels marginally and 17-P robustly. The stimulation was in the order cAMP > IBMX > theophylline > heparin > BAPTA-AM. The incubation of the follicles with VT stimulated P 4 , 17-P, MIH, GVBD and ovulation in a duration-dependent manner. The co-incubations with VT and the test compounds inhibited the VT-induced stimulation of P 4 , 17-P and MIH levels in a time-dependent manner in the order heparin > BAPTA-AM > cAMP > IBMX > theophylline. Concurrently, the VT-induced stimulation of GVBD and ovulation were also inhibited by the test compounds in the same order. The results show that VT induces FOM and ovulation preferentially acting through Ca 2+ pathway and a crosstalk between Ca 2+ and cAMP signaling pathways seems to integrate the processes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Estrogen hydroxylases (EHs) are cytochrome P450 Family 1 (Cyp1, Clan 2) proteins involved in estrogen hydroxylations at 2-, 4- or 16- carbon positions to form catecholestrogens. EHs are encoded by CYP1A1, CYP1A2 and CYP1B1 in mammals. In the catfish Heteropneustes fossilis, cyp1a1 and cyp1b1 cDNAs were cloned and characterized from liver and ovary. The cyp1a1 cDNA is 2071 bp long and codes for a 518 amino acids (aa) long protein. The cloned cyp1b1 cDNA is 1927 bp long and codes for a 509 residue protein. The deduced proteins clustered distinctly into teleost Cyp1a1 and Cyp1b1 clades, distinct from the tetrapod clusters and featured common function domains and homology with other teleost proteins. In the qPCR assay, the transcripts were the most abundant in the liver, followed by brain and ovary, and moderate in gill, kidney and muscle. Evidence was presented to show the involvement of the genes in reproduction. Expression of brain and ovarian transcripts showed significant seasonal variations with the highest abundance in the spawning phase. In situ hybridization showed the transcripts in the follicular layer (theca and granulosa) of the ovarian follicles. Periovulatory changes in the expression cyp1a1 and cyp1b1 were obtained during final oocyte maturation (FOM) and ovulation induced by human chorionic gonadotropin (hCG), both in vivo and in vitro, and by 2-hydroxyestradiol-17β (catecholestrogen) in vitro. In the brain, the transcript levels increased with time but in the ovary, the increase was maximal at 16 h and decreased at 24 h. The periovulatory activation of the cyp1 genes was reported in this study and discussed on the basis of complex regulation of FOM and ovulation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In seasonally breeding vertebrates, extrinsic factors like photoperiod and temperature are major determinants, controlling the annual reproductive cycle. In teleosts, kisspeptin, which occurs in two molecular forms: kisspeptin1 (Kiss1) and kisspetin2 (Kiss2), has been reported to alter gonadotropin (Lh and Fsh) secretion but its effect on gonadotropin-releasing hormone (Gnrh) secretion is not unequivocally proved. In the catfish Heteropneustes fossilis, we isolated and characterized kiss2 and gnrh2 cDNAs and the present work reports effects of altered photo-thermal conditions and melatonin (MT, a pineal hormone) on their expressions in the brain. The exposure of the catfish to long photoperiod (LP, 16 h light) at normal temperature (NT) or high temperature (HT, 28 °C) at normal photoperiod (NP) for 14 or 28 days stimulated both kiss2 and gnrh2 expression in both gonad resting and preparatory phases with the combination of LP + HT eliciting maximal effects. Short photoperiod (SP, 8 h light) under NT or HT altered the gene expression according to the reproductive phase and temperature. MT that mediates photo-thermal signals to the brain inhibited brain kiss2 and gnrh2 gene expression in the NP + HT, LP + NT, and SP + NT groups. The altered photo-thermal conditions elicited changes in steroidogenic pathway as evident from changes in plasma E 2 , progesterone, and testosterone levels. The results show that brain kiss2-gnrh2 signaling is involved in photo-thermal-mediated mechanisms controlling reproduction.

Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93-405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17β (E 2 ; 0.5 μg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E 2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E 2 -treated fish. Testosterone (T) decreased, and progesterone (P 4 ) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E 2 increased, and TMX, T and P 4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gonadotropin-releasing hormone 2 (Gnrh2) is one of the three classes of Gnrh distributed in vertebrates and is highly conserved. In the present study, the cDNA encoding Gnrh2 was isolated and characterized in the ostariophysan catfish Heteropneustes fossilis (hf). The cDNA is 611 bp long with an open reading frame (ORF) of 261 bp that encodes a highly conserved protein of 86 amino acids. The deduced Gnrh2 precursor protein clustered with the vertebrate Gnrh2 type. The sequence identity of hfgnrh2 is 94% with African catfish (Clarias gariepinus) gnrh2 mRNA (accession no. X78047). The hfgnrh2 transcripts were expressed only in the brain and gonads with a higher expression in the female brain and ovary in both resting and prespawning phases. The expression was higher in the prespawning phase than the resting phase. The gnrh2 expression in the brain and ovary showed significant seasonal variations but with opposite patterns. In the brain, the expression was the highest in the preparatory phase, decreased progressively to low levels in the postspawning and resting phases. In the ovary, the transcript level was low in the resting and preparatory phases, increased sharply in the prespawning phase reaching the peak level in the spawning phase and declined sharply in the postspawning phase. The gnrh2 mRNA showed the highest expression in the hind brain-medulla oblongata and moderate to low expression in forebrain regions and pituitary. Ovariectomy resulted in a duration-dependent inhibition of hfgnrh2 mRNA levels in the resting and prespawning phases. Steroid (E 2 , testosterone and progesterone) replacement treatments (0.5 μg/g body weight) in the 3- week ovariectomized fish restored the inhibition due to ovariectomy, elevated the expression over and above the sham level in the resting phase (E 2 group), and raised the levels almost to that of the sham group (testosterone and progesterone groups) in the prespawning phase. In the sham control groups, the steroid replacement resulted in a significant reduction in the mRNA levels. The expression of the gnrh2 mRNA in the brain-pituitary-gonadal axis and its regulation by gonadal steroids suggest that Gnrh2 may have a reproductive role in the catfish., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by immune tissues such as monocytes/macrophages and have pro-inflammatory/anti-inflammatory and neuroendocrine actions. In this study, we report the modulatory effects of stress hormones, the cortisol agonist dexamethasone and catecholamines on lipopolysaccharide (LPS) - induced stimulation of head kidney IL-6 in the catfish Heteropneustes fossilis. In the in vivo study, the intraperitoneal administration of LPS stimulated, and dexamethasone time-dependently inhibited IL-6 level. In the in vitro study, the incubation of macrophage cultures with LPS stimulated IL-6 level significantly in all incubation times. Dexamethasone did not alter the basal IL-6 level but inhibited time-dependently the LPS-induced stimulation. Likewise, catecholamines did not alter the basal level of IL-6. Both epinephrine and norepinephrine inhibited the LPS-induced stimulation of IL-6. Dopamine, on the other hand, was ineffective. The results indicate that IL-6 is a useful marker of head kidney macrophage activity for studying endocrine-immune interactions in the catfish., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the catfish Heteropneustes fossilis, three vasotocin (VT) receptor subtype genes, v1a1, v1a2, and v2a, were cloned and characterized previously. In the present study, using RNA probes, we localized the distribution of the gene transcripts in the brain-pituitary-gonadal (BPG) axis. The V1a-type receptor, v1a1 and v1a2, genes showed similar and overlapping distribution in the brain. The gene paralogs are distributed in the radial glial cells (RGCs) of the telencephalic ventricle and around the third ventricle in the hypothalamus and thalamus, olfactory tract, nucleus preopticus, nucleus lateralis tuberis, nucleus recessus lateralis and posterioris, nucleus saccus vasculosi, thalamic nuclei, habenular nucleus, habenular commissure, basal part of pineal stalk, accessory pretectal nucleus, optic tectum, corpus and valvula of the cerebellum, and facial and vagal lobes. The V2a receptor gene (v2a) has restricted distribution and is largely confined to the anterior subependymal region of the telencephalon. The localization pattern shows that the V1a-type receptors are distributed in major sensorimotor processing centers and the neuroendocrine/reproductive centers of the brain. In the pituitary, the receptor genes were localized differentially in the three divisions with the V1a-type receptor genes strongly expressed in the rostral pars distalis compared to the v2a paralog. In the ovary, the V1a-type receptor genes were localized in the follicular layer while v2a was localized in the oocyte membrane. In the testis, v1a2 and v2a are densely distributed in the interstitial tissue and seminiferous epithelium but the v1a1 is lowly expressed. The results suggest that the VT receptor genes have an extensive but differential distribution in the BPG axis. Future experimental studies are required to correlate the cellular localizations with specific functions of VT in the BPG axis.

The present study reports the molecular cloning of a previously uncharacterised neurohypophyseal nonapeptide precursor cDNA in two catfish species: Heteropneustes fossilis and Clarias batrachus. The deduced nonapeptide is CYISNCPVG ([V8] isotocin), which has not been reported in any vertebrate till date. Phylogenetic and conserved synteny analyses showed the gene to have originated from the isotocin precursor (pro-it) gene by fish-specific whole genome duplication (3R). The two isotocin lineages have been designated as pro-ita (new gene) and pro-itb (conventional it gene). All teleost groups may not possess both pro-ita and pro-itb and the pattern of losses/retention was found to be lineage-specific. Quantitative reverse transcriptase-polymerase chain reaction studies showed the expression of the pro-ita gene in the brain and ovary of H. fossilis. In situ hybridisation studies localised the pro-ita transcripts in the nucleus preopticus of the hypothalamus and the follicular layer (theca-granulosa) of oocytes, comprising tissues in which pro-itb and vasotocin precursor (pro-vt) mRNA expression was previously reported. The transcript levels varied with the reproductive stage and a high abundance was found in both brain and ovary during the breeding phase. The substitution of valine in place of isoleucine at the eighth position in Ita may have modified the ligand-receptor interaction, leading to sub-functionalisation and the retention of the gene in catfishes., (© 2018 British Society for Neuroendocrinology.)

This study was aimed to elucidate in vitro the effects of endogenous catecholamines: dopamine (DA), noradrenaline (NA) and adrenaline (A), and the β-adrenergic blocker propranolol on induction of final oocyte maturation (FOM) in the catfish Heteropneustes fossilis. With this aim, post vitellogenic follicles from sexually mature gravid female catfish were incubated with each of DA, NA and A in a concentration range of 5-250 μM, and propranolol in a concentration range of 1-200 μg/mL at time points varying from 0 to 30 h). Translucent follicles without germinal vesicle (GV) and opaque follicles with GV were scored separately for the calculation of percentage germinal vesicle breakdown (GVBD), an index of FOM. Data were analyzed by one-way ANOVA and were considered statistically significant when P values were less than 0.05. The analysis of the data showed that the incubation with NA only stimulated GVBD in a concentration - and time-dependent manner. Though the incubation with propranolol decreased total follicular cAMP level significantly at and above 10 μg/mL concentrations, a significant effect of the GVBD increase was noticed at 50 μg/mL or higher. However, the 10 μg/mL concentration of propranolol was effective to inhibit the NA-induced GVBD significantly albeit at a low level (39%). The present study suggests that final oocyte maturation is modulated by NA through a β-adrenergic mechanism, implicating a neural control of oocyte maturation and ovulation in teleosts., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the present study, in vitro effects of synthetic vasotocin (VT), isotocin (4Ser, 8Ile- oxytocin; ITb) and the recently cloned IT gene paralog product (8Val-Isotocin, ITa) were studied on the expression of pituitary gonadotropin (GtH) subunit mRNA levels. In male pituitaries of early (preparatory phase) and late (prespawning phase) recrudescing catfish, Heteropneustes fossilis, VT (10 nM, 100 nM and 1000 nM) stimulated fshβ expression dose-dependently. But in females, the dose-dependent effect was found only in the preparatory phase. In males, VT stimulated lhβ expression only at higher doses. In females, VT produced a significant dose-dependent increase of the lhβ expression only in the prespawning phase. VT stimulated the expression of gpα, dose-dependently in the preparatory phase in males and in the prespawning phase in females. The incubation of the pituitaries with ITb did not alter the fshβ expression in either sex in both preparatory and prespawning phases. In males, ITb stimulated the expression of lhβ and gpα only at the highest concentration (1000 nM) in both phases. In females, ITb stimulated both lhβ and gpα expression only at 1000 nM in the preparatory phase and dose-dependently in the prespawning phase. The incubation of the pituitaries with ITa produced effects similar to ITb on the expression of fshβ, lhβ, and gpα. The results show that the basic peptide VT modulates both fshβ and lhβ expressions, which are influenced by the sex and reproductive stage. The neutral peptide ITA/ITb exerts an insignificant effect on the fshβ expression regardless of sex or season. Both VT and ITa/ITb elicit a significant effect on the lhβ expression in late recrudescent phase especially in females., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The commercial fish spawning inducer Ovaprim (OVP) containing a salmon gonadotropin-releasing hormone analogue and domperidone (a dopamine receptor-2 antagonist) has been widely used as an effective spawning inducer in artificial breeding of fishes. It induces a preovulatory LH surge resulting in final oocyte maturation (FOM) and ovulation through a mechanism involving a steroidogenic shift to secrete a maturation-inducing steroid (MIS). In the present study, a 0.5μL/g body weight dose of OVP each injected at 0h and 24h intraperitoneally into gravid female catfish, Heteropneustes fossilis resulted in periovulatory changes in gonadotropin (GtH) subunit gene expression and steroid hormone levels. The OVP injections induced ovulation time-dependently from 6h onwards with 100% ovulation recorded from 24h to 48h. The fertilization rate was high from 6h to 18h and declined from 24h onwards. The OVP treatment up regulated the expression of GtH subunit genes differentially. The expression of glycoprotein-α (GPα) and luteinizing hormone (LHβ) peaked at 6h and 12h, and declined at 18h and 24h after the first injection. The second OVP injection at 24h elicited only a transient increase in the GPα expression at 6h and a sustained increase in the LHβ expression from 6h to 18h after the second injection, but both transcripts decreased subsequently. The follicle-stimulating hormone (FSHβ) expression responded to the OVP treatment from 12h onwards and maintained a constant level from 18h to 36h after the first injection; the second dose had little effect. Plasma steroids were differentially altered: the levels of estradiol-17β decreased while that of the MIS 17,20β-dihydroxy-4-pregnen-3-one; 17,20β-DP increased, causing the steroidogenic shift preceding FOM and ovulation. The present results indicate that LHβ expression coincides with the ovulation response and the late induction and maintenance of the FSH expression may be related to post-ovulatory events in the ovary., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Differential effects of photoperiod and temperature on the temporal modulation of gonadotrophin subunit genes (glycoprotein α, gpα), follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) expression were investigated in the stinging catfish Heteropneustes fossilis. Female H. fossilis were exposed to varying photoperiod and temperature conditions for 14 and 28 days in the early preparatory phase of the annual reproductive cycle. Gonadotrophin subunit gene expression, gonado-somatic index (I G ), ovarian histology and plasma steroid hormone levels were evaluated. The exposure of H. fossilis to long photoperiod (LP) of 16 h light or high temperature (HT) at 28 ± 2° C (mean ± s.e.), alone or in combination, resulted in significant increases in gpα, fshβ and lhβ messenger (m)RNA levels, I G , plasma oestradiol-17β (E 2 ), testosterone (T) and progesterone (P 4 ) levels. The ovaries were filled with advanced yolky oocytes. On the other hand, the short photoperiod (SP) of 8 h light exposure decreased the transcript levels with higher inhibition in the normal temperature (NT) group at 18 ± 2° C (mean ± s.e.) than the HT group at 28 ± 2° C. Furthermore, the inhibition reached the highest level in total darkness (TD) of 24 h light deprivation under NT conditions at 18 ± 2° C. Consequently, the SP and TD treatments inhibited the I G , plasma E 2 and T levels and ovarian development. The exposure to high temperature at 28 ± 2° C also modified the short photoperiod effect by elevating plasma E 2 level. The plasma T level changed only mildly while the plasma P 4 level showed the greatest fluctuations; the level reached the nadir in the SP + HT group but increased in the SP + NT group on day 28. A two-way ANOVA of the data showed differential effects of photoperiod and temperature; photoperiod produced a highly significant effect on fshβ expression while temperature had a highly significant effect both on lhβ and gpα levels. Thus, the differential expression of the gpα by the environmental variables ensures temporal synchronization of ovarian development and spawning., (© 2017 The Fisheries Society of the British Isles.)

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE 2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ovaprim (OVP), a commercial formulation of a salmon GnRH analogue and the dopamine receptor-2 blocker domperidone, is a successful spawning inducer for fish breeding. It induces a preovulatory surge in LH, which stimulates the synthesis of a maturation-inducing steroid (MIS, 17,20β-dihydroxy-4-pregnen-3-one) that initiates germinal vesicle breakdown (GVBD) and ovulation. Coincidently, the OVP treatment also stimulates vasotocin (VT) secretion in the brain and ovary of the catfish Heteropneustes fossilis that also stimulates the synthesis of the MIS. VT mediates its effect through V1- and V2-type receptors. In the present study in the catfish, we report that OVP stimulates the expression of VT receptor genes v1a1, v1a2 and v2a in the brain and ovary. A single intraperitoneal administration of OVP (0.5μL/g body weight) or incubation of post-vitellogenic ovarian follicles with 5μL/mL OVP, for 0, 4, 8, 12, 16, and 24h stimulated ovulation and GVBD, respectively, in a time-dependent manner. The OVP treatment in vivo stimulated brain VT receptor transcript levels 4h onwards. The peak expression was noticed at 12h (v1a1), 8 and 12h (v1a2), and 8, 12 and 16h (v2a), coinciding with FOM and ovulation. The VT receptor genes are expressed in the ovarian follicles compartmentally; both v1a1 and v1a2 are expressed in the isolated follicular layer (theca and granulosa) but absent in denuded oocytes. V2a is expressed in the denuded oocytes and not in the follicular layer. The OVP injection stimulated the v1a1 and v1a2 expression from 4h onwards in both intact follicle and isolated follicular layer, the peak expression was observed at 16h. The v2a expression was up-regulated in both intact follicles and denuded oocytes at 4h (denuded oocytes) or 8h (intact follicle) onwards with the peak expression at 12h and 16h (denuded oocytes) or at 16h (intact follicles). Under in vitro conditions, the OVP incubations elicited similar pattern of changes with the peak stimulation at 16h for all the genes. In conclusion, the VT receptor genes are differentially expressed in the ovarian follicles and OVP induced periovulatory stimulation of the VT receptor genes, coinciding with FOM and ovulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The article presents an overview of the comparative distribution, structure and functions of the nonapeptide hormones in chordates and non chordates. The review begins with a historical preview of the advent of the concept of neurosecretion and birth of neuroendocrine science, pioneered by the works of E. Scharrer and W. Bargmann. The sections which follow discuss different vertebrate nonapeptides, their distribution, comparison, precursor gene structures and processing, highlighting the major differences in these aspects amidst the conserved features across vertebrates. The vast literature on the anatomical characteristics of the nonapeptide secreting nuclei in the brain and their projections was briefly reviewed in a comparative framework. Recent knowledge on the nonapeptide hormone receptors and their intracellular signaling pathways is discussed and few grey areas which require deeper studies are identified. The sections on the functions and regulation of nonapeptides summarize the huge and ever increasing literature that is available in these areas. The nonapeptides emerge as key homeostatic molecules with complex regulation and several synergistic partners. Lastly, an update of the nonapeptides in non chordates with respect to distribution, site of synthesis, functions and receptors, dealt separately for each phylum, is presented. The non chordate nonapeptides share many similarities with their counterparts in vertebrates, pointing the system to have an ancient origin and to be an important substrate for changes during adaptive evolution. The article concludes projecting the nonapeptides as one of the very first common molecules of the primitive nervous and endocrine systems, which have been retained to maintain homeostatic functions in metazoans; some of which are conserved across the animal kingdom and some are specialized in a group/lineage-specific manner., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the catfish Heteropneustes fossilis, the anterior kidney is a hemopoietic tissue which surrounds the adrenal homologues, interrenal (IR) and chromaffin tissues corresponding to the adrenal cortical and adrenal medulla of higher mammals. The IR tissue is arranged in cell cords around the posterior cardinal vein (PCV) and its tributaries and secretes corticosteroids. The chromaffin tissue is scattered singly or in nests of one or more cells around the epithelial lining of the PCV or blood capillaries within the IR tissue. They are ferric ferricyanide-positive. Leukemia-inhibitory factor (LIF)-like reactivity was noticed in the lining of the epithelium of the IR cell cords and around the wall of the PCV and blood capillaries. No staining was observed in the hemopoietic cells. IL-1β- and TNF-α-like immunoreactivity was seen in certain cells in the hemopoietic tissue but not in the IR region. Macrophages were identified with mammalian macrophage-specific MAC387 antibodies and are present in the hemopoietic mass but not in the IR tissue. Pigments accumulate in the hemopoietic mass as melano-macrophage centers (MMCs) and are PAS-, Schmorl's- and Perls'-positive. The pigments contain melanin (black), hemosiderin (blue) and lipofuscin/ceroid (oxidized lipid, yellowish tan), as evident from the Perls' reaction. The MMCs were TUNEL-positive as evident from FITC fluorescence, indicating their apoptotic nature. The MMCs showed significant seasonal variation with their density increasing to the peak in the postspawning phase. Melanins were characterized spectrophotometrically for the first time in fish anterior kidney. The predominant form is pheomelanin (PM), followed by eumelanin (EM) and alkali-soluble melanin (ASM). Melanins showed significant seasonal variations with the level low in the resting phase and increasing to the peak in the postspawning phase. Under in vitro conditions, lipopolysaccharide (10 µg/mL) treatment increased significantly the levels of PM and EM levels both at 16 and at 32 h and the ASM level at 32 h. On the other hand, the synthetic glucocorticoid dexamethasone (100 nM) decreased significantly the levels of EM, PM and ASM time-dependently. The results indicate that the anterior kidney is an important site of immune-endocrine interaction.

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 28 days 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 28 days 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.

Gonadotropins are heterodimeric glycoproteins secreted by the pituitary, and consist of a common glycoprotein hormone alpha (GPα) and the function-specific follicle-stimulating hormone beta subunit (FSHβ) or luteinizing hormone beta subunit (LHβ). In the present study, the subunit protein genes were cloned and characterized from the pituitary of the catfish Heteropneustes fossilis. Full-length cDNAs of GPα, FSHβ, and LHβ are 511 base pairs (bp), 659 bp and 660 bp long, and encode 92, 108, and 112 aminoacids long mature proteins, respectively. GPα has 10 cysteines with 2 N-linked glycosylation sites while LHβ contains 12 cysteines with a single N-linked glycosylation site. In contrast, FSHβ has 13 cysteines, 1 additional over the conserved 12 cysteines of other vertebrates, and a single glycosylation site between Cys 3 and Cys 4. Phylogenetic analyses of the deduced proteins confirm their homology and relationships with the respective gonadotropin subunit proteins of gnathostome vertebrates. Tissue expression analysis by semi-quantitative RT-PCR shows that GPα mRNA is expressed only in the pituitary while both FSHβ and LHβ mRNA are expressed in extra-pituitary sites. The subunit mRNAs show both seasonal and sex dimorphic variations especially in the expression of FSHβ and LHβ transcripts. In the sexually quiescent phase, the transcript expression is low while in the recrudescent phase, the expressions are differential, high, and varied with regard to sex and reproductive phase. In situ hybridization of the mRNAs gave positive signals in gonadotropes in the pars distalis of the pituitary, which exhibited seasonal variation in staining intensity and numbers., (© 2015 Wiley Periodicals, Inc.)

Cytochrome P450 aromatase (Cyp19arom) is the rate-limiting enzyme controlling estrogen biosynthesis, coded by Cyp19a1 in most gnathostomes. Most teleosts have two forms expressed differentially in ovary (cyp19a1a) and neural tissue (cyp19a1b). In this study, full length cDNAs of 2006 bp and 1913 bp with ORFs of 1575 bp and 1488 bp were isolated from the brain and ovary, respectively, of the catfish Heteropneustes fossilis, an air-breathing species with high aquaculture potential. The ORFs encode predicted proteins of 495 and 524 amino acid residues, respectively. The proteins show 62% identity with each other and cluster in two distinct clades (the brain type and ovary type) in the teleost taxon, separated from the tetrapod type. In the in situ localization study, both cyp19a1a and cyp19a1b transcripts were localized in the brain but the signal intensity was higher for the brain type paralog. The transcript signals were observed in the radial glial cells and in neuronal populations of the dorso-lateral region of the telencephalon, pre-tectum, hypothalamus and medulla oblongata. In the ovary, both paralogs were expressed in the follicular layer with a high signal intensity of the ovarian type (cyp19a1a). The differential expression of the gene paralogs was evident from qPCR analysis. Cyp19a1b has relatively a high abundance in the female brain, followed by other peripheral tissues (gonads, liver, gill, kidney and muscle). On the other hand, cyp19a1a has relatively a high transcript abundance in the ovary and female brain, followed by the testis and male brain, and female liver and muscle. The expression was low in male liver and muscle, and the lowest in the gill and kidney. The expression of the two paralogs exhibit brain regional differences; both types have relatively a high transcript abundance in telencephalon-preoptic area with the cyp19a1b expression higher in females than males. In hypothalamus, the expression of both types is higher in males than females. In medulla, the expression of the cyp19a1b is higher than cyp19a1a, and the transcript abundance of the ovarian type is higher in females than males. The expression of the gene paralogs elicits significant seasonal variations in the ovary and brain. In both tissues, the expression increases from the resting to preparatory phases, and decreases through the prespawning phase to low levels in spawning phase. In vivo and/or in vitro treatments with human chorionic gonadotropin (hCG) stimulated the expression of the gene paralogs in the brain and ovary, time-dependently. In conclusion, both paralogs have an overlapping distribution at different levels of the brain-pituitary-gonad axis and may function as a single functional unit as far as the estrogen synthesis is concerned., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this article, the physiological role of ovarian vasotocin (VT) on fish final oocyte maturation (FOM) and ovulation is reviewed based on the studies mainly available in the catfish Heteropneustes fossilis. The VT system is characterized in the follicular layer of the oocytes by both immunocytochemical and in situ hybridization techniques. The distribution was confirmed in isolated follicular layer preparations by HPLC characterization and quantification. Three VT receptor subtype genes are identified: V1a1 and V1a2 subtypes are distributed in the follicular layer and V2 subtype is present along the granulosa-oocyte membrane junction. The expression of peptide, VT precursor gene and VT receptor genes shows seasonal and periovulatory changes in the ovary. VT secretion is modulated by E2 differentially in a season-specific manner, and by progestin steroids positively. VT modulates E2 in a biphasic manner in early recrudescent phase and induces a steroidogenic shift inhibiting E2 and stimulating progestin steroid (P4, 17P4 and 17,20β-DP) pathways in the late recrudescent phase. VT stimulates prostaglandin secretion, germinal vesicle breakdown (GVBD), oocyte hydration and ovulation. VT acts through different receptors to stimulate these processes. It uses the V1 type receptor to stimulate GVBD and ovulation, and the V2 type to stimulate oocyte hydration. VT acts as an important link in the cascade of gonadotropin control of FOM and ovulation. More research is required in other species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gonadotrophins (GTHs) play a central role in the regulation of gametogenesis and spawning. The structural duality of the GTHs [luteinising hormone (LH) and follicle-stimulating hormone (FSH)] is established in fishes with the exception of ancestral vertebrates. Most studies indicate that, in teleosts, the GTHs are secreted in separate cells. Phylogenetic analysis shows that the common α-subunit of the GTHs (and also of thyroid-stimulating hormone) and LHβ are highly conserved in fishes, as in tetrapods. However, FSHβ shows considerable divergence in teleosts. There may be 12 or 13 cysteine residues, with an additional one near the N-terminus. There may be one or two N-linked glycolsyation sites. In catfishes, there are 13 cysteine residues and one N-linked glycosylation site. In an extreme situation, a potential glycosylation site is lacking in some fishes. Both FSH and LH receptors are characterised in teleosts. The FSH receptor is promiscuous and can be cross-activated by LH. By contrast, the LH receptor is highly selective, being activated by its natural ligand or by heterologous ligands (e.g. human chorionic gonadotrophin). Consequently, teleosts show different patterns of LH and FSH secretion. In catfishes, in the absence of native FSH protein, LH controls all aspects of reproduction, from early gametogenesis to spawning., (© 2015 British Society for Neuroendocrinology.)

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-17β 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.

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.

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.

In the present study, α-methylparatyrosine (α-MPT), a tyrosine hydroxylase inhibitor was used to impair ovarian catecholaminergic activity in vitro. The consequent effects on catecholamine (CA) levels were correlated with follicular steroid production. l-dihdroxyphenylalanine (l-DOPA, the precursor of CA) and human gonadotropin (hCG) were supplemented to reverse the effect of α-MPT. The experiments were conducted in two reproductive phases, namely preparatory and pre-spawning phases in female catfish Heteropneustes fossilis. The incubation with α-MPT inhibited ovarian l-DOPA, dopamine (DA), norepinephrine (NE) and epinephrine (EP) levels and the l-DOPA supplementation compensated the inhibitory effect. The level of tyramine (TR) was increased by the α-MPT treatment but inhibited by the l-DOPA supplementation. α-MPT produced stage-specific (seasonal) effects on ovarian estradiol-17β (E2); in the preparatory phase, E2 was decreased significantly at both 12 and 24h and in the pre-spawning phase, the level was stimulated over the respective control groups. The changes were higher at 24h in both phases. l-DOPA and hCG increased the E2 level significantly in the preparatory phase and reversed the inhibitory effect of α-MPT in the co-incubation groups. In the pre-spawning phase, α-MPT-stimulated the E2 level compared to the control groups, which was reversed by l-DOPA, hCG, or by both, in co-incubations. In contrast, the α-MPT treatment decreased progesterone (P4), 17-hydroxyprogesterone and 17,20β-dihydroxy-4-prenen-3-one (17,20β-DP) in a duration-dependent manner while the co-incubations with l-DOPA, hCG, or by both, significantly reversed the inhibitory effect. These results suggest that ovarian CAs (DA, NE and EP) may exert differential and stage-specific effects on E2, inhibition in the preparative phase and stimulation in the pre-spawning phase. The progestin steroids appear to be stimulated by CAs. In conclusion, this study highlights a possible direct/causal functional interaction between CA activity and gonadotropin on steroidogenic activity, and that CAs may be involved in regulating temporal secretion of the hormones through causing the shift in steroidogenic pattern., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ovaprim (OVP) is used as an effective spawning inducer for artificial breeding of fishes and contains a salmon gonadotropin-releasing hormone analogue and a dopamine receptor-2 antagonist, domperidone. Previously, we have shown that vasotocin (VT) stimulates ovarian final oocyte maturation, hydration, and ovulation through a mechanism involving induction of a steroidogenic shift, favouring the production of a maturation-inducing hormone (MIH). In the present study, we demonstrated that OVP stimulated brain, plasma and ovarian VT levels, suggesting multiple sites of action, apart from its well established role in the induction of a preovulatory LH surge. An intraperitoneal injection of 0.5μL/g body weight of OVP for different time intervals (0, 4, 8, 12, 16 and 24h) induced ovulation as well as increased significantly brain and plasma VT levels in a time-dependent manner. Plasma steroids were differentially altered; the levels of estradiol-17β (E2) and testosterone (T) decreased, and the MIH (17, 20β-dihydroxy-4-pregnen-3-one; 17, 20β-DP) level increased time-dependently. In order to demonstrate whether OVP acts at the level of the ovary directly, in vitro experiments were conducted. The incubation of ovarian slices/follicles with OVP (1, 5 and 10μL/mL) for different time points (0, 4, 8, 12, 16 and 24h) induced germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. Ovarian VT increased significantly in a concentration- and time-dependent manner with a maximal increment at 16h. Ovarian T and E2 levels decreased concurrently with the rise in the MIH level, dose- and duration-dependently. The results show that OVP stimulates VT at the brain and ovarian level. The direct OVP-VT cascade has the potential to stimulate FOM and ovulation, sidelining the pituitary glycoprotein hormone (LH) surge., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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)., (© 2013.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Functional interactions between vasotocin (VT) and prostaglandins (PGs) in the regulation of final oocyte maturation (FOM) and ovulation were investigated in the catfish Heteropneustes fossilis. Incubation of post-vitellogenic follicles with VT resulted in significant increases of both PGF2α and PGE2 at 8 and 16h intervals. The rise was higher at 16h except in the 1000nM VT group, in which the PG levels decreased compared to the 100nM group (biphasic effect). VT was more effective to increase the PG levels in comparison to hCG or IT. The co-incubation of the follicles with both hCG (20IU/ml) and VT (100nM) increased significantly PGF2α level at 8h, higher than that elicited by each when incubated alone. Pre-incubation of the follicles with V1 receptor antagonist, alone or in co-incubation with VT, significantly inhibited the VT-stimulated PGF2α and PGE2 levels. Under similar conditions, V2 receptor antagonist did not affect the PGE2 levels. Both VT (100nM) and PGs stimulated FOM (germinal vesicle breakdown) and ovulation in a dose- and duration dependent manner, PGF2α was more effective. Incubation of postvitellogenic follicles with indomethacin (a non selective cyclooxygenase inhibitor) per se did not affect FOM and ovulation but significantly decreased VT and PG effects upon pre-incubation. The results suggest that the VT stimulation of PGs may be mediated mainly through the V1 receptor though the involvement of V2 receptor cannot be excluded. The article also discussed the positive interplay of gonadotropin, maturation-inducing steroid, VT and PG during FOM and ovulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Alkylphenol polyethoxylates (APEs), which are widely used in detergents, paints, herbicides, insecticides, and in many other formulations, have been widely detected in aquatic environments. 4-Nonylphenol (NP) is an important APE detected at microgram levels per litre (0.1-336 μg/L) in water. The objective of the present study was to evaluate NP's toxic effects at low and high sublethal concentrations (0.1 and 1 μg/L) on embryonic development of the catfish Heteropneustes fossilis at different time intervals. The data show that fertilization rate was decreased and cleavage and blastula were severely affected leading to complete mortality of embryos. NP exposure resulted in various body malformations in larvae, such as vertebral deformations, e.g., fin blistering/necrosis, axial deformities (lordosis, kyphosis, and scoliosis) of the spine in the abdominal and caudal region, tail curved completely backward, shortened body, severe spinal and yolk sac malformations, C-shaped severe spinal curvature, cranial malformation with undeveloped head, and failure of eye development. The level of body malformations increased with the concentration and exposure time. After 72 h of exposure, all larvae were dead at both concentrations. Scanning electron microscope study showed that epidermal cells (keratinocytes) were severely damaged in both low- and high-dose treatments throughout development, leading to development of numerous depressions representing sinking holes on the skin. Mucous glands increased significantly in treatment groups compared with control groups. The present study highlights the severe teratogenic effects of NP. The prevalence of the contaminant, if not checked, can lead to decreased population and ultimate disappearance of the species.

Seasonal, periovulatory and 2-hydroxyestradiol-17β (2-OHE(2))-induced changes on ovarian prostaglandin (PG) E(2) and F(2α) were investigated under in vivo or in vitro in the female catfish Heteropneustes fossilis. Both PGE(2) and PGF(2α) increased significantly during ovarian recrudescence with the peak levels in spawning phase. The PGs showed periovulatory changes with the peak levels at 16 h after the hCG treatment. Incubation of postvitellogenic ovary fragments with estradiol-17β (E(2)), 2-OHE(2) or 2-methoxyE(2) produced concentration-dependent increases in PG levels; 2-OHE(2) was more effective. In order to identify the receptor mechanism involved in the 2-OHE(2)-induced PG stimulation, the ovarian pieces were incubated with phentolamine (an α-adrenergic antagonist), propranolol (a β-adrenergic antagonist) or tamoxifen (an estrogen receptor blocker) alone or in combination with 2-OHE(2). The incubation of the tissues with the receptor blockers alone did not produce any significant effect on basal PG levels. However, co- and pre-incubation of the tissues with the blockers resulted in inhibition of the stimulatory effect of 2-OHE(2) on the PGs. Phentolamine was more effective than propranolol. The signal transduction pathway(s) involved in the 2-OHE(2)-induced PG secretion was investigated. The incubation of the ovarian pieces with 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor), chelerythrine (a protein kinase C inhibitor) and PD098059 (a mitogen-activated protein kinase inhibitor) significantly lowered the basal secretion of PGF(2α) and PGE(2). In contrast, H89 (a protein kinase A inhibitor) increased the basal secretion of PGs at 1 and 5 μM concentration and decreased it at 10 μM concentration. The co- or pre-incubation with IBMX, H89, chelerythrine and PD098059 significantly inhibited the stimulatory effect of 2-OHE(2) on PGF(2α) and PGE(2) levels. The inhibition was higher in the pre-incubation groups. Chelerythrine was the most effective followed by PD098059, IBMX and H89. The results suggest that 2-OHE(2) may employ both adrenergic and estrogen receptors, or a novel receptor mechanism having properties of both adrenergic and estrogen receptors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ovarian pieces containing postvitellogenic follicles were incubated in vitro with different concentrations of the catecholestrogen 2-hydroxyestradiol-17β (2-OHE(2)) to evaluate its effects on steroid production and germinal vesicle breakdown (GVBD) in the catfish Heteropneustes fossilis. The incubation with 2-OHE(2) induced a shift in steroidogenic pattern: the C(19) and C(18) steroids testosterone (T) and estradiol-17β (E(2)), respectively were significantly decreased with a concomitant significant increase in the C(21) steroids progesterone (P(4)), 17-hydroxyprogesterone (17-OHP), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-DP), 17,20α-dihydroxy-4-pregnen-3-one (17,20α-DP) and cortisol (F). Concomitantly, the catecholestrogen induced dose-dependently GVBD response, the first sign of meiosis resumption. The co- and pre-incubations of the ovarian pieces with 2-OHE(2), and adrenergic (phentolamine, α-blocker and propranolol, β-blocker) or estrogen (tamoxifen) receptor blockers resulted in inhibition of the stimulatory effect of the catecholestrogen on C(21) steroids and reversed the inhibition of testosterone and E(2). The α-blocker was more effective than the β-blocker. Our results suggest that 2-OHE(2) appears to employ both adrenergic (α-type) and estrogen receptor mechanisms in mediating the effects. The co- or pre-incubation of ovarian pieces with IBMX (a cAMP elevating drug), H89 (a protein kinase A inhibitor), and PD098059 (a MAP kinase kinase inhibitor) significantly inhibited the stimulatory effect of 2-OHE(2) on the C(21) steroids. The effect of chelerythrine (a protein kinase C inhibitor), on the other hand, varied with the incubation condition. In the co-incubation, the steroids showed varied effects: 17,20β-DP, testosterone and E(2) were elevated, and P(4) and 17-OHP were decreased. In the pre-incubation set up, all the steroids were inhibited except E(2). The inhibition by the blockers was higher in the pre-incubation groups. Taken together, the data suggest the involvement cAMP-protein kinase A, protein kinase C and MAP kinase pathways in the modulation of the steroidogenic activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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-17β (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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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-17β (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.

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.

The study reports for the first time vasotocin (VT) induction of final oocyte maturation and ovulation through the production of the maturation-inducing steroid 17, 20β-dihydroxy-4-pregnen-3-one (MIS, 17, 20β-DP). Post-vitellogenic follicles of the catfish Heteropneustes fossilis were incubated with different concentrations of VT (1, 10, 100 and 1000 nM) for different time periods. Germinal vesicle breakdown [GVBD, as a marker of final oocyte maturation (FOM)] and ovulation were scored. In another series of experiments, the follicles were incubated with VT alone or in combination with VT receptor (V(1) and V(2)) antagonists, and GVBD and ovulation were increased with progesterone, 17-hydroxy-4-pregnene-3, 20-dione (17-P) and 17, 20β-DP levels. VT stimulated both GVBD and ovulation in a concentration and time-dependent manner, and the responses were inhibited to varying degrees in groups incubated with the VT receptor antagonists. The V(1) antagonist inhibited the responses by 2- to 3-fold and more than the V(2) antagonist, and the combination was more potent than the separate incubation. Progestins increased time-dependently in the VT groups and the fold increase was greater for the MIS. The VT-induced steroid stimulation was significantly inhibited to near the control levels in co-incubations with both V(1) and V(2) receptor antagonists, in the order 17, 20β-DP > 17-P > P(4). The inhibition by the V(1) receptor antagonist was greater than that with the V(2) blocker, and followed the same order of inhibition described above. The results suggest that VT induces FOM and ovulation mainly through the V(1) receptors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neonatal asphyxia is a primary contributor to neonatal mortality and neuro-developmental disorders. It progresses in two distinct phases, as initial primary process and latter as the secondary process. A dynamic relationship exists between excitotoxicity and mitochondrial dysfunction during the progression of asphyxic injury. Study of status of glutamate and mitochondrial function in tandem during primary and secondary processes may give new leads to the treatment of asphyxia. Neonatal asphyxia was induced in rat pups on the day of birth by subjecting them to two episodes (10min each) of anoxia, 24h apart by passing 100% N(2) into an enclosed chamber. The NMDA antagonist ketamine (20mg/kg/day) was administered either for 1 day or 7 days after anoxic exposure. Tissue glutamate and nitric oxide were estimated in the cerebral cortex, extra-cortex and cerebellum. The mitochondria from the above brain regions were used for the estimation of malondialdehyde, and activities of superoxide dismutase and succinate dehydrogenase. Mitochondrial membrane potential was evaluated by using Rhodamine dye. Anoxia during the primary process increased glutamate and nitric oxide levels; however the mitochondrial function was unaltered in terms of succinate dehydrogenase and membrane potential. Acute ketamine treatment reversed the increase in both glutamate and nitric oxide levels and partially attenuated mitochondrial function in terms of succinate dehydrogenase activity. The elevated glutamate and nitric oxide levels were maintained during the secondary process but however with concomitant loss of mitochondrial function. Repeated ketamine administration reversed glutamate levels only in the cerebral cortex, where as nitric oxide was decreased in all the brain regions. However, repeated ketamine administration was unable to reverse anoxia-induced mitochondrial dysfunction. The failure of glutamate antagonism in the treatment of asphyxia may be due to persistence of mitochondrial dysfunction. Therefore, additionally targeting mitochondrial function may prove to be therapeutically beneficial in the treatment of asphyxia., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cell lines provide an important biological tool for carrying out investigations into physiology, virology, toxicology, carcinogenesis and transgenics. Teleost fish cell lines have been developed from a broad range of tissues such as ovary, fin, swim bladder, heart, spleen, liver, eye muscle, vertebrae, brain, skin. One hundred and twenty-four new fish cell lines from different fish species ranging from grouper to eel have been reported since the last review by Fryer and Lannan (J Tissue Culture Methods 16: 87-94, 1994). Among the cell lines listed, more than 60% were established from species from Asia, which contributes more than 80% of total fish production. This includes 59 cell lines from 19 freshwater, 54 from 22 marine and 11 from 3 brackish water fishes. Presently, about 283 cell lines have been established from finfish around the world. In addition to the listing and a scientific update on new cell lines, the importance of authentication, applications, cross-contamination and implications of overpassaged cell lines has also been discussed in this comprehensive review. The authors feel that the review will serve an updated database for beginners and established researchers in the field of fish cell line research and development.