Browsing by Author "R. Chaube"

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Air sac and gill vasotocin receptor gene expression in the air-breathing catfish Heteropneustes fossilis exposed to water and air deprivation conditions
(Springer Science and Business Media B.V., 2022) A. Rawat; R. Chaube; K.P. Joy
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.
An in vitro study on catecholamine modulation of ovarian steroidogenic activity in the catfish Heteropneustes fossilis
(Academic Press Inc., 2014) K.P. Joy; V. Singh; R. Chaube
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. © 2013 Elsevier Inc.
Annual and periovulatory changes in tyrosine hydroxylase activity in the ovary of the catfish Heteropneustes fossilis
(Academic Press Inc., 2010) T.K. Chourasia; R. Chaube; V. Singh; K.P. Joy
In the present study, tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine synthesis) activity was demonstrated in the ovary of the catfish to elucidate the possible physiological role of catecholamines in the gonad. The ovary is innervated by seven pairs of nerves, originating from the paired sympathetic chain lying dorsal to the posterior kidney. Ovarian TH activity showed a significant annual variation (P< 0.001, one-way ANOVA), correlating with gonado-somatic index. Activity was low from December to February (resting phase), increased from March to July (recrudescent phase) and then decreased in post-spawning phase (August-November). The annual pattern was similar to that of the brain. An intraperitoneal injection of 100. IU hCG/fish induced significant periovulatory changes in TH activity with the peak rise at 16 h, and the activity decreased after egg-stripping (P<0.001, one-way ANOVA). Brain TH activity showed similar periovulatory changes. The results suggest that catecholamine synthesis is increased during both ovarian recrudescence and spawning of the annual reproductive cycle, implying a functional role in ovarian growth, maturation and ovulation. © 2009 Elsevier Inc.
Brain steroid contents in the catfish Heteropneustes fossilis: Sex and gonad stage-specific changes
(2012) R. Chaube; S. Mishra
Neurosteroids are those which are synthesized in the central nervous system independently of supply by peripheral endocrine glands. In the present study, brain contents of the steroid hormones, estradiol-17β (E 2), testosterone (T), corticosteroids, and progestins were investigated in both male and female catfish Heteropneustes fossilis in prespawning (vitellogenic) and spawning (post-vitellogenic) phases using ELISA or HPLC. The data show that the measured steroid hormones showed both stage-specific and sex-related variations. Brain E 2 was significantly higher in males in the prespawning phase and in females in the spawning phase. Testosterone was significantly higher in males in comparison with females in the prespawning phase. Cortisol was significantly higher in the prespawning and spawning phases in males than in females. Corticosterone level was low in the brain. 21-deoxycortisol and deoxycorticosterone were significantly higher in the prespawning phase than in the spawning phase. Male brain recorded the highest concentration of deoxycorticosterone. Progesterone (P 4) was high in the prespawning phase and low in the spawning phase in both sexes. Levels of 17-hydroxy-4-pregnene-3,20-dione and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-DP) and the metabolites of P 4 were the highest in females in the prespawning phase. The stage-specific and sexual differences in the content of the steroids suggest their biosynthesis in the brain, which may have implications in brain functions, in addition to reproductive regulation. © 2011 Springer Science+Business Media B.V.
Brain tyrosine hydroxylase in the catfish Heteropneustes fossilis: Annual and circadian variations, and sex and regional differences in enzyme activity and some kinetic properties
(Academic Press Inc., 2003) R. Chaube; K.P. Joy
Dynamics of tyrosine hydroxylase (TH) was studied in various brain regions and pituitary in relation to annual/seasonal and circadian variations in either sex of the catfish Heteropneustes fossilis. α-Methylparatyrosine inhibited TH activity significantly in a time - (in vivo study) and concentration - (in vitro study) dependent manner. The inhibition was higher in resting phase than preparatory phase and in females than males (time-course study). The apparent kinetic constant of inhibition (Ki) varied both seasonally and regionally. The values were higher in resting phase and in the forebrain regions (telencephalon and hypothalamus) than medulla oblongata. No significant sex difference was noticed in the Ki values in the same season. TH activity showed significant annual/seasonal variations with telencephalon and hypothalamus showing higher activity than medulla oblongata. The females showed significantly higher enzyme activity than males with a distinct activity peak in June (prespawning phase). Such an activity peak was apparently absent in males. TH activity showed significant circadian/diurnal variations, the highest activity was noticed at 12h and the lowest at 24h. The apparent Michaelis-Menten constant (Km) values (hypothalamic TH) for substrate (L-tyrosine) and cofactor (DMPH4) showed seasonal variations with the highest values in the resting phase, decreasing through preparatory and prespawning phases, to the lowest values in spawning phase. Vmax was the lowest in the resting phase and highest in the spawning phase. TH activity was low in the pituitary and could be detected in pooled samples from March (preparatory phase) to July (spawning phase). Activity showed significant variations, which could be correlated with the gonadosomatic index. The results show that TH activity could be positively correlated with the annual reproductive cycle. The enzyme activity could be also correlated with seasonal, sex, and regional variations in the apparent Km and Vmax values suggesting apparent differences in the affinity of the enzyme towards substrate and cofactor. © 2002 Elsevier Science (USA). All rights reserved.
Catecholaminergic control of LH secretion: E2 modulation of tyrosine hydroxylase activity in female catfish Heteropneustes fossilis
(2003) K.P. Joy; R. Chaube
Tyrosine hydroxylase (TH) which catalyses the rate - limiting step in catecholamine (CA) synthesis shows significant annual variations with activity and kinetics increasing with the progress of gonad recrudescence up to spawning and decreasing thereafter. Estradiol-17 β (E2) exerts biphasic effects on in vivo and in vitro enzyme activity and kinetics: low dosages/concentrations stimulated, and high dosages/concentrations inhibited them. Preincubations of hypothalamic enzyme preparations with low (10-9 M) or high (10-3 M) E2 for 15 min at 30 °C, followed by cAMP (1.0 mM) for 10 min at 30 °C produced differential effects: an additive effect in the low concentration group and an inhibitory response in the high concentration group. The stimulatory or inhibitory effects on TH activity could be related to changes in apparent Km and Vmax of the enzyme for substrate and cofactor. The results suggest that TH activity and kinetics are influenced by the circulating titer of E2 and the steroid interacts with the cAMP signaling pathway in the acute regulation of TH. © 2004 Kluwer Academic Publishers.
Catfish Gonadotrophins: Cellular Origin, Structural Properties and Physiology
(2015) R. Chaube; K.P. Joy; A. Acharjee
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.
Cloning and characterization of estrogen hydroxylase (cyp1a1 and cyp1b1) genes in the stinging catfish Heteropneustes fossilis and induction of mRNA expression during final oocyte maturation
(Elsevier Inc., 2021) R. Chaube; A. Rawat; R.M. Inbaraj; K.P. Joy
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. © 2020 Elsevier Inc.
Cyclic AMP-protein kinase A and protein kinase C mediate in vitro T3 activation of brain tyrosine hydroxylase in the female catfish heteropneustes fossilis
(2005) R. Chaube; K.P. Joy
The present in vitro study demonstrates an involvement of both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signal transduction mechanisms in the triiodothyronone (T3)-activation of forebrain (telencephalon and hypothalamus) tyrosine hydroxylase (TH) activity in the female catfish Heteropneustes fossilis. Incubations of the enzyme preparations with different concentrations of T3 (0.15-2.4 ng/ml) stimulated TH activity over the concentrations. Similarly, coincubations of the enzyme preparations with T3 and cAMP (1.0 mM) or cAMP-elevating drugs such as 1-methyl-3-isobutylxanthine (1.5 mM) or theophylline (1.5 mM) increased TH activity significantly over that of T3. The stimulatory effect of TH activity with T3 or cAMP was coincident with a low apparent Km and high Vmax for the cofactor, suggesting a higher affinity of the enzyme. Incubation of the enzyme preparations with PKA (H-89) and PKC (calphostin-C) inhibitors decreased basal enzyme activity significantly, with the inhibition being greater in the former group. The incubations of the enzyme preparations with T3 or T3 + cAMP, followed by the different inhibitors, also decreased enzyme activity. Although T3 could not reverse the inhibitory effect of H-89, it could over-ride the effect of calphostin-C to some extent. The suppressive effect of the inhibitors could be related to a high apparent Km and low Vmax for the cofactor. The evidence strongly suggests a nongenomic action of T3 on TH activity via the cell signalling pathways, for which the cAMP-dependent PKA appears to be the major regulatory mechanism. © 2005 Blackwell Publishing Ltd.
Effects of altered photoperiod and temperature on expression levels of gonadotrophin subunit mRNAs in the female stinging catfish Heteropneustes fossilis
(Blackwell Publishing Ltd, 2017) A. Acharjee; R. Chaube; K.P. Joy
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 (IG), 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, IG, plasma oestradiol-17β (E2), testosterone (T) and progesterone (P4) 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 IG, plasma E2 and T levels and ovarian development. The exposure to high temperature at 28 ± 2° C also modified the short photoperiod effect by elevating plasma E2 level. The plasma T level changed only mildly while the plasma P4 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
Effects of altered photoperiod and temperature, serotonin-affecting drugs, and melatonin on brain tyrosine hydroxylase activity in female catfish, Heteropneustes fossilis: A study correlating ovarian activity changes
(John Wiley and Sons Inc., 2002) R. Chaube; K.P. Joy
Exposure of female catfish, Heteropneustes fossilis to 30-day regimes of long photoperiod (16L), elevated temperature (28 ± 2°C), or a combination of both stimulated brain tyrosine hydroxylase (TH) activity significantly over that of control fish held in natural conditions in gonad resting (10.5L:13.5D, 10 ± 2°C) and preparatory (12.5L:11.5D,18 ± 2°C) phases. The response was high in the combination group in both phases. The increase in TH activity was higher in forebrain regions (telencephalon and hypothalamus) than medulla oblongata. Exposure of the fish to short photoperiod (8L:16D) and total darkness decreased the enzyme activity significantly in both resting and preparatory phases regardless of the temperature. The inhibition was high in fish held under total darkness. Gonadosomatic index (GSI) was significantly elevated in long photoperiod and high temperature groups, alone or in combination, and decreased significantly in short photoperiod (only in preparatory phase) and total darkness groups. Administration of the serotonin precursor 5-hydroxytryptophan (5-HTP; 5 mg/100 g body weight [BW], three daily intraperitoneal [i.p.] injections prior to sacrificing) stimulated TH activity in fish held under long and normal photoperiods in both phases. Three daily injections of the serotonin blocker parachlorophenylalanine (p-CPA; 10 mg/100 g BW) and melatonin (75 μg/100 g BW) prior to sacrificing inhibited brain TH activity significantly in both phases. GSI was significantly stimulated by 5-HTP, and inhibited by both p-CPA and melatonin injections. Changes in TH activity and GSI can be correlated and explained on the basis of previous reports on changes in catecholamine activity that modulates gonadotropin secretion in the catfish. Further, the photoperiod and temperature-induced changes in TH activity may be modulated by alterations in serotonergic activity. © 2002 Wiley-Liss, Inc.
Effects of ovaprim, a commercial spawning inducer, on vasotocin and steroid hormone profiles in the catfish Heteropneustes fossilis: In vivo and in vitro studies
(Academic Press Inc., 2014) R. Chaube; R.K. Singh; K.P. Joy
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. © 2013 Elsevier Inc.
Effects of ovariectomy and oestradiol-17β replacement on brain tyrosine hydroxylase in the catfish Heteropneustes fossilis: Changes in in vivo activity and kinetic parameters
(2002) R. Chaube; K.P. Joy
In Heteropneustes fossilis, ovariectomy inhibited in vivo brain (hypothalamus-pituitary, telencephalon and medulla oblongata) tyrosine hydroxylase (TH) activity with significant effects in weeks 2, 3, 4 and 5 of the gonadal resting phase and in weeks 3, 4 and 5 of the prespawning phase (P<0.05, Tukey's test). Oestradiol-17β (OE2) replacement in 3-week ovariectomised fish produced biphasic responses in both seasons; the low dosages of 0.05 and 0.5 μg/g body weight (BW) elevated TH activity, whereas the high dosages of 1.0 and 2.0 μg/g BW decreased it. The magnitude of the inhibition was higher in the resting phase than in the prespawning phase. The inhibitory effect of ovariectomy may be produced by elevating the apparent Km values (decreased affinity) of the enzyme for both L-tyrosine (substrate) and dimethyltetrahydropteridine (cofactor) and consequently decreasing the Vmax. Significant changes (P<0.05) in both these parameters were noticed but showed minor differences with regard to the length of ovariectomy, season or brain regions. The biphasic effects of OE2 replacement on TH activity seemed to be produced by differential effects on apparent Km and Vmax. The stimulatory effect of the low dosages of OE2 coincides with a decrease in the apparent Km values (increased affinity) for both substrate and cofactor and an increase in the Vmax of the enzyme. The inhibitory effect of the high dosages of OE2 correlated with an increase in the apparent Km, values (decreased affinity) for both substrate and cofactor, and a decrease in the Vmax compared with the lower dosage groups. The results strongly suggested that OE2 can modulate brain catecholaminergic activity at the level of tyrosine hydroxylation which, in turn, may alter gonadotrophin secretion. OE2 may elicit biphasic effects by differentially altering the enzyme affinity towards the substrate and cofactor.
Effects of the fish spawning inducer ovaprim on vasotocin receptor gene expression in brain and ovary of the catfish Heteropneustes fossilis with a note on differential transcript expression in ovarian follicles
(Academic Press Inc., 2017) A. Rawat; R. Chaube; K.P. Joy
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 (0.5 μL/g body weight) or incubation of post-vitellogenic ovarian follicles with 5 μL/mL OVP, for 0, 4, 8, 12, 16, and 24 h stimulated ovulation and GVBD, respectively, in a time-dependent manner. The OVP treatment in vivo stimulated brain VT receptor transcript levels 4 h onwards. The peak expression was noticed at 12 h (v1a1), 8 and 12 h (v1a2), and 8, 12 and 16 h (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 4 h onwards in both intact follicle and isolated follicular layer, the peak expression was observed at 16 h. The v2a expression was up-regulated in both intact follicles and denuded oocytes at 4 h (denuded oocytes) or 8 h (intact follicle) onwards with the peak expression at 12 h and 16 h (denuded oocytes) or at 16 h (intact follicles). Under in vitro conditions, the OVP incubations elicited similar pattern of changes with the peak stimulation at 16 h 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. © 2016 Elsevier Inc.
Estrogen regulation of in vitro brain tyrosine hydroxylase activity in the catfish Heteropneustes fossilis: Interactions with cAMP-protein kinase A and protein kinase C systems in enzyme activation
(Academic Press Inc., 2005) R. Chaube; K.P. Joy
In the present in vitro study, interactions of both cAMP-protein kinase A (PKA) and protein kinase C (PKC) systems were investigated in the estradiol-17β (E2) regulation of forebrain (hypothalamus and telencephalon) tyrosine hydroxylase (TH) activity in the female catfish Heteropneustes fossilis in vitellogenic phase. E2 produced biphasic effects on TH activity: low concentrations (10-12-10-5M) stimulated, and high concentrations (10-3-10-4 M) inhibited enzyme activity (Tukey's test, P < 0.05). Co-incubations of the enzyme preparations with cAMP (1.0 mM), IBMX (1.5 mM) or theophylline (1.5 mM) and a low concentration of E2 (10-9 M) increased TH activity significantly. However, the co-incubations with a high concentration of E2 (10-3 M) decreased it significantly. Pre-incubations of the enzyme preparations with cAMP (0.1 mM), followed by different concentrations of E2 (10-12, 10-9, 10 -4, and 10-3 M) produced concentration-dependent biphasic effects. The pre-incubations with a low concentration of E2 (10 -9 M), followed by different concentrations of cAMP (0.05-1.0 mM) produced a significant concentration-dependent stimulation of TH activity and that with a high concentration of E2 (10-3 M) produced a significant decrease in TH activity. Co-incubations of high and low E 2, with or without cAMP, and PKA inhibitor (H-89) decreased TH activity significantly. The incubations with H-89 abolished the stimulatory effect of low E2 or low E2 + cAMP and intensified the inhibitory effect of high E2 or high E2 + cAMP combination. Co-incubations with PKC inhibitor (calphostin C) did not influence the stimulatory effect of low E2 but lowered the stimulatory effect of low E2 + cAMP treatment. Kinetic studies showed that the stimulatory effect of a low E2 concentration was due to a decrease in apparent Km and an increase in apparent Vmax for both cofactor and substrate, and the inhibitory effect of a high E2 concentration was due to reverse changes in the kinetics. The stimulatory effect of cAMP alone or in combination with low E2 was related to decreased Km and increased Vmax for the cofactor. The inhibitory effect of PKA and PKC blockers, alone or in combination with E2 and/or cAMP was due to increased Km and decreased Vmax of the enzyme for the cofactor. The present data suggest that E2 modulates the short-term activation of brain TH activity differentially and may involve mainly the cAMP-PKA system. © 2005 Elsevier Inc. All rights reserved.
Expression profile of kisspeptin2 and gonadotropin-releasing hormone2 mRNA during photo-thermal and melatonin treatments in the female air-breathing catfish Heteropneustes fossilis
(Springer Science and Business Media B.V., 2020) R. Chaube; S. Sharma; B. Senthilkumaran; S.G. Bhat; K.P. Joy
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 E2, progesterone, and testosterone levels. The results show that brain kiss2-gnrh2 signaling is involved in photo-thermal-mediated mechanisms controlling reproduction. © 2020, Springer Nature B.V.
Foreword
(Kluwer Academic Publishers, 2013) K.P. Joy; R. Moses Inbaraj; R. Kirubagaran; R. Chaube
[No abstract available]
Gene network analysis of Diabetic Nephropathy and its associated diseases- An in silico study
(World Research Association, 2022) P. Shree; N. Pandey; R. Chaube; N. Garg; Y.B. Tripathi
Metabolic syndrome (MetS) is a cluster of conditions including Diabetic nephropathy (DN), Hypertension (HT), Type 2 Diabetes mellitus in obese and obesity. Network biology has been developed as a way to review gene interactions. Herein an effort was made to identify common genes between the above MetS using Cytoscape3.0. Further molecular docking study has been performed to identify potential inhibitors for common genes using active phytoconstituents of Pueraria tuberosa (PTY-2). AGT (angiotensinogen) and ACE (angiotensinconverting enzyme) were found to be common genes between DN and HT. From the molecular docking study, tuberosin (9.19 kcal/mol) and robinin (11.28 kcal/mol) showed highest binding affinity with AGT and ACE and followed all drug-like properties as per Lipinski's rule of five and Admet prediction. Hence, based on our findings we may suggest that by inhibiting AGT and ACE, the downstream functioning pathways are responsible for these diseases and their associated complications can be normalized. © 2022 World Research Association. All rights reserved.
Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17β on mRNA expression
(Academic Press Inc., 2017) R. Chaube; A. Rawat; R.M. Inbaraj; J. Bobe; Y. Guiguen; A. Fostier; K.P. Joy
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 1442 bp with an open-reading frame (ORF) of 792 bp, 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 24 h in the brain and at 16 h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16 h. 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. © 2016 Elsevier Inc.
Identification of kisspeptin2 cDNA in the catfish Heteropneustes fossilis: Expression profile, in situ localization and steroid modulation
(Academic Press Inc., 2020) R. Chaube; S. Sharma; B. Senthilkumaran; S.G. Bhat; K.P. Joy
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 (E2; 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 E2 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 + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 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. © 2020 Elsevier Inc.