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Nitric oxide (NO) is a gaseous molecule that regulates various reproductive functions. It is a well-recognized regulator of GnRH-FSH/LH-sex steroid secretion in vertebrates including fish. Kisspeptin is a recently discovered neuropeptide which also regulates GnRH secretion. Nitrergic and kisspeptin neurons are reported in close physical contact in the mammalian brain suggesting their interactive role in the release of GnRH. The existence of kisspeptin and NOS is also demonstrated in vertebrate gonads, but information on their reciprocal relation in gonads, if any, is obscure. Therefore, attempts were made to evaluate the functional reciprocal relation between nitric oxide and kisspeptin in the catfish gonads, if any, by administering the nitric oxide synthase (NOS) inhibitor, L-NAME {N(G)-nitro-l-arginine methyl ester}, which reduces NO production, and kisspeptin agonist (KP-10) and assessing their impacts on the expressions of kisspeptin1, different NOS isoforms, NO and steroid production in the gonadal tissue. The results revealed that L-NAME suppressed the expression of kiss1 in gonads of the catfish establishing the role of NO in kisspeptin expression. However, KP-10 increased the expression of all the isoforms of NOSs (iNOS, eNOS, nNOS) and concurrently NO and steroids in the ovary and testis. In vitro studies also indicate that kisspeptin stimulates the production of NO and estradiol and testosterone levels in the gonadal explants and medium. Thus, in vivo results clearly suggest a reciprocal interaction between kisspeptin and NO to regulate the gonadal activity of the catfish. The in vitro findings further substantiate our contention regarding the interactive role of kisspeptin and NO in gonadal steroidogenesis. [ABSTRACT FROM AUTHOR]

Neurokinin B (NKB), a recently discovered neuropeptide, plays a crucial role in regulating the kiss-GnRH neurons in vertebrate's brain. NKB is also characterized in gonadal tissues; however, its role in gonads is poorly understood. Therefore, in the present study, the effects of NKB on gonadal steroidogenesis and gametogenesis through in vivo and in vitro approaches using NKB antagonist MRK-08 were evaluated. The results suggest that the NKB antagonist decreases the development of advanced ovarian follicles and germ cells in the testis. In addition, MRK-08 further reduces the production of 17β-estradiol in the ovary and testosterone in the testis under both in vivo and in vitro conditions in a dose-dependent manner. Furthermore, the in vitro MRK-08 treatment of gonadal explants attenuated the expression of steroidogenic marker proteins, i.e., StAR, 3β-HSD, and 17β-HSD dose-dependently. Moreover, the MAP kinase proteins, pERK1/2 & ERK1/2 and pAkt & Akt were also downregulated by MRK-08. Thus, the study suggests that NKB downregulates steroidogenesis by modulating the expressions of steroidogenic marker proteins involving ERK1/2 & pERK1/2 and Akt/pAkt signalling pathways. NKB also appears to regulate gametogenesis by regulating gonadal steroidogenesis in the catfish. [ABSTRACT FROM AUTHOR]

Asprosin is an orexigenic adipokine that regulates appetite and glucose homeostasis in mammals. To date, only fragmentary findings are reported regarding its role in testicular activities. In the current investigation, immunolocalization and direct action of asprosin in adult mice testis was evaluated. Immunohistochemical and immunoblot studies were performed to analyse the testicular expression of asprosin. Intratesticular treatment of asprosin (0.1 μg and 1.0 μg per testis) was given to evaluate its direct action on testicular functions. Sertoli and Leydig cells were found to be immuno‐positive for asprosin. Intratesticular administration of asprosin resulted into a significant increase in glucose and lactate levels along with enhanced expression of asprosin receptor OLFR734, insulin receptor (IR), glucose transporter 8 (GLUT 8), lactate dehydrogenase (LDH) activity and monocorboxylate transporters (MCT2 and 4). In addition, asprosin administration increased the testicular expression of cell proliferation (proliferating cell nuclear antigen: PCNA), cell survival (B cell lymphoma 2: Bcl2) and decreased germ cell apoptosis (Cysteine aspartic acid protease 3: Caspase 3) leading to increased sperm counts. Further, asprosin treatment resulted into increased level of total cholesterol, testosterone and steroidogenic markers (steroidogenic acute regulatory protein: StAR; 3beta‐hydroxysteroid dehydrogenases: 3β HSD and 17beta‐hydroxysteroid dehydrogenases: 17β HSD). Asprosin treatment promotes testicular glucose uptake and lactate synthesis to provide energy for steroidogenesis and spermatogenesis. The significant correlation between the asprosin‐induced increased IR expression and increased testosterone, glucose and lactate levels suggests its role in increased survival and proliferation but decrease in germ cell apoptosis. This study proposed asprosin's role as an autocrine/paracrine regulator of testicular functions in adult mice. [ABSTRACT FROM AUTHOR]

Psychological stress is now widely recognized as one of the major risk factors for male fertility. Its impact on the dynamics of testicular germ cells, however, has yet to be fully investigated. Therefore, we used the rat restraint stress (RS) model as a psychological stressor to assess the impact of psychological stress on testicular germ cell dynamics. Adult male SD rats were exposed to sub‐chronic RS for 1.5 and 3 h per day for 30 days. The quality of cauda epididymis spermatozoa was adversely affected by RS exposure, and the frequency of spermatozoa with tail abnormalities was higher than that of spermatozoa with head abnormalities. RS exposure adversely affected testicular daily sperm production by disturbing the meiotic and post meiotic germ cell kinetics in the testis. The histomorphology of the testis was altered by loosening and vacuolization in the seminiferous epithelium, germ cell exfoliation and the presence of giant cells. Seminiferous tubules of stage I–VI and VII–VIII were severely affected in rats exposed to RS for 3 h. By interfering with steroidogenic enzymes, RS exposure disrupts testosterone biosynthesis. The testicular oxidative balance was also disturbed by RS exposure, which disrupted the levels/activities of lipid peroxidation, Nrf‐2, superoxide dismutase and catalase. There was also an increase in caspase‐3 activity and a decrease in the Bax‐Bcl2 ratio. In conclusion, our findings suggest that psychological stressors like RS impair testicular functions in rats by disrupting germ cell dynamics, downregulating testicular androgenesis and increasing oxidative stress and apoptosis. [ABSTRACT FROM AUTHOR]

Photo-thermal regulation of reproduction in seasonally breeding vertebrates, including teleost fishes, is established. The photo-thermal cues are relayed to the higher brain centers, where from command, neurosecretions are released to the hypothalamus to activate the hypothalamo-pituitary–gonadal axis to secrete GnRH-FSH/LH-sex steroids in general. GnRH being a pivotal regulator of reproduction, plenty of studies have been carried out to investigate the regulators of GnRH secretion. Kiss is the recently established upstream regulator of GnRH. However, studies on the regulation of kiss are highly ambiguous in vertebrates, including fishes. Therefore, attempts were made to examine the role of photoperiod and temperature in the regulation of kiss1 expression in the gonad during the late-quiescence phase of the reproductive cycle of the catfish, Clarias batrachus, employing the techniques like histology, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) along with analysis of data through analysis of variance (ANOVA) followed by a post hoc test and Duncan's multiple range test at P < 0.05 for comparisons amongst different groups. The study reveals that long photoperiod and high temperature stimulate the expression of kiss1 significantly (P < 0.05), while short photoperiod and low temperature suppress its expression in fish gonads (P < 0.05). Photoperiod and temperature were almost equally effective in regulating the kiss1 expression, but when long photoperiod and temperature are given in combination, their stimulatory role in kiss1 expression is amplified significantly (P < 0.05). The long photoperiod and high temperature also increase the steroid levels in gonads (P < 0.05) and stimulate gametogenesis. [ABSTRACT FROM AUTHOR]

The present field study evaluates the health status of the catfish Clarias batrachus reared in coal mine effluent (CME)-fed pond water at Rajrappa mining complex using biochemical, haematological and histopathological parameters. Simultaneously, risk assessment along with recovery response of the CME intoxicated fish following their treatment with CME-free freshwater was also studied. The CME-fed pond water fish revealed significant decrease in biomolecules concentrations and considerable increase in activities of several enzymes along with metallothionein level as compared to control. The impaired regulation of metabolic function was also revealed by blood parameters showing significant decrease in haemoglobin content (8.78 ± 0.344 g/100 mL) and red blood cells count (1.77 ± 0.12 × 106 mm3) while substantial elevation in white blood cells (187.13 ± 9.78 × 103 mm3). The histopathological study also confirmed the changes including hypertrophy of club cells of skin, swelling of secondary lamella of gills, extensive fibrosis in liver and glomerular shrinkage with increased Bowman's space in kidney. Potential health risk assessments based on estimated daily intake and target hazard quotient indicated health risks associated with the consumption of such fishes. The CME-contaminated fish when transferred to CME-free freshwater exhibited decreased metal content accompanied by eventual recovery response as evident by retrieval in biochemical and haematological parameters. Withdrawal study also revealed restoration in the activity of different marker enzymes in fish tissues including blood as well as recovery in their cellular architecture. The results of the present study validate the depuration process as an effective practice for detoxification of fish contaminated with effluent. [ABSTRACT FROM AUTHOR]

Growth hormone (GH), a key regulator of somatic and reproductive growth in vertebrates, has been extensively studied, although primarily in female fish. Despite numerous reports about sex- and species-specific growth patterns in fish, to our knowledge, there is no report about the 24 h rhythm of plasma GH in male fish. Thus, we aimed to investigate temporal variations in plasma GH levels and the existence of any rhythms therein during the reproductively active months of March to August in the male walking catfish, Clarias batrachus. We also aimed to compare the secretory temporal patterns of GH in male–female specimens of C. batrachus to decipher sexual dimorphism in GH secretions in fish. After 14 days of acclimation to the natural environment, male catfish (N = 240 in total) were sorted and randomly divided into eight groups for study at ZT0 (sunrise ~06:00 h), 3, 6, 9, 12, 15, 18, and 21. During each month, physical parameters like duration of photoperiod and water temperature were measured. Male catfish (n = 40/month) in all eight groups were sampled (n = 5/group) at each time point under the natural time-of-year 24 h light-dark (LD) cycle. Male catfish were anesthetized and blood was collected through a caudal puncture, centrifuged, and plasma isolated. Plasma GH was measured using a competitive homologous enzyme-linked immunosorbent assay. Further, testes were removed, weighed, and the gonadosomatic index (GSI) was calculated. A significant effect of time and season (p ˂ 0.05, two-way ANOVA) on plasma GH level was detected. Cosinor analyses verified the existence of statistically significant (p ˂ 0.05) ultradian (12 h) and 24 h rhythms of plasma GH in male C. batrachus, with the higher values of Mesor (time series mean) and amplitude (one-half peak-to-trough difference) of the periodicities from March to July. Mapping of the acrophases (peak times) showed two ultradian and one 24 h acrophase of GH during the early photophase and early scotophase from March to August. Distinct sexual-dimorphism in plasma GH Mesors and acrophases was noticed between male and female catfish. GSI values of male catfish indicate males mature a little earlier than females in terms of size and reproductive activity. The findings that plasma GH show 24 h and seasonal fluctuations in a sex-specific manner collectively demonstrate the importance of considering the effect of biological 24 h and seasonal time and sex on the GH level in regulating the physiology of somatic growth and reproduction in catfish. [ABSTRACT FROM AUTHOR]

In the present study, a multi-biomarker approach was used to assess the toxicity of the coal mine effluent (CME) generated at the Rajrappa coal mine on the catfish Clarias batrachus. A core of biomarkers indicative of nutritional value, oxidative stress, and histopathology was selected to illustrate the toxic effects of CME-containing different heavy metals and other toxicants. The results of metal bioaccumulation in CME-exposed fish tissues revealed the highest metal concentration in liver (1.34–297.68 mg/kg) while lowest in muscles (1.47–23.26 mg/kg) as compared to other tissues and so was the metallothionein level. The high value of bioaccumulation observed in liver, kidney, and gills reflects their affinity for metals. In addition, the values of metal pollution index (MPI) of different fish tissues further affirmed that liver followed by kidney and gills are at greater risk than brain, skin, and muscles. Significant alterations in the activity of certain enzymes (aspartate amino transferase, alanine amino transferase, alkaline phosphatase) as well as oxidative stress markers (superoxide dismutase, catalase and lipid peroxidation) were detected in the tissues of CME-exposed fish. The tissue-specific metal accumulation and increased metallothionein levels may be associated with the biochemical and physiological activity of an organ and its constitutive antioxidant defenses. The histopathological changes in the various tissues of the CME-exposed fish justify the high metal accumulation and biochemical alterations. Overall results indicate that the Rajrappa coal mine effluent is very toxic having adverse health impact on the fish and might also affect the human health when consumed. [ABSTRACT FROM AUTHOR]

The original article can be found online at https://doi.org/10.1007/s00441-023-03788-0. Superscripts *, ** and *** are used for p44 and superscripts #, ## & ### for p42 while superscripts $ and $$ for pAkt (Fig. Superscripts *, ** and *** are used for StAR, while superscripts # & ### are used for 3 -HSD and superscripts $, $$ and $$$ for 17 -HSD, respectively (Fig. [Extracted from the article]

In the present study, three aquatic macrophytes, Eichhornia crassipes, Salvinia molesta, and Pistia stratiotes were used to assess their relative efficacies in decontamination of a fish culture pond, regularly fed with coal mine effluent (CME). The level of metals like Fe, Mn, Ni, Zn, Cu, Pb, Cr, and Cd were much higher in CME-fed pond water than their recommended limits in drinking water set by the Bureau of Indian standards and in effluents by the Environmental Protection Agency. The levels of metal were lowered substantially in CME-fed pond water after exposure of the above plants to such water, however, metal levels in the plants increased tremendously. The increased metal levels in plants severely damaged their physiological and biochemical processes. The contents of chlorophyll a, b and carotenoid were reduced by 63.2, 64.2, and 46.3%, respectively, in E. crassipes, 41, 57.4, and 57.8% in S. molesta, and 42, 62, and 61% in P. stratiotes. The accumulating metals also generated oxidative stress in plants, as evident from the increased superoxide dismutase and catalase activities and enhanced malondialdehyde content. The E. crassipes was the most potent in absorbing the metals from the CME-fed pond water, followed by S. molesta and P. stratiotes. [ABSTRACT FROM AUTHOR]

Intense immunoreactivity was observed in several neurons of the nucleus preopticus (NPO) located in the preoptic area (POA), in addition to several GH cells in the proximal pars distalis (PPD) of the pituitary gland of Clarias batrachus. The immunoreactive cells were located in the paraventricular as well as supraoptic subdivisions of the NPO. GH immunoreactive fibers projecting from the neurons were traced caudally to the pituitary gland via the conspicuous preoptico-hypophysial tract (PHT) in the ventral tuberal area to the neurohypophysis of the pituitary. Apart from these immunoreactive fibers in the preoptico-hypophysial tract, some fine caliber fiber probably arising from the neurons located dorsally in the NPO also showed GH immunoreactivity, and these fibers constituted an independent tract. Bilaterally, it extended caudally through the dorsal hypothalamus almost as far as the saccus vasculosus where it curved sharply to descend into the caudal tuberal region and finally entered into the pituitary gland. The fibers of this tract were mainly distributed in the rostral pars distalis (RPD). This tract is quite distinct from the preoptico-hypophysial tract and herein called as the accessory preoptico-hypophysial tract (APHT). Expression of GH mRNA in the NPO was found 65-fold more than that of the control region, rostral cerebellum. These results altogether suggest that GH secreted by NPO neurons might serve as a neuro-modulatory role in the brain of C. batrachus. Transportation of GH to the pituitary via two independent tracts may represent the duality of neuroendocrine function. The present study underscores the possibility that GH in the brain of vertebrates may be a phylogenetically conserved phenomenon and provide clues to our understanding of the evolutionary course taken by the hormone. [ABSTRACT FROM AUTHOR]

The present study was undertaken to understand the physiological significance of the existence of nitric oxide synthase (NOS)/nitric oxide (NO) system in fish ovary. For this, two doses of NO donor, sodium nitroprusside (SNP, 25 µg and 50 µg) and NOS inhibitor, N-nitro-l-arginine methyl ester (l-NAME, 50 µg and 100 µg)/100 g body weight were administered during the two reproductive phases of reproductive cycle of the Clarias batrachus. During the late-quiescence phase, high dose of l-NAME decreased the NO, testosterone, 17β-estradiol, vitellogenin contents in serum and ovary and activities of 5-ene-3β-hydroxysteroid dehydrogenases (3β-HSD) and 17β-hydroxysteroid dehydrogenases (17β-HSD) in ovary, whereas higher dose of SNP increased these parameters. l-NAME also reduced oocytes-I but increased perinucleolar oocytes in the ovary, whereas SNP treatment increased the number of advanced oocytes (oocytes-I and II) than the perinucleolar oocytes when compared with control ovary. During the midrecrudescence phase, both doses of SNP increased NO, testosterone, 17β-estradiol and vitellogenin in serum and ovary; however, l-NAME treatment lowered their levels. The activities of ovarian 3β-HSD and 17β-HSD were also stimulated by SNP, but l-NAME suppressed their activities compared to the control. The SNP-treated ovaries were dominated by oocyte-II and III stages, whereas l-NAME-treated ovary revealed more perinucleolar oocytes and oocytes-I and practically no advanced oocytes. Expression of endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) was augmented by the SNP and declined by l-NAME treatments as compared to the control. This study, thus, provides distinct evidence of NO-stimulated steroidogenesis, vitellogenesis and folliculogenesis in fish. [ABSTRACT FROM AUTHOR]

A cell line designated as HFB-ES was established from blastula stage embryos of H. fossilis (Singhi). The embryonic cells were harvested and maintained in Leibovitz's medium supplemented with 15% fetal bovine serum. The cell line had been subcultured for more than 90 passages in a period of 24 months. HFB-ES cells were able to grow at temperatures between 25 and 35℃ with an optimum temperature of 28℃. The growth rate of HFB-ES was proportional to FBS concentration, with optimum growth seen at 15% FBS concentration. The originality of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I (COI), cytochrome b gene, and microsatellite DNA profile. Results of chromosome complements of HFB showed normal karyomorphology with 56 (2«) diploid number of chromosomes after 40 passages which indicated that the developed cell line is chromosomally stable. The pluripotency of HFB was demonstrated by alkaline phosphatase activity and Oct-4 gene expression. Expression of GFP reporter gene was successful in HFB-ES. These results indicated that HFB-ES could be utilized for future gene expression studies. [ABSTRACT FROM AUTHOR]

The plasma profile of growth hormone (GH) was monitored in the freshwater catfish, Clarias batrachus over a single 24-h transverse timescale in the middle of each month starting from March to August. This period included different physiological status of the fish and also varying environmental photoperiod and ambient temperature. Statistically significant ultradian rhythm in GH was detected in all the months of study. Of the two ultradian peaks, one was located invariably in the early photophase with the other one in the early scotophase. During the period of intense reproductive activity (June and July), circadian rhythm in GH was also recorded in addition to ultradian rhythm. The 24-h average (Mesor) and amplitude of ultradian rhythm of GH were high in March and April coinciding with the increase in photoperiod, temperature and feeding activity. Mesor increased further in May, reaching the maximum in June with a further rise in the water temperature and intense gonadal activity, but the amplitude declined during these months. In the breeding month of July, Mesor declined moderately and amplitude declined further. In August, Mesor and amplitude both attained minimum levels. In July and August one of the ultradian peaks overlapped with the circadian peak of the GH rhythm and the circadian peaks appeared in the night, while in other months it appeared in the early morning or at midday. Synchronization of one of the ultradian peaks with that of the circadian peak in GH in the night of July and August might provide physiological advantage for the execution of breeding at the coolest part of the day. The rhythmic patterns of GH secretion might have a strong correlation with the changing photoperiod, temperature, and feeding and reproductive activity. The observed temporal organization in GH in C. batrachus may have physiological consequences that might provide suitable milieu for the regulation of its somatic and gonadal growth, and execution of reproductive activity. [ABSTRACT FROM AUTHOR]

Analysis of changes in the plasma levels of GTH II over a 24-h day/nightcycle revealed statistically significant circadian or ultradian variationor both in its circulating level during the gonadally active months of thereproductive cycle. The 24-h average and amplitude of circadian or ultradianrhythm in GTH II increased with the advancement of ovarian development andmaturation. In April, a single peak in GTH II level was noticed in the nightat 20.6 h. In contrast, in May, June and July a biphasic pattern (ultradianrhythm) was noticed with two characteristic peaks, one in the photophase andthe other in the scotophase. In addition, in May a statistically significantcircadian rhythm in plasma GTH II was validated with a peak located at 20.1h. These rhythms seem to have physiological significance. The ultradian rhythmwith two peaks during the reproductively active phase appears to provide asuitable physiological milieu for the temporally different yet synchronouspopulation of oocytes for the secretion of steroids. Thus, the observed temporalorganization in GTH II may have physiological consequences leading to accomplishmentof reproductive process at appropriate time of the year. [ABSTRACT FROM AUTHOR]

In the present investigation, daily variations in plasma levels of melatonin,testosterone and estradiol-17β were analysed in the tropical freshwatercatfish, Clarias batrachus during early and late-preparatory, pre-spawning,late-spawning and post-spawning phases of its annual reproductive cycle. Plasmamelatonin levels exhibited a significant circadian rhythm during all the reproductivephases but peaks were invariably at night. The amplitude of melatonin washigh during the early-preparatory phase and low in the pre-spawning phase.Testosterone also displayed significant rhythm during all the studied phasesexcept post spawning phase. Estradiol-17β revealed diurnal rhythm onlyduring preparatory and pre-spawning phases. The peak in the levels of plasmatestosterone and estradiol-17β were recorded during the photophase. Duringthe late-preparatory phase, the rhythm of testosterone coincided with therhythm of estradiol-17β. [ABSTRACT FROM AUTHOR]

Coal mining generates huge quantity of toxic effluent which consistently pollutes the neighboring wetlands where the local inhabitants regularly cultivate edible fishes. In the present study the concentration of heavy metals Fe, Zn, Cu, Mn, Ni, Cd, Pb and Cr were analyzed in the water and various tissues of edible catfish Clarias batrachus reared in a pond receiving effluents from Rajrappa coal mine, Jharkhand, India. The metal concentrations in the pond water were dramatically higher (Fe 350%, Zn 423%, Cu 12%, Mn 7029%, Ni 713%, Cd 1700%, Pb 4333% and Cr 588%) than the safe limit of Environmental Pollution Agency (2003) as well as the control tap water. Excessive amounts of metals in effluent caused their substantial transfer to the different tissues of the catfish reared in such ponds. Results showed that accumulation of metals in fish tissues were in the following order: liver > kidney > air breathing organ (ABO) > gills > skin > brain > muscles. Among the various tissues the highest accumulation of most of the metals was recorded in the liver (2.05-271.28 mg/kg dry weight) and lowest in the muscles (1.39-30.27 mg/kg dry weight), while the concentration of metals in other tissues ranged in between. The accumulation of heavy metals in tissues appears to cause remarkable histopathological alterations in skin, gills, ABO, liver and kidney that might be leading to deleterious effect on fish physiology and consequently impact the consumers of such fishes. [ABSTRACT FROM AUTHOR]