Correlation of testicular melatonin and cellular stress in an annual spermatogenic cycle of Clarias batrachus

Correlation of testicular melatonin and cellular stress in an annual spermatogenic cycle of Clarias batrachus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Correlation of testicular melatonin and cellular stress in an annual spermatogenic cycle of Clarias batrachus Akash Acharyya, Kazi Nurul Hasan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3943922/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Melatonin, primarily produced by pineal gland, shows a rhythmic pattern of synthesis in response to environmental cues. Hitherto, the role of testicular melatonin in the regulation of annual spermatogenic cycle is indeterminate in any fish species. Present study aims to explore a correlation, for the first time, of melatonin with cellular stress levels in accordance to the rhythmic progression of spermatogenesis through six distinct reproductive phases of an annual gonadal cycle of catfish, Clarias batrachus under natural photo-thermal conditions. We assessed gonadosomatic index (GSI), testicular melatonin concentrations, relative percentage of haploid cells by using DNA dye in the seminiferous tubules, and different intracellular stress markers. The concentration of testicular melatonin displayed a distinct seasonal pattern, reaching their peak during the “phase of functional maturity”. Interestingly, the levels of 2’,7’–dichlorofluorescein [reactive oxygen species (ROS) marker], malondialdehyde (MDA) and RNS (total nitrate) were found significantly lowered at the verge of “mature state”. Correlation and LOESS regression analyses showed a striking positive relationship between testicular melatonin concentration and the activity of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Principal component (PC) analysis also identified the key components of “mature state” viz., GSI, testicular melatonin, haploid cell population (spermatid and spermatozoa), and the activity of enzymatic antioxidants. Cumulatively, melatonin may have a role in growth and maturation of spermatogenic cells by reducing the cellular stress to augment the quality of germ cells required for seed production in the culture of any fish species. Clarias batrachus Melatonin Testis Antioxidants ROS Cell cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Melatonin, N -acetyl-5-methoxy-tryptamine, is a chronobiotic molecule derived rhythmically from L-tryptophan and predominantly produced within the pineal gland (Lerner et al. 1958). It reaches a crescendo during the nocturnal embrace of darkness, and remarkably synchronizes with external environmental cues such as photoperiod, water temperature, rainfall and more (Falcón et al. 2010). The level of melatonin experiences synchronized rhythmic fluctuations both in the pineal gland and in the bloodstream, a pattern observed consistently across various vertebrates. In addition to pineal gland, several other non-pineal tissues like retina, gut, liver, testis, and ovary contribute significantly to the synthesis of this hormone in all vertebrates, including fish (Hasan et al. 2016; Acharyya et al. 2023). Ubiquitous presence of melatonin in every living species studied to date suggests that it is a phylogenetically ancient molecule and believed to have evolved as a scavenger of free radicals, and over the course of evolution, it acquired diverse functions within various living systems (Maitra and Hasan 2016). Numerous scientific reports have provided compelling evidence highlighting the crucial role of melatonin in the regulation of reproduction across different fish species (Das et al. 2022; Hasan et al. 2023). The correlation of melatonin with gonadal cycle is studied mostly in female fish but such information is very scarce in male fish species. The hormone significantly regulates oocyte development in an annual ovarian cycle of carp depending upon the reproductive status of the animal (Maitra et al. 2005). Study also revealed that exogenous melatonin causes precocious or tardy effects by modulating ovarian and testicular steroidogenesis in fish (Bhattacharya et al. 2007; Lombardo et al. 2012). The involvement of melatonin in the growth and maturation of germ cells is mediated through hypothalamo-hypophyseal-gonadal (HPG) axis as a hormone or as an antioxidant (Maitra and Hasan 2016; Acharyya et al. 2021). In zebrafish, a 10-day exposure to melatonin (100 nM and 1 µM) in tank water significantly influences seasonal reproduction through kisspeptin mediated hypothalamic gonadotropin-releasing hormone (GnRH) production, and directly regulating the transcription of mprα and mprβ genes in the gonads involve in oocyte competence and maturation (Carnevali et al. 2011). Endocrine action of melatonin primarily operated through the regulation of HPG axis as suggested by the presence of G-protein coupled receptors [MT1 (Mel1a), MT2 (Mel1b), and Mel1c] in different target sites namely suprachiasmatic nucleus (SCN), pars tuberalis (PT) of pituitary gland, testis and ovary (Klosen et al. 2019). Rhythmic expression of ovarian melatonin receptors throughout an annual cycle in fish also highlighted its involvement in oocyte growth and maturation by interacting with HPG axis (Moniruzzaman and Maitra 2012). Several seasonal and non-seasonal breeding animal species express testicular receptor proteins, indicating melatonin may also regulate receptor mediated, a component of HPG axis, reproductive functions (Yang et al. 2014; Kozioł et al. 2020). Further, the amphiphilic nature of melatonin enables it to readily penetrate cellular barriers, allowing it to function as a widespread direct scavenger of free radicals and an indirect antioxidant (Tan et al. 1993). This characteristic has unveiled a new realm of possibilities, suggesting that besides its receptor-mediated hormonal actions, melatonin may also exert receptor-independent effects in regulating reproduction (Gao et al. 2022). Melatonin critically scavenges oxygen free radicals during oocyte maturation and ovulation, mitigating the detrimental effects of reactive oxygen species (ROS) and protecting oocytes from oxidative damage (Silva et al. 2024). Ovarian melatonin follows a seasonal pattern in carp, reaching its peak at spawning and this temporal variation shows a significant negative correlation with malondialdehyde (MDA) level, an indicator of intracellular oxidative stress (Hasan et al. 2014). This observation also suggests that melatonin minimizes oxidative stress in the ovary by augmenting the activities of antioxidative enzymes, specifically superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) throughout the phases of oocyte growth and maturation. Currently, study also suggested exogenous melatonin mediated meiotic maturation through heightening of antioxidative capacity in late-vitellogenic oocytes of turbot ( Scophthalmus maximus ) (Zhang et al. 2023). To our knowledge, no controlled studies are available to demonstrate the role of testicular melatonin as an antioxidant in the regulation of spermatogenic cycle in any fish species. A very recent study delves into the intricate dynamics of melatonin levels, reproductive parameters, and environmental factors in adult male catfish ( Clarias batrachus ) throughout an annual reproductive cycle (Acharyya et al. 2023). Notably, both intra-testicular melatonin concentrations and the dynamics of spermatogenic cells are synchronized with different meteorological factors like photoperiod, temperature, and rainfall. However, there is a dearth of information regarding the influence of melatonin on the regulation of cellular stress levels in testis over the course of an annual cycle in any seasonally breeding animals. Consequently, the present study endeavours to elucidate the developmental dynamics of male germ cells through the analysis of changes in ploidy (based on genomic DNA content) during the course of cell cycle progression in Catfish, Clarias batrachus . Additionally, it also aims to discern any relation of melatonin in the regulation of intra-testicular oxidative (ROS) and nitrosative (total nitrate) stress during spermatogenesis under natural photo-thermal conditions, encompassing six distinct phases within the annual reproductive cycle. Materials and methods Chemicals and regents Melatonin ELISA kit was purchased from Fine Test (EU0199), Wuhan Fine Biotech Co., Ltd. Wuhan, China. Nicotinamide Adenine Dinucleotide, reduced (NADH), nitro blue tetrazolium (NBT), and glutathione, reduced (GSH) were purchased from SRL, Mumbai, India. 5-methylphenazinium methyl sulphate (PMS), phenylmethylsulfonyl fluoride (PMSF), 1-chloro-2, 4-dinitrobenzen (CDNB), o-phenylenediamine (OPD), trichloro acetic acid (TCA), thiobarbituric acid (TBA), 5, 5’-dithiobis (2-nitrobenzoate) (DTNB), 2’,7’-dichlorofluorescin diacetate (DCFH 2 DA), Propidium iodide (PI), RNase A, vanadium (III) chloride (VCl 3 ), sulfanilamide, N-(1-napthyl) ethylenediamine dihydrochloride (NEDD) and other chemicals were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA unless otherwise specified. Collection of fish To ensure the reliability and consistency of our findings, we gathered adult male catfish Clarias batrachus (males and females were separated on the basis of sexual dimorphic characteristics), weighing about 210 ± 25 g, from the water bodies near the university campus, Purulia, India [23°35' N, 86°33' E], in each month of an annual cycle. The testicular development consists of six distinct phases, phase of slow spermatogenesis (January to February) or Phase-I, early phase of rapid spermatogenesis (March to April) or Phase-II, late phase of rapid spermatogenesis (May to June) or Phase-III, phase of functional maturity (July to mid-August) or Phase-IV, phase of depletion (late-August to mid-October) or Phase-V and phase of relaxation and rehabilitation (mid-October to December) or Phase-VI (Acharyya et al. 2023). Immediately following capture in each month, five adult male catfish ( N = 5) were promptly transported to the laboratory and allowed to acclimate for seven days in a glass aquarium under natural photo-thermal conditions. This acclimation period aimed to reduce the stress caused during the collection and transportation of the fish before they were sacrificed. The fish were provided with commercial fish feed pellets, Growfin, from Growel Feeds Pvt. Ltd., (Andhra Pradesh, India), enriched with 40% crude protein along with 12% moisture and crude fat, fibre (6% and 3%, respectively). The Institutional Animal Ethics Committee (IAEC), Department of Zoology (Reg. No. 1973/GO/Re/S/17/CPCSEA), Sidho Kanho Birsha University, reviewed and approved animal care, hygiene, and laboratory procedures in accordance with the guidelines of CPCSEA, New Delhi, Govt. of India. Collection and preparation of testicular samples Following the dissection, the testes from each fish were rinsed using phosphate buffer saline (PBS) solution (100 mM; pH 7.4). Subsequently, tissue samples were carefully rid of any additional tissue fragments and placed on blotting paper to remove excess moisture. Each sample was individually weighed to determine the gonadosomatic index (GSI). A portion of each pair of testes was promptly immersed in Bouin’s fixative for histological analysis. Another portion of testicular fragment was minced in sterile PBS (1X, pH 7.4) to obtain a single cell suspension for the evaluation of cell cycle status and reactive oxygen species (ROS) level by flow cytometer. The residual portion of each testis was mixed with Tris-HCl buffer (50 mM, pH 7.4 with 1 mM EDTA, 1 mM PMSF, 100 mM sucrose, 1% leupeptin hemisulfate) and homogenized at 4°C followed by a brief sonication to obtain a 10% tissue homogenate for the measurement of melatonin concentration, levels of malondialdehyde (MDA), total nitrate and the levels of different enzymatic or non-enzymatic antioxidants. The homogenates were centrifuged at 13,000 g for 20 min at a temperature of 4°C. The supernatants were stored at a temperature of − 80°C for subsequent analysis (Hasan et al. 2014). Histological study of the testes under microscope Following dehydration of fixed testis tissue fragments, 5 µm thick sections were prepared using an automated microtome (Medimeas MRM-AT). The sections were stained with haematoxylin-eosin (H/E) stain. The relative percentage of different spermatogenic cells was analysed by calculating the count of seminiferous tubules containing a specific germ cell stage divided by the count of total seminiferous tubules, and then multiplying by 100 (Bhattacharya et al. 2007). The slides were visualized using a Leica DM2500 microscope equipped with a photo-micrographic device and image analysis software, LAS v4.9. Spermatogenic cells were examined by evaluating approximately 100 sections of seminiferous tubules from five histological slides per fish (20 sections for each slide), each taken at a scale of 100 µm. Abundance of spermatogenic haploid cells through flow cytometry analysis Percentage of haploid cells, maturational marker, in testis was determined by flow cytometric analysis in CytoFlex (Beckman Coulter, USA). Testicular fragments were minced in 1 mL sterile PBS (1X, pH 7.4) and filtered through a 40 µm cell strainer for single cell suspension. Following centrifugation at 2,500 g for 5 min at 4°C, cells were fixed in methanol for 15 min at RT and mixed with RNase A (20 mg/mL) after a chill shock at − 20°C for 2 min and kept at 4°C for overnight. Cells were stained with a red-fluorescent DNA dye, propidium iodide (PI) (10 µL of 1 mg/mL stock solution) for 35–40 min. Fluorescence signals from 10000 cells were recorded from the FL2 channel, measured at 565–595 nm. The results were further analysed by using CytExpert v2.4.0.28 software. Forward (FSC) and side scatter (SSC) plots were procured from both unstained and stained samples. A histogram plot, obtained from the ‘events’ within the polygon gate representing sample cells, measures the DNA content. To get the relative percentage of haploid cells, ‘line segment’ gating for respective phases viz. haploid, G 0 -G 1 , S and G 2 -M was further analyzed in different samples (Nóbrega et al. 2015). Estimation of intra-testicular melatonin concentration Testicular melatonin concentration was determined by ELISA kit (EU0199) following the instructions provided by the manufacturer (sensitivity: < 4.688 pg/mL, detection range: 7.813 to 500 pg/mL). In brief, 50 µL of test sample was mixed with an equal volume of biotin-labelled anti-melatonin antibody in each well for a period of 45 min at 37°C. 100 µL of HRP-streptavidin conjugated secondary antibody was added. Afterward, 90 µL of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was introduced. The absorbance was promptly measured at 450 nm after adding the stop solution using a microplate absorbance reader (Bio-Rad, iMark™), and the concentration was determined by interpolating the data with the help of a standard curve (Acharyya et al. 2023). Estimation of intra-testicular oxidative stress Generation of reactive oxygen species (ROS) Prepared testicular single cell suspension was provided with fluorescent probe DCFH 2 DA (ROS sensitive) and incubated for 15–20 min in dark and examined through a flow cytometer, CytoFlex (Beckman Coulter, USA) to measure the amount of oxidised DCF, indicator of intra-testicular ROS. Data were analysed by using CytExpert v2.4.0.28 software. A histogram plot was taken from the ‘events’ selected in polygon gate, where the X-axis representing the intensity of the probe and Y-axis representing the number of cells (count) containing the probe or DCF + cells. From the histogram plot, mean fluorescence intensity (MFI) of the probe for each sample was obtained to compare the amount of ROS generated in different samples (Aitken et al. 2013). Malondialdehyde (MDA) level Testicular homogenates were centrifuged at 3000 g for 15 min at 4°C (Tarsons, Spinwin, MC-05-R) and the supernatant was mixed with reagent containing TCA (20%), TBA (0.5%), and HCl (2.5 N). The mixture was incubated at boiling water bath for 20 min following centrifugation at 500 g for 15 min at room temperature (RT) and the absorbance of the supernatant was recorded at 532 nm in a microplate absorbance reader (Bio-Rad, iMark™) to measure MDA level, a marker of lipid peroxidation (Mondal et al. 2017). Estimation of intra-testicular nitrosative stress Total nitrate level, a marker of nitrosative stress, was measured in testicular sample by incubating with VCl 3 and Griess reagent containing sulphanilamide (1%) and NEDD (0.1%) for 30–45 min in dark condition. The absorbance was taken at 540 nm and concentration was determined from a standard curve of sodium nitrate (NaNO 3 ) (Miranda et al. 2001). Estimation of activity/level of enzymatic and non-enzymatic antioxidants Enzymatic antioxidants Superoxide dismutase activity (SOD) Testicular homogenate (25 µL) was mixed with 200 µL of phosphate buffer (50 mM, pH 7.4) containing reaction mixture 0.1 mM EDTA, 62 µM NBT, 98 µM NADH and 33 µM PMS. The absorbance was recorded at 560 nm in a microplate reader (Moniruzzaman et al. 2016). Catalase activity (CAT) The activity of intra-testicular catalase was measured by spectrophotometric method (Aebi 1984). Briefly, 20 µL of tissue homogenate was mixed with assay buffer, containing 50 mM Tris-Cl (pH ~ 8), 0.25 mM EDTA and 9 mM H 2 O 2 to the final volume of 1 mL in a quartz cuvette and kinetic study was performed at 240 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) for 1 min at 15 seconds intervals. Glutathione peroxidase activity (GPx) Briefly, 100 µL of testicular homogenate was mixed with OPD (0.4 mg/mL in phosphate citrate buffer, pH ~ 5) in presence of a co-substrate H 2 O 2 (0.013%). After 30 min of incubation at RT, the reaction was terminated with 3N H 2 SO 4 and absorbance was measured at 492 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) (Hasan et al. 2014). Glutathione S transferase activity (GST) 100 µL of sample was mixed with 900 µL of phosphate buffer (50 mM, pH 6.5) containing 100 mM CDNB and 100 mM GSH, and after 5 min of incubation at 30°C absorbance was measured at 340 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) at every 60 seconds for 5 min to measure the GST activity (Mondal et al. 2017). Non-enzymatic antioxidant Reduced glutathione (GSH) Testicular samples were mixed with equal volume of perchloric acid (5%) and centrifuged at 800 g for 10 min at 4°C (Tarsons, Spinwin, MC-05-R) followed by an incubation of the supernatant (100 µL) with phosphate buffer (100 mM, pH 8.0) and DTNB (4%) for 3 min at RT for the estimation of GSH level. Absorbance was measured at 412 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) at every 60 seconds for 5 min (Hasan et al. 2020). Statistical analysis The mean differences were compared by conducting a one-way ANOVA, as all the datasets passed the normality test ( p > 0.01) after subjecting the monthly mean data (mean ± S.E.M) of all the variables (GSI, germ cell profiles, percentages of haploid cell population, melatonin concentration, stress levels, enzymatic and non-enzymatic antioxidants) to a Shapiro-Wilks test for assessing normality. During the annual reproductive cycle, the monthly data for each parameter were pooled and expressed as the mean ± S.E.M. of the number of fish samples ( N = 5). This pooling was done because no significant differences were observed for each parameter between the consecutive months corresponding to a particular reproductive phase. In cases where the F values indicated statistical significance, a post hoc Duncan’s multiple range test (DMRT) was conducted at a significance level of p < 0.05 to compare the means. The data was analysed using GraphPad Prism v8.4.3, and presented graphically. Coefficient of correlation and regression (linear and LOESS) analyses Pearson’s correlation analysis was conducted to explore a potential relationship between melatonin concentration, relative percentage of spermatogenic cells, and biochemical parameters of testis (two variables at a time). Additionally, linear regression analysis was employed to assess the functional relationship between the response variable and independent (predictor) variable, specifically between two variables in isolation. A significance level of p < 0.05 was used as the threshold for determining statistical significance in both the analyses. Furthermore, the LOESS (locally estimated scatterplot smoothing) regression method was employed due to its flexibility in capturing the relationship between two variables without making any prior assumptions. A smoothing parameter of 0.66 was chosen to implement the LOESS regression method. Principal component analysis (PCA) Principal Components Analysis (PCA) was conducted to identify underlying patterns and relationships among various seasonal parameters within the dataset and to achieve dimension reduction. This approach was chosen based on the significant correlations observed through correlation and linear regression analyses among the independent variables. To interpret the total multidimensional dataset, coordinate or loading scores (coefficients of correlation), contribution values, and squared cosine values for each variable (representing the studied parameters) or individual (representing each studied sample) were performed. The loading values of each individual or variable for each considered principal component (PC) were also analyzed to extract the key variables that contribute significantly to the dataset interpretation. To assess the quality of representation, contribution of each individual or variable in the principal components (PCs) was determined by dividing 100 by the total number of individuals or variables. Additionally, squared cosine values were calculated to evaluate the quality of representation for each individual or variable across the PCs (Acharyya et al. 2023). Results Seasonal variations of gonadosomatic index (GSI) The GSI value, which reflects testicular growth and development, displayed significant ( F = 70.95; p < 0.001) rhythmic changes throughout the annual reproductive cycle. The highest seasonal peak value (0.254 ± 0.018, p < 0.001) was observed during the phase of ‘functional maturity’ (phase-IV). Following this peak, there was a gradual decrease in the GSI value, eventually reaching a significant lowest value (0.054 ± 0.003, p < 0.001) during the ‘relaxation and rehabilitation’ phase (phase-VI). The GSI value exhibited a consistent upward trend across the subsequent phases i.e., ‘slow spermatogenesis’ (phase-I), as well as the ‘early’ (phase-II) and ‘late’ (phase-III) phases of rapid spermatogenesis ( Fig. 1 ) . Seasonal distribution (%) of spermatogenic germ cells in the seminiferous tubules The seminiferous tubules exhibited temporal variations in the relative percentage (%) of developing germ cells [spermatogonia (SG), spermatocytes (SC), spermatids (ST), and spermatozoa (SZ)] in an annual cycle ( Fig. 2 ) . During phase-I, the relative abundance of SC containing seminiferous tubules was dominant significantly ( F = 386.5, p < 0.001) over SG, whereas, the mature stages (ST and SZ) were absent. ST were started appearing in phase-II, but SC was found most abundant germ cell type ( F = 1620, p < 0.001). In phase-III, four types of cells were observed including most mature SZ. In the subsequent phase-IV, seminiferous tubules containing SZ was increased significantly ( F = 173, p < 0.001) in compare to other germ cells. Depletion in the germ cells within the lobules was observed in phase-V along with empty seminiferous tubules. Succeeding phase-VI, mature stages (ST and SZ) were absent and SG was most abundant germ cell population ( F = 488.24, p < 0.001). Seasonal variations of haploid spermatogenic cells Observation of genomic DNA content by flow cytometer technique revealed specific haploid and diploid cell populations with definite ploidy number made us enable to detect the different stages through the progression of a cell cycle. Analysis identified testicular cells undergoing through G 0 -G 1 , S and G 2 -M phases of a cell cycle (diploid) along with quantification of the abundance of most mature cells ST and SZ (haploid) in different reproductive phases (Fig. 3 ) . The present study revealed a significant pattern of seasonal variation in the haploid germ cell population ( F = 5441.71, p < 0.001) and reached its annual peak at phase-IV (72.13 ± 0.07) ( Fig. 3 ) . Seasonal variations of intra-testicular melatonin concentration The results of the one-way ANOVA analysis showed a significant seasonal variations testicular melatonin concentration ( F = 33.55; p < 0.001). It gradually increased during phase-II and III and at the ‘phase of functional maturity’ (phase-IV), the highest amplitude was observed (115.9 ± 6.5 pg/g tissue). Subsequently, there was a rhythmic decrease in concentration as the reproductive phases progressed, reaching the lowest values during the ‘phase of slow spermatogenesis’ (phase-I) in testis (24.6 ± 3.5 pg/g tissue) ( Fig. 4 ) . Seasonal variations of intra-testicular oxidative stress Level of reactive oxygen species (ROS) Relative intensity of the ROS marker (2’,7’–dichlorofluorescein) in testicular cells were quantified as MFI in a flow cytometer in six reproductive phases. It showed significant seasonal variations ( F = 140.24, p < 0.001) ( Figs. 5 , 6 a ) with its peak during phase-III (MFI: 22710 ± 1529.08) and lowest value during phase-I (MFI: 4688.24 ± 110.8). Level of malondialdehyde (MDA) Intra-testicular oxidative status was determined by measuring of MDA level, which showed significant variations ( F = 175.27, p < 0.001) throughout an annual cycle ( Fig. 6 b ) . The level was found highest (46.42 ± 1.00 nM/mg protein) during phase-III and lowest during phase-VI (15.93 ± 0.73 nM/mg protein). Seasonal variations of intra-testicular total nitrate content Intra-testicular total nitrate content was found significantly variable ( F = 580.01, p < 0.001) throughout the annual cycle. The value found highest during phase-III (84.1 ± 1.45 µM/g tissue) and lowest during phase-I (15.12 ± 1.67 µM/g tissue) ( Fig. 6 c ) . Seasonal variations of intra-testicular antioxidants Enzymatic antioxidants Activities of SOD ( F = 275.98, p < 0.001), CAT ( F = 116.29, p < 0.001), GPx ( F = 385.28, p < 0.001) and GST ( F = 205.24, p < 0.001) were found identical variation throughout the annual testicular cycle. Lowest activity of each enzyme was found in phase-I, except GPx, which was lowest during phase-VI and then the activities of all the enzymes increased gradually and reached their respective peak during phase-IV ( Fig. 6 d-f ) . Non-enzymatic antioxidant (GSH) The seasonal pattern of GSH content ( F = 22.57, p < 0.001) was not identical like enzymatic antioxidants. The value found highest during phase-V (3.6 ± 0.06 µM/mg protein) and lowest during phase-II (2.02 ± 0.07 µM/mg protein) ( Fig. 6 f ) . Correlation between the studied variables Intra-testicular melatonin showed a significant positive correlation with the activities of enzymatic antioxidants (SOD, CAT, GPx and GST). The variables like GSI and mature germ cells (ST and SZ) also showed a significant positive correlation with the levels of intra-testicular MDA, total nitrate, enzymatic and non-enzymatic (GSH) antioxidants. Significant negative correlation was observed for the immature cells (SG and SC) with the levels of enzymatic antioxidants and GSH content. The level of significance for each correlation was mentioned in Table 1 . Regression (linear and LOESS) analysis between the studied variables Linear regression analysis showed that level of each stress marker (MDA, ROS and total nitrate) and enzymatic antioxidant (SOD, CAT, GPx and GST) was positively correlated with GSI and relative percentage (%) of seminiferous tubules containing mature germ cells, ST and SZ. Further, a positive correlation was also evidenced in the regression analysis between enzymatic antioxidant and testicular melatonin concentration ( Fig. 7 a and b) . Although, stress indicating markers regressed non-linearly with the percentage of seminiferous tubules containing mature germ cell and testicular melatonin content, so, we executed LOESS analysis. Smoothed bell-shaped regression curve of these parameters was identified taking percentage of SZ or testicular melatonin as an independent variable ( Fig. 8 ) . Principal component analysis (PCA) of the studied variables The first three principal components (PC1, PC2, and PC3) with eigenvalues greater than 1 (8.94, 2.83, and 1.41, respectively) collectively explained over 85% of the total variance. PC1 accounted for 59.62%, PC2 for 18.88%, and PC3 for 9.42%. Mean factor scores, contributions, and squared cosine values for each component were derived from PCA ( Table 2 ) . The expected average contribution was calculated to be 3.33% based on the presence of thirty individuals across all six phases. PC1 primarily differentiated phase-IV from I and VI, while PC2 and PC3 separated phase-III and II from other phases, respectively. Further analysis identified crucial variables for the differentiation of each phase ( Table 3 ) . The expected average contribution for each variable, calculated as 6.67% based on the total number of variables being 15, represents the minimum threshold value that indicates the significance of each variable in relation to their respective principal components (PCs). PC1, correlated with phase-IV, highlighted GSI, percentage of seminiferous tubules containing mature SZ, haploid cell population, melatonin concentration, and activities of enzymatic antioxidants. PC2, correlated with phase-III, emphasized levels of ST, MDA, ROS, and total nitrate. PC3, linked to phase-II, focused on SC and total nitrate levels. Collectively, PCA revealed PC1 encompassed the regulatory elements for phases-I, IV, and VI; PC2 for phase-III; and PC3 for phase-II ( Fig. 9 ) . Discussion To the best of our knowledge, current study in economically important Clarias batrachus , provides the first information of the correlation between melatonin and cellular stress in testis during six distinct phases of an annual gonadal cycle in any fish species under natural photo-thermal conditions. Intra-testicular melatonin concentration followed a significant circa-annual rhythmic pattern as it increased gradually and reached its peak during the phase of ‘functional maturity’ (phase-IV) and declined afterwards. Meanwhile, GSI level and the increasing changes (~ 5 folds) in the relative percentage of spermatids (ST) to spermatozoa (SZ) (most mature spermatogenic cell type) was also observed in same reproductive phase, indicating the onset of maturity in the annual cycle. High level of melatonin (~ 1.5 folds) in phase-IV compared to preceding phase demonstrating its probable involvement in the regulation of annual spermatogenic events (Acharyya et al. 2023) (Fig. 1 , 2 , 4 ). We also analysed the data gained by highly sensitive flow cytometry for demonstrating the cell cycle status throughout the seasons. It revealed that the haploid cells (ST and SZ) population was highest (72.13 ± 0.07) in phase-IV and ~ 3 folds higher than the earlier phase. This critical observation validated the present histological data on relative dynamics of spermatogenic haploid cell types. The dominancy of SZ at phase-IV and its positive association with testicular melatonin was significantly established by the study on same fish species (Acharyya et al. 2023). The observation was also supported by the PCA data that PC1 corresponding to phase-IV correlated with GSI, mature germ cell (SZ), haploid cell population and melatonin concentration (Fig. 9 , Table 2 , 3 ). Previous research has also argued the mitotic proliferation of spermatogonial stem cells for subsequent spermatocytes formation, and the initiation of meiosis leading to the development of spermatids and spermatozoa during the mature phase in catfish (Pathak and Lal 2010). In testis, dual role of reactive oxygen species (ROS) is crucial during the complex process of spermatogenic development: it helps in functional activation and also causes detrimental effects in mature spermatozoa depending upon its load (Martin-Hidalgo et al. 2019). Often elevated ROS interacts with the methylene groups abundant in the polyunsaturated fatty acids in the membrane of ST and SZ (Len et al. 2019) resulting oxidative damage during sperm maturation by higher degree of lipid peroxidation (Figueroa et al. 2018). However, there is only limited literature regarding the effects of melatonin on fish reproductive performance in male against oxidative damage (Félix et al. 2023). Melatonin exerts its antioxidative properties by effectively neutralizing a diverse range of free radicals such as hydrogen peroxide, hydroxyl and peroxy radicals, singlet oxygen, peroxynitrite anion, nitric oxide, and lipid peroxidation products (Tamura et al. 2020). In present in vivo study, we showed that level of ROS and MDA was ~ 3 folds and ~ 2 folds higher in phase-III compared to phase-IV, respectively (Fig. 6 a-b). Interestingly, these parameters showed significant positive correlation with GSI and percentage of seminiferous tubule containing ST (Table 1 ). The data possibly indicating melatonin mediated negative regulation of ROS and MDA and the transition from pro-oxidative to antioxidative status while shifting from immature to mature phase. In carp Catla catla , parallel observation also supported our findings that melatonin regulates MDA content during oocyte development in the mature phase (Hasan et al. 2014). Quality of stored sperm was improved and shown to increase after melatonin administration (0.5µM) in paddlefish ( Polyodon spathula ) through the inhibition of ROS generation (Gao et al. 2019). While there is scarcity of scientific literature regarding seasonal oxidative status associated with melatonin concentration in testis in any fish, a very few experimental observations suggested its antioxidative role in protecting spermatozoa (Acharyya et al. 2021). In addition of ROS, some researchers in mammalian studies suggested that nitrate (a form of NO), a useful marker of nitrosative stress, may stimulate spermatogenesis and the evidence in fish is supported by few studies (Wilson-Leedy and Ingermann 2011, Barman et al. 2013). In present work, total nitrate content was significantly varied throughout an annual cycle revealed that it reached its peak during phase-III (~ 2.5 folds higher than phase-IV) and showed significant positive correlation with GSI and percentage of seminiferous tubules containing ST and SZ (Table 1 ). The data was also well documented in PCA that ST, MDA, ROS, and total nitrate correlated with PC 2 corresponding to phase-III (Fig. 9 ). For the time being, influence of melatonin on testicular nitric oxide level throughout an annual cycle in any seasonally breeding animal including fish is not documented so far. Our study provided the pattern of inter-relationship between these two parameters suggested melatonin may regulate testicular nitrate in the quality control mechanism in sperm. In male Wistar rats with varicocele-induced elevated nitric oxide melatonin was found to counteract this effect (Semercioz et al. 2003). Further studies demonstrated the ability of melatonin to directly scavenge NO and detoxify peroxynitrite in cell-free systems as well as in human spermatozoa (Reiter et al. 2007; du Plessis et al. 2010). Administration of sodium nitroprusside increased GSI, the activities of testicular steroidogenic enzymes (3β-HSD and 17β-HSD) as well as concentration of testosterone supporting the stimulatory role of NO in sperm maturation (Singh and Lal 2017). A very recent study on frozen pig sperm revealed that melatonin increased sperm viability and motility by decreasing NO production (Lee and Lee 2023). Collectively, these findings suggested that melatonin may influence the seasonal variability in total nitrate content in the testis of fish but the specific mechanism requires carefully controlled experimental observation. The membrane of fish spermatozoa is particularly susceptible to ROS resulting impairment of its motility and fertility (Figueroa et al. 2018; Len et al. 2019). In order to mitigate cellular stress, a specific defense system consisting of both enzymatic and non-enzymatic antioxidants aiming to establish a biological equilibrium with the generation of free radicals in testis (Acharyya et al. 2021; Félix et al. 2023). SOD coverts superoxide anion into hydrogen peroxide which is independently neutralized by heme-containing CAT, and selenium-containing GPx enzyme into water (Hasan et al. 2014; Lu et al. 2015). In concert with SOD, both CAT and GPx constitute the primary enzyme defense mechanism against the harmful effects of pro-oxidants (Mondal et al. 2017). A crucial enzyme in the glutathione redox cycle is GST which catalyzes the reaction involving the conjugation of non-enzymatic GSH with xenobiotic substrates for detoxification. However, GSH is a vital tripeptide that combats oxidative stress and sustains the reduced cellular environment (Maitra and Hasan 2016). The activities of SOD, CAT, GPx and GST gradually increased and reached peak in ‘mature phase’ in an annual reproductive cycle (Fig. 6 ). Concomitantly, testicular melatonin in the present investigation was shown to positively correlate with the activities of different enzymatic antioxidants SOD, CAT, GPx and GST arguing its potential role in the regulation of oxidative balance (Fig. 7 a, Table 1 ). Some interesting observations like increase of ~ 2.5 folds for SOD and ~ 1.5 folds for CAT, GPx and GST activities were evidenced in phase-IV compared to the preceding phase-III. GSI, seminiferous tubule percentage of tubules containing ST and SZ had significant positive correlation with the activities of SOD, CAT, GPx and GST suggesting the establishment of antioxidative milieu during the maturation of spermatozoa (Félix et al. 2023) (Fig. 7 b ) . During the annual reproductive cycle, the levels of reduced glutathione (GSH) in testicular tissue extracts remained stable without significant seasonal variations. Notably, GSH levels were high consistently through phases III to V of the cycle. This may be due to abundant GSH was crucial to the maturation of spermatozoa and its protection against oxidative stress. The suggested hypothesis also evidenced in an experiment on Oreochromis niloticus , showed that protection of sperm was confirmed by more GSH production in testis (Hamed et al. 2016). Further, addition of 0.1mM melatonin significantly decreased H 2 O 2 (100 µM)-induced oxidative damage by the elevation in GSH content and activities of SOD, CAT and GPx in rabbit sperm (Zhu et al. 2019). However, in order to justify the correlation of testicular melatonin with various regulatory variables involved in spermatogenesis, contribution of non-linear LOESS regression data was significant along with linear regression and PCA. LOESS analysis of ROS, MDA and total nitrate with seminiferous tubules (%) containing SZ or intra-testicular melatonin revealed identical smooth bell-shaped curve. Collectively, these observations suggested melatonin possibly exerts important negative regulation on oxidative and/or nitrosative stress during germ cell maturation to SZ through the up-regulation of the enzymatic activities of SOD, CAT, GPx and GST in the course seasonal cycle (Fig. 8 ). Endogenous or exogenous administration of melatonin acts as an antioxidant by activating essential antioxidative enzymes in conjunction with the maturation of carp oocytes (Hasan et al. 2014). It also resulted in a significant reduction in malondialdehyde levels, a marker for intracellular oxidative stress (Moniruzzaman et al. 2016; Mondal et al. 2017). But in the present scenario, stress scavenging activity of melatonin in testicular cycle in any fish remains obscure. Addition of 0.1 mM melatonin significantly increased sperm viability and membrane integrity followed by the decrease of intracellular reactive oxygen species and lipid peroxidation in cryopreserved human sperm (Deng et al. 2017). A similar study in Mithun ( Bos frontalis ) observed that implantation of melatonin (18 mg/50 kg bw) significantly improved sperm quality parameters associated with increased total antioxidant capacity in freeze thawed sperm (Perumal et al. 2018). A recent study also proposed a potential correlation between melatonin levels, steroidogenic activity, and antioxidants during sperm maturation highlighting a possible role in testicular function in fish gilthead seabream ( Sparus aurata ), European seabass ( Dicentrarchus labrax ), and Senegalese sole ( Solea senegalensis ) (Félix et al. 2023). Conclusions In summary, this study represents the pioneering effort to examine the annual rhythmicity of testicular melatonin and its correlation with gonadal development indices (GSI and the relative abundance of lobules containing different stages of growing germ cells) as well as oxidative status in a commercially significant catfish, Clarias batrachus . The investigation will be conducted across six distinct reproductive phases under natural photo-thermal conditions. The significant of this study is to warrant a possibility to unveil the issue that intra-testicular melatonin concentrations vary rhythmically in an annual testicular cycle in accordance with the sexual status of the fish. The study also demonstrated that melatonin concentration positively correlated with GSI, relative percentage of most advanced germ cell stages (ST and SZ), and the activities of antioxidative enzymes, indicating that the hormone may have a positive effect in maintenance of cellular stress during spermatogenesis. The significance of this study lies in its potential to shed light by examining the rhythmicity of melatonin levels within the testes. This research attempts to provide valuable insights into the regulatory mechanisms governing annual testicular function and its association with melatonin. Looking ahead, the application of in vitro experiment holds immense promise in unravelling the complex network governing melatonin mediated regulation of spermatogenesis. This holistic approach may unveil intricate mechanisms in reproductive processes and making it a fascinating avenue for exploration in fish reproduction. Declarations Acknowledgements The award of SRF [F. No. 09/1156(0006)/2018-EMR-I] to AA from Council of Scientific and Industrial Research (CSIR), India is appreciatively acknowledged. The authors are also thankful to Mr. Joydeep Das and Mr. Sayan Mukherjee for their academic support. Author contribution statement AA: Conceptualization, Methodology, Validation, Investigation, Formal analysis, Writing- original draft, review & editing. KNH: Supervision, Funding acquisition, Conceptualization, Methodology, Data Analysis, Writing- review & editing. Funding This work was financially supported by University Grants Commission (UGC) [F.30- 448/2018(BSR)] to KNH, DST-FIST [N.SR/FST-LS1/2018/173], Govt. of India. Data availability Data of the experiment will be available upon request to the corresponding author. Ethical approval The Institutional Animal Ethics Committee (IAEC), Department of Zoology (Reg. No. 1973/GO/Re/S/17/CPCSEA), Sidho Kanho Birsha University, reviewed and approved animal care, hygiene, and laboratory procedures in accordance with the guidelines of CPCSEA, New Delhi, Govt. of India. Competing interest The authors declare that there is no competing interest. References Acharyya A, Das J, Hasan KN (2021) Melatonin as a multipotent component of fish feed: basic information for its potential application in aquaculture. Front Mar Sci 8:734066. https://doi.org/10.3389/fmars.2021.734066 Acharyya A, Das J, Hasan KN (2023) Rhythmicity in testicular melatonin and its correlation with the dynamics of spermatogenic cells in an annual reproductive cycle of Clarias batrachus under natural photo-thermal conditions. Theriogenology 208:15-27. https://doi.org/10.1016/j.theriogenology.2023.06.001 Aebi H (1984) Catalase in vitro . 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Cryobiolog y 88:1-8. https://doi.org/10.1016/j.cryobiol.2019.04.009 Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.doc Table2.doc Table3.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3943922","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272596829,"identity":"7aca5b99-2f92-47f5-83d7-3fec941a9acd","order_by":0,"name":"Akash Acharyya","email":"","orcid":"","institution":"Sidho-Kanho- Birsha University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akash","middleName":"","lastName":"Acharyya","suffix":""},{"id":272596830,"identity":"ac3b8e91-b0d6-42a0-8e9b-34733783fcee","order_by":1,"name":"Kazi Nurul Hasan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACCSA+DGbJHz4A4suQoEWCLQFE8hClhRnC4jEAUYS1SLYff3i4oKZO3ly65/OrGzUWPAzsh49uwKdFmich4fCMY4cNd845u8065xjQYTxpaTfwaZFjSDhwmIftAOOGA7nbjHPYgFokeMzwa+F/2HCY51+d/YYDOc+Mc/4RoUVaIpnhMG8bc+KGGznMj3PbiNAiOeMZUEvf4eQNZ46ZMef2SfCwEfKLxPn0x595vtXZbjje/Phzzrc6OX72w8fwakEGbBJgkljlIMD8gRTVo2AUjIJRMHIAALfvSi9dU+MpAAAAAElFTkSuQmCC","orcid":"","institution":"Sidho-Kanho- Birsha University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kazi","middleName":"Nurul","lastName":"Hasan","suffix":""}],"badges":[],"createdAt":"2024-02-09 19:04:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3943922/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3943922/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51175716,"identity":"8ee95d59-4fba-4ead-878c-76564025c7ce","added_by":"auto","created_at":"2024-02-15 12:34:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19506,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical presentation of the seasonal values (mean ± S.E.M., \u003cem\u003eN\u003c/em\u003e = 5) of gonadosomatic index (GSI) during six reproductive phases (phase I-VI) in catfish \u003cem\u003eClarias batrachus\u003c/em\u003e. Uppercase alphabets indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) among six phases following one-way ANOVA and \u003cem\u003epost hoc\u003c/em\u003e Duncan’s multiple range test (DMRT). Same letter denotes no significant differences.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/4bafb6aa939d2be2e8323279.jpg"},{"id":51175913,"identity":"7a35d5a1-d46a-4c88-acfe-93a49c2611cd","added_by":"auto","created_at":"2024-02-15 12:42:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82874,"visible":true,"origin":"","legend":"\u003cp\u003eGraphic demonstration of the values (mean ± S.E.M., \u003cem\u003eN\u003c/em\u003e = 5) of relative percentage (%) of seminiferous tubules containing four types of germ cells viz. spermatogonia (SG), spermatocytes (SC), spermatids (ST) and spermatozoa (SZ) during six reproductive phases in catfish \u003cem\u003eClarias batrachus\u003c/em\u003e. Uppercase alphabets indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) among four germ cell types in each reproductive phase using one-way ANOVA followed by DMRT. Same letter represents no significant differences. ND: Not detected.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/8d722fa5d43f3b2e6d6392eb.jpg"},{"id":51175717,"identity":"f1b03ba4-57e8-4cab-8a21-b8d196f010bd","added_by":"auto","created_at":"2024-02-15 12:34:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106569,"visible":true,"origin":"","legend":"\u003cp\u003eA histogram plot, obtained from the ‘events’ within the polygon gate representing the status of the cell cycle progression by measuring the DNA content viz. haploid, G\u003csub\u003e0\u003c/sub\u003e-G\u003csub\u003e1\u003c/sub\u003e, S and G\u003csub\u003e2\u003c/sub\u003e-M of testicular germ cells analysed in a flow cytometer in each phase after propidium iodide (PI) staining. The percentage of each stage during progression of cell cycle was mentioned in the parenthesis. Bar diagram summarizing the seasonal values (mean ± S.E.M., \u003cem\u003eN\u003c/em\u003e = 5) of only haploid cells (ST and SZ) population during six reproductive phases (phase I to VI) in catfish \u003cem\u003eClarias batrachus\u003c/em\u003e. Uppercase alphabets indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) among six phases following one-way ANOVA and \u003cem\u003epost hoc\u003c/em\u003e Duncan’s multiple range test (DMRT). ND: Not detected.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/24ab606e24408944a1585c64.jpg"},{"id":51175722,"identity":"931c7b0c-3acb-4ac2-a54a-d14b06f90331","added_by":"auto","created_at":"2024-02-15 12:34:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20030,"visible":true,"origin":"","legend":"\u003cp\u003eBox and whisker plot showing seasonal variations in testicular melatonin concentrations represented as quartiles: median, 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentile (\u003cem\u003eN\u003c/em\u003e = 5) during different reproductive phases (phase I to VI). Different uppercase alphabets denote statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) among six phases following one-way ANOVA and DMRT. Same letter indicates no significant differences.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/7dcafd6423ea88fc9bf2fdd9.jpg"},{"id":51175723,"identity":"7680078b-9694-48bb-a595-0f5657801169","added_by":"auto","created_at":"2024-02-15 12:34:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78618,"visible":true,"origin":"","legend":"\u003cp\u003eHistograms representing flow cytometric determination of ROS generation in testicular cells after DCFH\u003csub\u003e2\u003c/sub\u003eDA staining during six reproductive phases (phase I to VI) in catfish \u003cem\u003eClarias batrachus\u003c/em\u003e. The X-axis and Y-axis represents the intensity and the number of cells (count) of the probe or DCF\u003csup\u003e+\u003c/sup\u003e cells, respectively. Fluorescent intensity was obtained from total 10000 records and statistically converted into mean fluorescence intensity (MFI) of the sample.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/c5935a1491ed59b5ce3f3647.jpg"},{"id":51175725,"identity":"fb950c46-77be-4173-9e1e-d841edbea7fd","added_by":"auto","created_at":"2024-02-15 12:34:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105408,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical presentation of the seasonal values (mean ± S.E.M., \u003cem\u003eN\u003c/em\u003e = 5) of \u003cstrong\u003e(a)\u003c/strong\u003e mean fluorescence intensity (MFI) of DCFH\u003csub\u003e2\u003c/sub\u003eDA, \u003cstrong\u003e(b)\u003c/strong\u003e MDA level, \u003cstrong\u003e(c)\u003c/strong\u003e total nitrate level, activity/content of \u003cstrong\u003e(d)\u003c/strong\u003e SOD, \u003cstrong\u003e(e)\u003c/strong\u003e CAT \u003cstrong\u003e(f)\u003c/strong\u003e GPx, GST and GSH in testis during six reproductive phases (phase I to VI) in catfish \u003cem\u003eClarias batrachus\u003c/em\u003e. Uppercase alphabets indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) of each parameter among six reproductive phases using one-way ANOVA followed by DMRT. Same letter denotes no significant differences.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/454f85352dc6e9c9b1c52697.jpg"},{"id":51175912,"identity":"3835a259-69ae-4a3d-905f-b935efee8740","added_by":"auto","created_at":"2024-02-15 12:42:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":129276,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plots showing linear regression analysis of different cellular stress markers and enzymatic antioxidants with \u003cstrong\u003e(a) \u003c/strong\u003eGSI and testicular melatonin and \u003cstrong\u003e(b)\u003c/strong\u003e relative percentage (%) of seminiferous tubules containing mature ST and SZ. r2 and \u003cem\u003ep\u003c/em\u003e values respectively represent the goodness-of-fit and the significant level for analysis of each pair of variables.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/32572ad7c2b2ca5e963a4cfc.jpg"},{"id":51175914,"identity":"066ec999-5d98-4e57-bed3-3f89b092ab65","added_by":"auto","created_at":"2024-02-15 12:42:42","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":141708,"visible":true,"origin":"","legend":"\u003cp\u003eLocally estimated scatterplot smoothing (LOESS) analysis correlated stress parameters (MDA, ROS and total nitrate) with \u003cstrong\u003e(a) \u003c/strong\u003erelative percentage (%) of seminiferous tubules containing most mature germ cell, SZ and \u003cstrong\u003e(b)\u003c/strong\u003e testicular melatonin.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/796604d7f63c012ea10f1ed0.jpg"},{"id":51175726,"identity":"0fbd09a5-1d9c-4633-ab99-b5c0e0ce885e","added_by":"auto","created_at":"2024-02-15 12:34:42","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":89393,"visible":true,"origin":"","legend":"\u003cp\u003eThe bi-plot resulting from principal component analysis (PCA) visually displayed both individuals (samples) and variables (parameters) in two principal components (PC1 and PC2). It groups individuals in different reproductive phases together within a 95% eclipse, and the pointed arrows indicated the pattern of contribution of each variable to their respective PCs. Scree plot (displayed as inset) represents the eigenvalues and cumulative percentage of variance in different principal components.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/e201fe394c98c922ed33b61f.jpg"},{"id":64336037,"identity":"4229e1df-4043-4895-921b-c7b258407d27","added_by":"auto","created_at":"2024-09-11 21:44:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1660558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/4ed411bf-0b82-41e7-8608-5b6de91b8390.pdf"},{"id":51175718,"identity":"c4ab3724-ad53-4ca3-b6dd-6a18110225b1","added_by":"auto","created_at":"2024-02-15 12:34:41","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34304,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.doc","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/c3f5b586d3915b22500f0a2b.doc"},{"id":51175720,"identity":"4d0bc437-53bd-45a0-9ef9-8613774858d3","added_by":"auto","created_at":"2024-02-15 12:34:42","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":36352,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.doc","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/a54da109099d43fafb915ccf.doc"},{"id":51175915,"identity":"ac6cd242-f81c-40b7-8f80-57b928e9ebf5","added_by":"auto","created_at":"2024-02-15 12:42:42","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":49152,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.doc","url":"https://assets-eu.researchsquare.com/files/rs-3943922/v1/0a98c443c0529dca29d456c6.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Correlation of testicular melatonin and cellular stress in an annual spermatogenic cycle of Clarias batrachus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMelatonin, \u003cem\u003eN\u003c/em\u003e-acetyl-5-methoxy-tryptamine, is a chronobiotic molecule derived rhythmically from L-tryptophan and predominantly produced within the pineal gland (Lerner et al. 1958). It reaches a crescendo during the nocturnal embrace of darkness, and remarkably synchronizes with external environmental cues such as photoperiod, water temperature, rainfall and more (Falc\u0026oacute;n et al. 2010). The level of melatonin experiences synchronized rhythmic fluctuations both in the pineal gland and in the bloodstream, a pattern observed consistently across various vertebrates. In addition to pineal gland, several other non-pineal tissues like retina, gut, liver, testis, and ovary contribute significantly to the synthesis of this hormone in all vertebrates, including fish (Hasan et al. 2016; Acharyya et al. 2023). Ubiquitous presence of melatonin in every living species studied to date suggests that it is a phylogenetically ancient molecule and believed to have evolved as a scavenger of free radicals, and over the course of evolution, it acquired diverse functions within various living systems (Maitra and Hasan 2016). Numerous scientific reports have provided compelling evidence highlighting the crucial role of melatonin in the regulation of reproduction across different fish species (Das et al. 2022; Hasan et al. 2023). The correlation of melatonin with gonadal cycle is studied mostly in female fish but such information is very scarce in male fish species. The hormone significantly regulates oocyte development in an annual ovarian cycle of carp depending upon the reproductive status of the animal (Maitra et al. 2005). Study also revealed that exogenous melatonin causes precocious or tardy effects by modulating ovarian and testicular steroidogenesis in fish (Bhattacharya et al. 2007; Lombardo et al. 2012). The involvement of melatonin in the growth and maturation of germ cells is mediated through hypothalamo-hypophyseal-gonadal (HPG) axis as a hormone or as an antioxidant (Maitra and Hasan 2016; Acharyya et al. 2021). In zebrafish, a 10-day exposure to melatonin (100 nM and 1 \u0026micro;M) in tank water significantly influences seasonal reproduction through kisspeptin mediated hypothalamic gonadotropin-releasing hormone (GnRH) production, and directly regulating the transcription of \u003cem\u003emprα\u003c/em\u003e and \u003cem\u003emprβ\u003c/em\u003e genes in the gonads involve in oocyte competence and maturation (Carnevali et al. 2011). Endocrine action of melatonin primarily operated through the regulation of HPG axis as suggested by the presence of G-protein coupled receptors [MT1 (Mel1a), MT2 (Mel1b), and Mel1c] in different target sites namely suprachiasmatic nucleus (SCN), pars tuberalis (PT) of pituitary gland, testis and ovary (Klosen et al. 2019). Rhythmic expression of ovarian melatonin receptors throughout an annual cycle in fish also highlighted its involvement in oocyte growth and maturation by interacting with HPG axis (Moniruzzaman and Maitra 2012). Several seasonal and non-seasonal breeding animal species express testicular receptor proteins, indicating melatonin may also regulate receptor mediated, a component of HPG axis, reproductive functions (Yang et al. 2014; Kozioł et al. 2020).\u003c/p\u003e \u003cp\u003eFurther, the amphiphilic nature of melatonin enables it to readily penetrate cellular barriers, allowing it to function as a widespread direct scavenger of free radicals and an indirect antioxidant (Tan et al. 1993). This characteristic has unveiled a new realm of possibilities, suggesting that besides its receptor-mediated hormonal actions, melatonin may also exert receptor-independent effects in regulating reproduction (Gao et al. 2022). Melatonin critically scavenges oxygen free radicals during oocyte maturation and ovulation, mitigating the detrimental effects of reactive oxygen species (ROS) and protecting oocytes from oxidative damage (Silva et al. 2024). Ovarian melatonin follows a seasonal pattern in carp, reaching its peak at spawning and this temporal variation shows a significant negative correlation with malondialdehyde (MDA) level, an indicator of intracellular oxidative stress (Hasan et al. 2014). This observation also suggests that melatonin minimizes oxidative stress in the ovary by augmenting the activities of antioxidative enzymes, specifically superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) throughout the phases of oocyte growth and maturation. Currently, study also suggested exogenous melatonin mediated meiotic maturation through heightening of antioxidative capacity in late-vitellogenic oocytes of turbot (\u003cem\u003eScophthalmus maximus\u003c/em\u003e) (Zhang et al. 2023). To our knowledge, no controlled studies are available to demonstrate the role of testicular melatonin as an antioxidant in the regulation of spermatogenic cycle in any fish species.\u003c/p\u003e \u003cp\u003eA very recent study delves into the intricate dynamics of melatonin levels, reproductive parameters, and environmental factors in adult male catfish (\u003cem\u003eClarias batrachus\u003c/em\u003e) throughout an annual reproductive cycle (Acharyya et al. 2023). Notably, both intra-testicular melatonin concentrations and the dynamics of spermatogenic cells are synchronized with different meteorological factors like photoperiod, temperature, and rainfall. However, there is a dearth of information regarding the influence of melatonin on the regulation of cellular stress levels in testis over the course of an annual cycle in any seasonally breeding animals. Consequently, the present study endeavours to elucidate the developmental dynamics of male germ cells through the analysis of changes in ploidy (based on genomic DNA content) during the course of cell cycle progression in Catfish, \u003cem\u003eClarias batrachus\u003c/em\u003e. Additionally, it also aims to discern any relation of melatonin in the regulation of intra-testicular oxidative (ROS) and nitrosative (total nitrate) stress during spermatogenesis under natural photo-thermal conditions, encompassing six distinct phases within the annual reproductive cycle.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and regents\u003c/h2\u003e \u003cp\u003eMelatonin ELISA kit was purchased from Fine Test (EU0199), Wuhan Fine Biotech Co., Ltd. Wuhan, China. Nicotinamide Adenine Dinucleotide, reduced (NADH), nitro blue tetrazolium (NBT), and glutathione, reduced (GSH) were purchased from SRL, Mumbai, India. 5-methylphenazinium methyl sulphate (PMS), phenylmethylsulfonyl fluoride (PMSF), 1-chloro-2, 4-dinitrobenzen (CDNB), o-phenylenediamine (OPD), trichloro acetic acid (TCA), thiobarbituric acid (TBA), 5, 5\u0026rsquo;-dithiobis (2-nitrobenzoate) (DTNB), 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescin diacetate (DCFH\u003csub\u003e2\u003c/sub\u003eDA), Propidium iodide (PI), RNase A, vanadium (III) chloride (VCl\u003csub\u003e3\u003c/sub\u003e), sulfanilamide, N-(1-napthyl) ethylenediamine dihydrochloride (NEDD) and other chemicals were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA unless otherwise specified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCollection of fish\u003c/h2\u003e \u003cp\u003eTo ensure the reliability and consistency of our findings, we gathered adult male catfish \u003cem\u003eClarias batrachus\u003c/em\u003e (males and females were separated on the basis of sexual dimorphic characteristics), weighing about 210\u0026thinsp;\u0026plusmn;\u0026thinsp;25 g, from the water bodies near the university campus, Purulia, India [23\u0026deg;35' N, 86\u0026deg;33' E], in each month of an annual cycle. The testicular development consists of six distinct phases, phase of slow spermatogenesis (January to February) or Phase-I, early phase of rapid spermatogenesis (March to April) or Phase-II, late phase of rapid spermatogenesis (May to June) or Phase-III, phase of functional maturity (July to mid-August) or Phase-IV, phase of depletion (late-August to mid-October) or Phase-V and phase of relaxation and rehabilitation (mid-October to December) or Phase-VI (Acharyya et al. 2023). Immediately following capture in each month, five adult male catfish (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) were promptly transported to the laboratory and allowed to acclimate for seven days in a glass aquarium under natural photo-thermal conditions. This acclimation period aimed to reduce the stress caused during the collection and transportation of the fish before they were sacrificed. The fish were provided with commercial fish feed pellets, Growfin, from Growel Feeds Pvt. Ltd., (Andhra Pradesh, India), enriched with 40% crude protein along with 12% moisture and crude fat, fibre (6% and 3%, respectively). The Institutional Animal Ethics Committee (IAEC), Department of Zoology (Reg. No. 1973/GO/Re/S/17/CPCSEA), Sidho Kanho Birsha University, reviewed and approved animal care, hygiene, and laboratory procedures in accordance with the guidelines of CPCSEA, New Delhi, Govt. of India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCollection and preparation of testicular samples\u003c/h2\u003e \u003cp\u003eFollowing the dissection, the testes from each fish were rinsed using phosphate buffer saline (PBS) solution (100 mM; pH 7.4). Subsequently, tissue samples were carefully rid of any additional tissue fragments and placed on blotting paper to remove excess moisture. Each sample was individually weighed to determine the gonadosomatic index (GSI). A portion of each pair of testes was promptly immersed in Bouin\u0026rsquo;s fixative for histological analysis. Another portion of testicular fragment was minced in sterile PBS (1X, pH 7.4) to obtain a single cell suspension for the evaluation of cell cycle status and reactive oxygen species (ROS) level by flow cytometer. The residual portion of each testis was mixed with Tris-HCl buffer (50 mM, pH 7.4 with 1 mM EDTA, 1 mM PMSF, 100 mM sucrose, 1% leupeptin hemisulfate) and homogenized at 4\u0026deg;C followed by a brief sonication to obtain a 10% tissue homogenate for the measurement of melatonin concentration, levels of malondialdehyde (MDA), total nitrate and the levels of different enzymatic or non-enzymatic antioxidants. The homogenates were centrifuged at 13,000 \u003cb\u003eg\u003c/b\u003e for 20 min at a temperature of 4\u0026deg;C. The supernatants were stored at a temperature of \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis (Hasan et al. 2014).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological study of the testes under microscope\u003c/h2\u003e \u003cp\u003eFollowing dehydration of fixed testis tissue fragments, 5 \u0026micro;m thick sections were prepared using an automated microtome (Medimeas MRM-AT). The sections were stained with haematoxylin-eosin (H/E) stain. The relative percentage of different spermatogenic cells was analysed by calculating the count of seminiferous tubules containing a specific germ cell stage divided by the count of total seminiferous tubules, and then multiplying by 100 (Bhattacharya et al. 2007). The slides were visualized using a Leica DM2500 microscope equipped with a photo-micrographic device and image analysis software, LAS v4.9. Spermatogenic cells were examined by evaluating approximately 100 sections of seminiferous tubules from five histological slides per fish (20 sections for each slide), each taken at a scale of 100 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAbundance of spermatogenic haploid cells through flow cytometry analysis\u003c/h2\u003e \u003cp\u003ePercentage of haploid cells, maturational marker, in testis was determined by flow cytometric analysis in CytoFlex (Beckman Coulter, USA). Testicular fragments were minced in 1 mL sterile PBS (1X, pH 7.4) and filtered through a 40 \u0026micro;m cell strainer for single cell suspension. Following centrifugation at 2,500 \u003cb\u003eg\u003c/b\u003e for 5 min at 4\u0026deg;C, cells were fixed in methanol for 15 min at RT and mixed with RNase A (20 mg/mL) after a chill shock at \u0026minus;\u0026thinsp;20\u0026deg;C for 2 min and kept at 4\u0026deg;C for overnight. Cells were stained with a red-fluorescent DNA dye, propidium iodide (PI) (10 \u0026micro;L of 1 mg/mL stock solution) for 35\u0026ndash;40 min. Fluorescence signals from 10000 cells were recorded from the FL2 channel, measured at 565\u0026ndash;595 nm. The results were further analysed by using CytExpert v2.4.0.28 software. Forward (FSC) and side scatter (SSC) plots were procured from both unstained and stained samples. A histogram plot, obtained from the \u0026lsquo;events\u0026rsquo; within the polygon gate representing sample cells, measures the DNA content. To get the relative percentage of haploid cells, \u0026lsquo;line segment\u0026rsquo; gating for respective phases viz. haploid, G\u003csub\u003e0\u003c/sub\u003e-G\u003csub\u003e1\u003c/sub\u003e, S and G\u003csub\u003e2\u003c/sub\u003e-M was further analyzed in different samples (N\u0026oacute;brega et al. 2015).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of intra-testicular melatonin concentration\u003c/h2\u003e \u003cp\u003eTesticular melatonin concentration was determined by ELISA kit (EU0199) following the instructions provided by the manufacturer (sensitivity: \u0026lt; 4.688 pg/mL, detection range: 7.813 to 500 pg/mL). In brief, 50 \u0026micro;L of test sample was mixed with an equal volume of biotin-labelled anti-melatonin antibody in each well for a period of 45 min at 37\u0026deg;C. 100 \u0026micro;L of HRP-streptavidin conjugated secondary antibody was added. Afterward, 90 \u0026micro;L of 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB) substrate was introduced. The absorbance was promptly measured at 450 nm after adding the stop solution using a microplate absorbance reader (Bio-Rad, iMark\u0026trade;), and the concentration was determined by interpolating the data with the help of a standard curve (Acharyya et al. 2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of intra-testicular oxidative stress\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eGeneration of reactive oxygen species (ROS)\u003c/h2\u003e \u003cp\u003ePrepared testicular single cell suspension was provided with fluorescent probe DCFH\u003csub\u003e2\u003c/sub\u003eDA (ROS sensitive) and incubated for 15\u0026ndash;20 min in dark and examined through a flow cytometer, CytoFlex (Beckman Coulter, USA) to measure the amount of oxidised DCF, indicator of intra-testicular ROS. Data were analysed by using CytExpert v2.4.0.28 software. A histogram plot was taken from the \u0026lsquo;events\u0026rsquo; selected in polygon gate, where the X-axis representing the intensity of the probe and Y-axis representing the number of cells (count) containing the probe or DCF\u003csup\u003e+\u003c/sup\u003e cells. From the histogram plot, mean fluorescence intensity (MFI) of the probe for each sample was obtained to compare the amount of ROS generated in different samples (Aitken et al. 2013).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde (MDA) level\u003c/h2\u003e \u003cp\u003eTesticular homogenates were centrifuged at 3000 \u003cb\u003eg\u003c/b\u003e for 15 min at 4\u0026deg;C (Tarsons, Spinwin, MC-05-R) and the supernatant was mixed with reagent containing TCA (20%), TBA (0.5%), and HCl (2.5 N). The mixture was incubated at boiling water bath for 20 min following centrifugation at 500 \u003cb\u003eg\u003c/b\u003e for 15 min at room temperature (RT) and the absorbance of the supernatant was recorded at 532 nm in a microplate absorbance reader (Bio-Rad, iMark\u0026trade;) to measure MDA level, a marker of lipid peroxidation (Mondal et al. 2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of intra-testicular nitrosative stress\u003c/h2\u003e \u003cp\u003eTotal nitrate level, a marker of nitrosative stress, was measured in testicular sample by incubating with VCl\u003csub\u003e3\u003c/sub\u003e and Griess reagent containing sulphanilamide (1%) and NEDD (0.1%) for 30\u0026ndash;45 min in dark condition. The absorbance was taken at 540 nm and concentration was determined from a standard curve of sodium nitrate (NaNO\u003csub\u003e3\u003c/sub\u003e) (Miranda et al. 2001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of activity/level of enzymatic and non-enzymatic antioxidants\u003c/h2\u003e \u003cp\u003eEnzymatic antioxidants\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSuperoxide dismutase activity (SOD)\u003c/h2\u003e \u003cp\u003eTesticular homogenate (25 \u0026micro;L) was mixed with 200 \u0026micro;L of phosphate buffer (50 mM, pH 7.4) containing reaction mixture 0.1 mM EDTA, 62 \u0026micro;M NBT, 98 \u0026micro;M NADH and 33 \u0026micro;M PMS. The absorbance was recorded at 560 nm in a microplate reader (Moniruzzaman et al. 2016).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCatalase activity (CAT)\u003c/h2\u003e \u003cp\u003eThe activity of intra-testicular catalase was measured by spectrophotometric method (Aebi 1984). Briefly, 20 \u0026micro;L of tissue homogenate was mixed with assay buffer, containing 50 mM Tris-Cl (pH\u0026thinsp;~\u0026thinsp;8), 0.25 mM EDTA and 9 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to the final volume of 1 mL in a quartz cuvette and kinetic study was performed at 240 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) for 1 min at 15 seconds intervals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGlutathione peroxidase activity (GPx)\u003c/h2\u003e \u003cp\u003eBriefly, 100 \u0026micro;L of testicular homogenate was mixed with OPD (0.4 mg/mL in phosphate citrate buffer, pH\u0026thinsp;~\u0026thinsp;5) in presence of a co-substrate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.013%). After 30 min of incubation at RT, the reaction was terminated with 3N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and absorbance was measured at 492 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) (Hasan et al. 2014).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGlutathione S transferase activity (GST)\u003c/h2\u003e \u003cp\u003e100 \u0026micro;L of sample was mixed with 900 \u0026micro;L of phosphate buffer (50 mM, pH 6.5) containing 100 mM CDNB and 100 mM GSH, and after 5 min of incubation at 30\u0026deg;C absorbance was measured at 340 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) at every 60 seconds for 5 min to measure the GST activity (Mondal et al. 2017).\u003c/p\u003e \u003cp\u003eNon-enzymatic antioxidant\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eReduced glutathione (GSH)\u003c/h2\u003e \u003cp\u003eTesticular samples were mixed with equal volume of perchloric acid (5%) and centrifuged at 800 \u003cb\u003eg\u003c/b\u003e for 10 min at 4\u0026deg;C (Tarsons, Spinwin, MC-05-R) followed by an incubation of the supernatant (100 \u0026micro;L) with phosphate buffer (100 mM, pH 8.0) and DTNB (4%) for 3 min at RT for the estimation of GSH level. Absorbance was measured at 412 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) at every 60 seconds for 5 min (Hasan et al. 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe mean differences were compared by conducting a one-way ANOVA, as all the datasets passed the normality test (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.01) after subjecting the monthly mean data (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M) of all the variables (GSI, germ cell profiles, percentages of haploid cell population, melatonin concentration, stress levels, enzymatic and non-enzymatic antioxidants) to a Shapiro-Wilks test for assessing normality. During the annual reproductive cycle, the monthly data for each parameter were pooled and expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. of the number of fish samples (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5). This pooling was done because no significant differences were observed for each parameter between the consecutive months corresponding to a particular reproductive phase. In cases where the F values indicated statistical significance, a \u003cem\u003epost hoc\u003c/em\u003e Duncan\u0026rsquo;s multiple range test (DMRT) was conducted at a significance level of \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 to compare the means. The data was analysed using GraphPad Prism v8.4.3, and presented graphically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCoefficient of correlation and regression (linear and LOESS) analyses\u003c/h2\u003e \u003cp\u003ePearson\u0026rsquo;s correlation analysis was conducted to explore a potential relationship between melatonin concentration, relative percentage of spermatogenic cells, and biochemical parameters of testis (two variables at a time). Additionally, linear regression analysis was employed to assess the functional relationship between the response variable and independent (predictor) variable, specifically between two variables in isolation. A significance level of \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was used as the threshold for determining statistical significance in both the analyses. Furthermore, the LOESS (locally estimated scatterplot smoothing) regression method was employed due to its flexibility in capturing the relationship between two variables without making any prior assumptions. A smoothing parameter of 0.66 was chosen to implement the LOESS regression method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis (PCA)\u003c/h2\u003e \u003cp\u003ePrincipal Components Analysis (PCA) was conducted to identify underlying patterns and relationships among various seasonal parameters within the dataset and to achieve dimension reduction. This approach was chosen based on the significant correlations observed through correlation and linear regression analyses among the independent variables. To interpret the total multidimensional dataset, coordinate or loading scores (coefficients of correlation), contribution values, and squared cosine values for each variable (representing the studied parameters) or individual (representing each studied sample) were performed. The loading values of each individual or variable for each considered principal component (PC) were also analyzed to extract the key variables that contribute significantly to the dataset interpretation. To assess the quality of representation, contribution of each individual or variable in the principal components (PCs) was determined by dividing 100 by the total number of individuals or variables. Additionally, squared cosine values were calculated to evaluate the quality of representation for each individual or variable across the PCs (Acharyya et al. 2023).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003eSeasonal variations of gonadosomatic index (GSI)\u003c/h2\u003e\n \u003cp\u003eThe GSI value, which reflects testicular growth and development, displayed significant (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;70.95; \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) rhythmic changes throughout the annual reproductive cycle. The highest seasonal peak value (0.254\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) was observed during the phase of \u0026lsquo;functional maturity\u0026rsquo; (phase-IV). Following this peak, there was a gradual decrease in the GSI value, eventually reaching a significant lowest value (0.054\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) during the \u0026lsquo;relaxation and rehabilitation\u0026rsquo; phase (phase-VI). The GSI value exhibited a consistent upward trend across the subsequent phases i.e., \u0026lsquo;slow spermatogenesis\u0026rsquo; (phase-I), as well as the \u0026lsquo;early\u0026rsquo; (phase-II) and \u0026lsquo;late\u0026rsquo; (phase-III) phases of rapid spermatogenesis \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eSeasonal distribution (%) of spermatogenic germ cells in the seminiferous tubules\u003c/h2\u003e\n \u003cp\u003eThe seminiferous tubules exhibited temporal variations in the relative percentage (%) of developing germ cells [spermatogonia (SG), spermatocytes (SC), spermatids (ST), and spermatozoa (SZ)] in an annual cycle \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. During phase-I, the relative abundance of SC containing seminiferous tubules was dominant significantly (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;386.5, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) over SG, whereas, the mature stages (ST and SZ) were absent. ST were started appearing in phase-II, but SC was found most abundant germ cell type (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1620, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). In phase-III, four types of cells were observed including most mature SZ. In the subsequent phase-IV, seminiferous tubules containing SZ was increased significantly (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;173, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) in compare to other germ cells. Depletion in the germ cells within the lobules was observed in phase-V along with empty seminiferous tubules. Succeeding phase-VI, mature stages (ST and SZ) were absent and SG was most abundant germ cell population (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;488.24, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001).\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eSeasonal variations of haploid spermatogenic cells\u003c/h2\u003e\n \u003cp\u003eObservation of genomic DNA content by flow cytometer technique revealed specific haploid and diploid cell populations with definite ploidy number made us enable to detect the different stages through the progression of a cell cycle. Analysis identified testicular cells undergoing through G\u003csub\u003e0\u003c/sub\u003e-G\u003csub\u003e1\u003c/sub\u003e, S and G\u003csub\u003e2\u003c/sub\u003e-M phases of a cell cycle (diploid) along with quantification of the abundance of most mature cells ST and SZ (haploid) in different reproductive phases (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. The present study revealed a significant pattern of seasonal variation in the haploid germ cell population (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5441.71, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) and reached its annual peak at phase-IV (72.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eSeasonal variations of intra-testicular melatonin concentration\u003c/h2\u003e\n \u003cp\u003eThe results of the one-way ANOVA analysis showed a significant seasonal variations testicular melatonin concentration (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;33.55; \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). It gradually increased during phase-II and III and at the \u0026lsquo;phase of functional maturity\u0026rsquo; (phase-IV), the highest amplitude was observed (115.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 pg/g tissue). Subsequently, there was a rhythmic decrease in concentration as the reproductive phases progressed, reaching the lowest values during the \u0026lsquo;phase of slow spermatogenesis\u0026rsquo; (phase-I) in testis (24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 pg/g tissue) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eSeasonal variations of intra-testicular oxidative stress\u003c/h2\u003e\n \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e\n \u003ch2\u003eLevel of reactive oxygen species (ROS)\u003c/h2\u003e\n \u003cp\u003eRelative intensity of the ROS marker (2\u0026rsquo;,7\u0026rsquo;\u0026ndash;dichlorofluorescein) in testicular cells were quantified as MFI in a flow cytometer in six reproductive phases. It showed significant seasonal variations (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;140.24, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) \u003cstrong\u003e(\u003c/strong\u003eFigs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cstrong\u003e)\u003c/strong\u003e with its peak during phase-III (MFI: 22710\u0026thinsp;\u0026plusmn;\u0026thinsp;1529.08) and lowest value during phase-I (MFI: 4688.24\u0026thinsp;\u0026plusmn;\u0026thinsp;110.8).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003eLevel of malondialdehyde (MDA)\u003c/h2\u003e\n \u003cp\u003eIntra-testicular oxidative status was determined by measuring of MDA level, which showed significant variations (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;175.27, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) throughout an annual cycle \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cstrong\u003e)\u003c/strong\u003e. The level was found highest (46.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 nM/mg protein) during phase-III and lowest during phase-VI (15.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 nM/mg protein).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSeasonal variations of intra-testicular total nitrate content\u003c/h3\u003e\n\u003cp\u003eIntra-testicular total nitrate content was found significantly variable (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;580.01, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) throughout the annual cycle. The value found highest during phase-III (84.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 \u0026micro;M/g tissue) and lowest during phase-I (15.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67 \u0026micro;M/g tissue) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003eSeasonal variations of intra-testicular antioxidants\u003c/h2\u003e\n \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\n \u003ch2\u003eEnzymatic antioxidants\u003c/h2\u003e\n \u003cp\u003eActivities of SOD (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;275.98, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001), CAT (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;116.29, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001), GPx (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;385.28, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) and GST (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;205.24, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) were found identical variation throughout the annual testicular cycle. Lowest activity of each enzyme was found in phase-I, except GPx, which was lowest during phase-VI and then the activities of all the enzymes increased gradually and reached their respective peak during phase-IV \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed-f\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cdiv id=\"Sec33\" class=\"Section4\"\u003e\n \u003ch2\u003eNon-enzymatic antioxidant (GSH)\u003c/h2\u003e\n \u003cp\u003eThe seasonal pattern of GSH content (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22.57, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) was not identical like enzymatic antioxidants. The value found highest during phase-V (3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u0026micro;M/mg protein) and lowest during phase-II (2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;M/mg protein) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n \u003ch2\u003eCorrelation between the studied variables\u003c/h2\u003e\n \u003cp\u003eIntra-testicular melatonin showed a significant positive correlation with the activities of enzymatic antioxidants (SOD, CAT, GPx and GST). The variables like GSI and mature germ cells (ST and SZ) also showed a significant positive correlation with the levels of intra-testicular MDA, total nitrate, enzymatic and non-enzymatic (GSH) antioxidants. Significant negative correlation was observed for the immature cells (SG and SC) with the levels of enzymatic antioxidants and GSH content. The level of significance for each correlation was mentioned in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eRegression (linear and LOESS) analysis between the studied variables\u003c/h3\u003e\n\u003cp\u003eLinear regression analysis showed that level of each stress marker (MDA, ROS and total nitrate) and enzymatic antioxidant (SOD, CAT, GPx and GST) was positively correlated with GSI and relative percentage (%) of seminiferous tubules containing mature germ cells, ST and SZ. Further, a positive correlation was also evidenced in the regression analysis between enzymatic antioxidant and testicular melatonin concentration \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea \u003cstrong\u003eand b)\u003c/strong\u003e. Although, stress indicating markers regressed non-linearly with the percentage of seminiferous tubules containing mature germ cell and testicular melatonin content, so, we executed LOESS analysis. Smoothed bell-shaped regression curve of these parameters was identified taking percentage of SZ or testicular melatonin as an independent variable \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003ch3\u003ePrincipal component analysis (PCA) of the studied variables\u003c/h3\u003e\n\u003cp\u003eThe first three principal components (PC1, PC2, and PC3) with eigenvalues greater than 1 (8.94, 2.83, and 1.41, respectively) collectively explained over 85% of the total variance. PC1 accounted for 59.62%, PC2 for 18.88%, and PC3 for 9.42%. Mean factor scores, contributions, and squared cosine values for each component were derived from PCA \u003cstrong\u003e(\u003c/strong\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. The expected average contribution was calculated to be 3.33% based on the presence of thirty individuals across all six phases. PC1 primarily differentiated phase-IV from I and VI, while PC2 and PC3 separated phase-III and II from other phases, respectively. Further analysis identified crucial variables for the differentiation of each phase \u003cstrong\u003e(\u003c/strong\u003eTable \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. The expected average contribution for each variable, calculated as 6.67% based on the total number of variables being 15, represents the minimum threshold value that indicates the significance of each variable in relation to their respective principal components (PCs). PC1, correlated with phase-IV, highlighted GSI, percentage of seminiferous tubules containing mature SZ, haploid cell population, melatonin concentration, and activities of enzymatic antioxidants. PC2, correlated with phase-III, emphasized levels of ST, MDA, ROS, and total nitrate. PC3, linked to phase-II, focused on SC and total nitrate levels. Collectively, PCA revealed PC1 encompassed the regulatory elements for phases-I, IV, and VI; PC2 for phase-III; and PC3 for phase-II \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, current study in economically important \u003cem\u003eClarias batrachus\u003c/em\u003e, provides the first information of the correlation between melatonin and cellular stress in testis during six distinct phases of an annual gonadal cycle in any fish species under natural photo-thermal conditions. Intra-testicular melatonin concentration followed a significant circa-annual rhythmic pattern as it increased gradually and reached its peak during the phase of \u0026lsquo;functional maturity\u0026rsquo; (phase-IV) and declined afterwards. Meanwhile, GSI level and the increasing changes (~\u0026thinsp;5 folds) in the relative percentage of spermatids (ST) to spermatozoa (SZ) (most mature spermatogenic cell type) was also observed in same reproductive phase, indicating the onset of maturity in the annual cycle. High level of melatonin (~\u0026thinsp;1.5 folds) in phase-IV compared to preceding phase demonstrating its probable involvement in the regulation of annual spermatogenic events (Acharyya et al. 2023) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We also analysed the data gained by highly sensitive flow cytometry for demonstrating the cell cycle status throughout the seasons. It revealed that the haploid cells (ST and SZ) population was highest (72.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) in phase-IV and ~\u0026thinsp;3 folds higher than the earlier phase. This critical observation validated the present histological data on relative dynamics of spermatogenic haploid cell types. The dominancy of SZ at phase-IV and its positive association with testicular melatonin was significantly established by the study on same fish species (Acharyya et al. 2023). The observation was also supported by the PCA data that PC1 corresponding to phase-IV correlated with GSI, mature germ cell (SZ), haploid cell population and melatonin concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Previous research has also argued the mitotic proliferation of spermatogonial stem cells for subsequent spermatocytes formation, and the initiation of meiosis leading to the development of spermatids and spermatozoa during the mature phase in catfish (Pathak and Lal 2010).\u003c/p\u003e \u003cp\u003eIn testis, dual role of reactive oxygen species (ROS) is crucial during the complex process of spermatogenic development: it helps in functional activation and also causes detrimental effects in mature spermatozoa depending upon its load (Martin-Hidalgo et al. 2019). Often elevated ROS interacts with the methylene groups abundant in the polyunsaturated fatty acids in the membrane of ST and SZ (Len et al. 2019) resulting oxidative damage during sperm maturation by higher degree of lipid peroxidation (Figueroa et al. 2018). However, there is only limited literature regarding the effects of melatonin on fish reproductive performance in male against oxidative damage (F\u0026eacute;lix et al. 2023). Melatonin exerts its antioxidative properties by effectively neutralizing a diverse range of free radicals such as hydrogen peroxide, hydroxyl and peroxy radicals, singlet oxygen, peroxynitrite anion, nitric oxide, and lipid peroxidation products (Tamura et al. 2020). In present \u003cem\u003ein vivo\u003c/em\u003e study, we showed that level of ROS and MDA was ~\u0026thinsp;3 folds and ~\u0026thinsp;2 folds higher in phase-III compared to phase-IV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). Interestingly, these parameters showed significant positive correlation with GSI and percentage of seminiferous tubule containing ST (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The data possibly indicating melatonin mediated negative regulation of ROS and MDA and the transition from pro-oxidative to antioxidative status while shifting from immature to mature phase. In carp \u003cem\u003eCatla catla\u003c/em\u003e, parallel observation also supported our findings that melatonin regulates MDA content during oocyte development in the mature phase (Hasan et al. 2014). Quality of stored sperm was improved and shown to increase after melatonin administration (0.5\u0026micro;M) in paddlefish (\u003cem\u003ePolyodon spathula\u003c/em\u003e) through the inhibition of ROS generation (Gao et al. 2019). While there is scarcity of scientific literature regarding seasonal oxidative status associated with melatonin concentration in testis in any fish, a very few experimental observations suggested its antioxidative role in protecting spermatozoa (Acharyya et al. 2021). In addition of ROS, some researchers in mammalian studies suggested that nitrate (a form of NO), a useful marker of nitrosative stress, may stimulate spermatogenesis and the evidence in fish is supported by few studies (Wilson-Leedy and Ingermann 2011, Barman et al. 2013). In present work, total nitrate content was significantly varied throughout an annual cycle revealed that it reached its peak during phase-III (~\u0026thinsp;2.5 folds higher than phase-IV) and showed significant positive correlation with GSI and percentage of seminiferous tubules containing ST and SZ (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The data was also well documented in PCA that ST, MDA, ROS, and total nitrate correlated with PC 2 corresponding to phase-III (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). For the time being, influence of melatonin on testicular nitric oxide level throughout an annual cycle in any seasonally breeding animal including fish is not documented so far. Our study provided the pattern of inter-relationship between these two parameters suggested melatonin may regulate testicular nitrate in the quality control mechanism in sperm. In male Wistar rats with varicocele-induced elevated nitric oxide melatonin was found to counteract this effect (Semercioz et al. 2003). Further studies demonstrated the ability of melatonin to directly scavenge NO and detoxify peroxynitrite in cell-free systems as well as in human spermatozoa (Reiter et al. 2007; du Plessis et al. 2010). Administration of sodium nitroprusside increased GSI, the activities of testicular steroidogenic enzymes (3β-HSD and 17β-HSD) as well as concentration of testosterone supporting the stimulatory role of NO in sperm maturation (Singh and Lal 2017). A very recent study on frozen pig sperm revealed that melatonin increased sperm viability and motility by decreasing NO production (Lee and Lee 2023). Collectively, these findings suggested that melatonin may influence the seasonal variability in total nitrate content in the testis of fish but the specific mechanism requires carefully controlled experimental observation.\u003c/p\u003e \u003cp\u003eThe membrane of fish spermatozoa is particularly susceptible to ROS resulting impairment of its motility and fertility (Figueroa et al. 2018; Len et al. 2019). In order to mitigate cellular stress, a specific defense system consisting of both enzymatic and non-enzymatic antioxidants aiming to establish a biological equilibrium with the generation of free radicals in testis (Acharyya et al. 2021; F\u0026eacute;lix et al. 2023). SOD coverts superoxide anion into hydrogen peroxide which is independently neutralized by heme-containing CAT, and selenium-containing GPx enzyme into water (Hasan et al. 2014; Lu et al. 2015). In concert with SOD, both CAT and GPx constitute the primary enzyme defense mechanism against the harmful effects of pro-oxidants (Mondal et al. 2017). A crucial enzyme in the glutathione redox cycle is GST which catalyzes the reaction involving the conjugation of non-enzymatic GSH with xenobiotic substrates for detoxification. However, GSH is a vital tripeptide that combats oxidative stress and sustains the reduced cellular environment (Maitra and Hasan 2016). The activities of SOD, CAT, GPx and GST gradually increased and reached peak in \u0026lsquo;mature phase\u0026rsquo; in an annual reproductive cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Concomitantly, testicular melatonin in the present investigation was shown to positively correlate with the activities of different enzymatic antioxidants SOD, CAT, GPx and GST arguing its potential role in the regulation of oxidative balance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Some interesting observations like increase of ~\u0026thinsp;2.5 folds for SOD and ~\u0026thinsp;1.5 folds for CAT, GPx and GST activities were evidenced in phase-IV compared to the preceding phase-III. GSI, seminiferous tubule percentage of tubules containing ST and SZ had significant positive correlation with the activities of SOD, CAT, GPx and GST suggesting the establishment of antioxidative milieu during the maturation of spermatozoa (F\u0026eacute;lix et al. 2023) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. During the annual reproductive cycle, the levels of reduced glutathione (GSH) in testicular tissue extracts remained stable without significant seasonal variations. Notably, GSH levels were high consistently through phases III to V of the cycle. This may be due to abundant GSH was crucial to the maturation of spermatozoa and its protection against oxidative stress. The suggested hypothesis also evidenced in an experiment on \u003cem\u003eOreochromis niloticus\u003c/em\u003e, showed that protection of sperm was confirmed by more GSH production in testis (Hamed et al. 2016). Further, addition of 0.1mM melatonin significantly decreased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;M)-induced oxidative damage by the elevation in GSH content and activities of SOD, CAT and GPx in rabbit sperm (Zhu et al. 2019).\u003c/p\u003e \u003cp\u003eHowever, in order to justify the correlation of testicular melatonin with various regulatory variables involved in spermatogenesis, contribution of non-linear LOESS regression data was significant along with linear regression and PCA. LOESS analysis of ROS, MDA and total nitrate with seminiferous tubules (%) containing SZ or intra-testicular melatonin revealed identical smooth bell-shaped curve. Collectively, these observations suggested melatonin possibly exerts important negative regulation on oxidative and/or nitrosative stress during germ cell maturation to SZ through the up-regulation of the enzymatic activities of SOD, CAT, GPx and GST in the course seasonal cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Endogenous or exogenous administration of melatonin acts as an antioxidant by activating essential antioxidative enzymes in conjunction with the maturation of carp oocytes (Hasan et al. 2014). It also resulted in a significant reduction in malondialdehyde levels, a marker for intracellular oxidative stress (Moniruzzaman et al. 2016; Mondal et al. 2017). But in the present scenario, stress scavenging activity of melatonin in testicular cycle in any fish remains obscure. Addition of 0.1 mM melatonin significantly increased sperm viability and membrane integrity followed by the decrease of intracellular reactive oxygen species and lipid peroxidation in cryopreserved human sperm (Deng et al. 2017). A similar study in Mithun (\u003cem\u003eBos frontalis\u003c/em\u003e) observed that implantation of melatonin (18 mg/50 kg bw) significantly improved sperm quality parameters associated with increased total antioxidant capacity in freeze thawed sperm (Perumal et al. 2018). A recent study also proposed a potential correlation between melatonin levels, steroidogenic activity, and antioxidants during sperm maturation highlighting a possible role in testicular function in fish gilthead seabream (\u003cem\u003eSparus aurata\u003c/em\u003e), European seabass (\u003cem\u003eDicentrarchus labrax\u003c/em\u003e), and Senegalese sole (\u003cem\u003eSolea senegalensis\u003c/em\u003e) (F\u0026eacute;lix et al. 2023).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study represents the pioneering effort to examine the annual rhythmicity of testicular melatonin and its correlation with gonadal development indices (GSI and the relative abundance of lobules containing different stages of growing germ cells) as well as oxidative status in a commercially significant catfish, \u003cem\u003eClarias batrachus\u003c/em\u003e. The investigation will be conducted across six distinct reproductive phases under natural photo-thermal conditions. The significant of this study is to warrant a possibility to unveil the issue that intra-testicular melatonin concentrations vary rhythmically in an annual testicular cycle in accordance with the sexual status of the fish. The study also demonstrated that melatonin concentration positively correlated with GSI, relative percentage of most advanced germ cell stages (ST and SZ), and the activities of antioxidative enzymes, indicating that the hormone may have a positive effect in maintenance of cellular stress during spermatogenesis. The significance of this study lies in its potential to shed light by examining the rhythmicity of melatonin levels within the testes. This research attempts to provide valuable insights into the regulatory mechanisms governing annual testicular function and its association with melatonin. Looking ahead, the application of \u003cem\u003ein vitro\u003c/em\u003e experiment holds immense promise in unravelling the complex network governing melatonin mediated regulation of spermatogenesis. This holistic approach may unveil intricate mechanisms in reproductive processes and making it a fascinating avenue for exploration in fish reproduction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe award of SRF [F. No. 09/1156(0006)/2018-EMR-I] to AA from Council of Scientific and Industrial Research (CSIR), India is appreciatively acknowledged. The authors are also thankful to Mr. Joydeep Das and Mr. Sayan Mukherjee for their academic support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAA:\u003c/strong\u003e Conceptualization, Methodology, Validation, Investigation, Formal analysis, Writing- original draft, review \u0026amp; editing. \u003cstrong\u003eKNH:\u003c/strong\u003e Supervision, Funding acquisition, Conceptualization, Methodology, Data Analysis, Writing- review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by University Grants Commission (UGC) [F.30- 448/2018(BSR)] to KNH, DST-FIST [N.SR/FST-LS1/2018/173], Govt. of India. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData of the experiment will be available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Institutional Animal Ethics Committee (IAEC), Department of Zoology (Reg. No. 1973/GO/Re/S/17/CPCSEA), Sidho Kanho Birsha University, reviewed and approved animal care, hygiene, and laboratory procedures in accordance with the guidelines of CPCSEA, New Delhi, Govt. of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no competing interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcharyya A, Das J, Hasan KN (2021) Melatonin as a multipotent component of fish feed: basic information for its potential application in aquaculture. Front Mar Sci 8:734066. https://doi.org/10.3389/fmars.2021.734066\u003c/li\u003e\n\u003cli\u003eAcharyya A, Das J, Hasan KN (2023) Rhythmicity in testicular melatonin and its correlation with the dynamics of spermatogenic cells in an annual reproductive cycle of \u003cem\u003eClarias batrachus\u003c/em\u003e under natural photo-thermal conditions. 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Anim Reprod Sci 147:10-16. https://doi.org/10.1016/j.anireprosci.2014.03.017\u003c/li\u003e\n\u003cli\u003eZhang J, Li F, Zhang X et al (2023) Melatonin improves turbot oocyte meiotic maturation and antioxidant capacity, inhibits apoptosis-related genes mRNAs \u003cem\u003ein vitro\u003c/em\u003e. Antioxidants 12:1389. https://doi.org/10.3390/antiox12071389\u003c/li\u003e\n\u003cli\u003eZhu Z, Li R, Lv Y, Zeng W (2019) Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress. Cryobiolog\u003cem\u003ey\u003c/em\u003e 88:1-8. https://doi.org/10.1016/j.cryobiol.2019.04.009\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Clarias batrachus, Melatonin, Testis, Antioxidants, ROS, Cell cycle","lastPublishedDoi":"10.21203/rs.3.rs-3943922/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3943922/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMelatonin, primarily produced by pineal gland, shows a rhythmic pattern of synthesis in response to environmental cues. Hitherto, the role of testicular melatonin in the regulation of annual spermatogenic cycle is indeterminate in any fish species. Present study aims to explore a correlation, for the first time, of melatonin with cellular stress levels in accordance to the rhythmic progression of spermatogenesis through six distinct reproductive phases of an annual gonadal cycle of catfish, \u003cem\u003eClarias batrachus\u003c/em\u003e under natural photo-thermal conditions. We assessed gonadosomatic index (GSI), testicular melatonin concentrations, relative percentage of haploid cells by using DNA dye in the seminiferous tubules, and different intracellular stress markers. The concentration of testicular melatonin displayed a distinct seasonal pattern, reaching their peak during the \u0026ldquo;phase of functional maturity\u0026rdquo;. Interestingly, the levels of 2\u0026rsquo;,7\u0026rsquo;\u0026ndash;dichlorofluorescein [reactive oxygen species (ROS) marker], malondialdehyde (MDA) and RNS (total nitrate) were found significantly lowered at the verge of \u0026ldquo;mature state\u0026rdquo;. Correlation and LOESS regression analyses showed a striking positive relationship between testicular melatonin concentration and the activity of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). Principal component (PC) analysis also identified the key components of \u0026ldquo;mature state\u0026rdquo; viz., GSI, testicular melatonin, haploid cell population (spermatid and spermatozoa), and the activity of enzymatic antioxidants. Cumulatively, melatonin may have a role in growth and maturation of spermatogenic cells by reducing the cellular stress to augment the quality of germ cells required for seed production in the culture of any fish species.\u003c/p\u003e","manuscriptTitle":"Correlation of testicular melatonin and cellular stress in an annual spermatogenic cycle of Clarias batrachus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 12:34:37","doi":"10.21203/rs.3.rs-3943922/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de7822e9-a5ee-463c-a592-a8e24e41f02f","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-11T21:36:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-15 12:34:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3943922","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3943922","identity":"rs-3943922","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}