Paternal chronic exposure to copper nanoparticles (CuNPs) impairs testicular androgen and estrogen signalling in adult male offspring in mice

Paternal chronic exposure to copper nanoparticles (CuNPs) impairs testicular androgen and estrogen signalling in adult male offspring in mice | 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 Paternal chronic exposure to copper nanoparticles (CuNPs) impairs testicular androgen and estrogen signalling in adult male offspring in mice Vanrohlu Nicy, Guruswami Gurusubramanian, Vikas Kumar Roy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8068308/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2026 Read the published version in Biological Trace Element Research → Version 1 posted 12 You are reading this latest preprint version Abstract At present, the use of copper nanoparticles (CuNPs) is very common for various human applications. Despite its uses to improve human welfare, the toxicity of CuNPs is well documented. It can cause toxicity to different vital organs, including the male reproductive organs, or testes. Whether the offspring of CuNP-treated males show testicular impairment has not been well documented. The present study investigated the effects of paternal exposure to CuNPs on the testes of male offspring (Swiss Albino mice) divided into the following four groups: a control group and 10 mg/kg, 100 mg/kg and 200 mg/kg exposure groups, whose only male parent was exposed to CuNPs at 0, 10, 100 and 200 mg/kg, respectively, for 70 days, which covered two spermatogenic cycles. The findings reveal that exposure of male parents to CuNPs at higher doses, 100 and 200 mg/kg, compromises spermatogenesis in the testes of male offspring due to decreased germ cell proliferation. Our results showed that oxidative stress was also elevated in the male offspring of male parents treated with a higher dose of CuNPs. However, elevated apoptosis (increased caspase3) was noted in the male offspring of all treated male parental groups. Circulating testosterone and estrogen levels were elevated in the F1 males of higher dose CuNP-treated male paternal groups; however, the expression of androgen receptor (AR), apelin receptor (APJ) and estrogen receptor-β (Erβ) was decreased in the male offspring from all treated parental groups. In conclusion, paternal exposure to CuNPs was found to disrupt spermatogenesis and steroid signalling function in the offspring of F1 males. Testis CuNPs F1 males Spermatogenesis Testosterone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction As human living standards rise quickly, thousands of chemical pollutants are released into the environment by industry, agriculture and pharmaceuticals, causing serious harm to the plants and animal health [ 1 , 2 ]. Copper metals have been used in various human applications [ 3 ], and copper is also a trace metal that regulates various biological functions in humans and animals [ 4 ]. Other forms of copper, such as the nano form and nano particles, are being used in water treatment, agriculture, livestock, wood preservation and textile manufacturing [ 5 ]. Despite being used in various applications, copper nanoparticles (CuNPs) have also been known to cause health hazards [ 6 ]. It has been shown that different concentrations (50, 100 and 200 mg/kg) of CuNPs damages the kidneys and liver; moreover, a higher dose of CuNPs showed necrosis in the proximal kidney tubules [ 7 ]. Previous studies from our group and another laboratory have also shown that CuNPs exposure impairs testicular functions in males [ 8 , 9 ]. Nanoparticles have been known to acts as endocrine disruptors, which alter the male reproductive system by affecting testicular structure, steroid hormone levels and spermatogenesis [ 10 ]. It has also been shown that the direct exposure of parental germ cells to toxic materials causes epigenetic changes and intergenerational inheritance [ 11 , 12 , 13 ]. The inheritance of transgenerational toxic effects through parents affects the physiological functions of offspring [ 11 ]. We have shown that exposure of male mice to CuNPs causes endocrine imbalances such as suppression of testosterone levels and decreased androgen receptor (AR) expression [ 8 , 9 ]. However, whether the F1 male offspring of treated male mice manifest reproductive dysfunction has not been investigated. It has been shown that parental exposure of C. elegans to CuNPs at a dose of 150 mg/L causes developmental and reproductive toxicity in their progeny [ 14 ]. It has also been suggested that paternal endocrine disruptors affect the offspring [ 11 , 15 ]. Other endocrine disruptors, like bisphenol A exposure at the adult or embryonic stage in male mice, led to abnormal offspring with decreased sperm quality and spermatogenesis [ 16 ]. Only recently, it was shown that CuNPs exposure for one spermatogenic cycle followed by termination of CuNPs treatment for another 35 days, induces oxidative stress, impairs spermatogenesis and compromises epigenetic modification, poor sperm quality and developmental anomalies in male offspring [ 17 ]. The above- mentioned evidence, which showed that paternal exposure to chemical toxicants and endocrine disruptors causes anomalies in the offspring, still needs further investigation. As in our previous studies, we showed compromised testicular functions along with altered reproductive parameters in mice [ 8 , 9 ], therefore, we hypothesised that male offspring of CuNPs-treated male parents would show compromised testicular functions. The aim of the present work was to investigate the effects of paternal CuNPs exposure on the testes of male offspring by evaluating various parameters, such as circulating steroids levels, proliferation, apoptosis and oxidative stress. 2. Material and methods 2.1 Animals and experimental design The animals used in this experiment were the male offspring of three-month-old Swiss albino ( Mus musculus ) male mice taken from an inbred colony of the Animal Housing Facility, Zoology Department, Mizoram University, Aizawl, Mizoram. Animals were housed under controlled conditions, on a 12 h light/dark cycle, with a temperature of 25 ± 2℃ and food and water ad libitum. All animal experiments were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. The animals experiment protocol was approved by the Institutional Ethical Committee (process number: MZU/IAEC/2020/10), Mizoram University, Aizawl, Mizoram, India and was compiled with ARRIVE guidelines. Four different experimental groups with five animals in each group were formed. Group 1 (the control) was given phosphate buffered saline (PBS), and groups 2, 3 and 4 were administered, by oral gavage, 10, 100 and 200 mg/kg bw, respectively, CuNPs dissolved in PBS for 70 consecutive days, according to previous experiments [ 8 , 18 , 19 ]. However, it has been documented that 10–12 mg/person/day is an acceptable copper intake besides natural intake of 1–2 mg/person/day [ 20 , 21 ]. Furthermore, based on this data, an acceptable upper exposure limits 0.15 mg/kgbw/day was fixed by European Food Safety Authority [ 22 ], and 0.2 mg/kgbw/day exposure has been permitted for adult. The dose in our study is 50, 500 and 1000 times higher than these permissible limits for 10, 100 and 200 mg/kg CuNPs respectively. After the treatment was over, the treated male mice were house with healthy female mice (1 male per 1 female mouse) and were allowed to breed. The number of pups was recorded, and the pups were separated from their parents at weaning and allowed to attain adulthood (three months old) to further investigate whether treatment of male parents with CuNPs showed a notable effect on the next generation. In the present study, we investigated the possible effects of CuNPs exposure of male parents on testicular activity in male offspring. Previously we have shown that 70 days of consecutive CuNPs treatment resulted in impaired spermatogenesis in male mice [ 8 ]. The male offspring of treated males were compared with offspring derived from untreated male parents. The male mouse offspring (n = 5, each individual from different treated or control male parents) were euthanized at three months of age by decapitation under mild anaesthesia (90 mg/kg ketamine and 4.5 mg/kg xylazine, by intraperitoneal injection of 1 ml/kg bw). The collected blood was centrifuged to separate the serum and was used for hormonal analysis. The sperm parameters were analysed immediately, the testis from one side was frozen at -20℃ while the other one was fixed with Bouins fluid (picric acid 75% + formaldehyde 25% + glacial acetic acid 5%) for 24 h and then kept in 70% alcohol for 24 h and processed for histopathological and immunohistochemical analysis. 2.2 Nanoparticles used The nanoparticles used in this experiment were Copper (II) oxide (Cat# 544868-5G), which was purchased from Sigma-Aldrich Chemicals Pvt Ltd (St. Louis, Missouri, United States). The nanoparticles were characterized by TEM analysis and were in nano-powder form; their details were previously described [ 8 ]. 2.3 Body weight and testis weight The mice were weighed and the weights recorded just before sacrifice. The testis weight was also recorded, and the following index was calculated: Gonado-somatic index = (testis weight/body weight)*100 2.4 Sperm parameter analysis The cauda epididymis was dissected out and minced in 250 µl PBS maintained at 37℃, and sperm motility was observed in 10 different areas of sperm homogenate dropped onto a clean slide. Sperm homogenate (20 µl) was further diluted in 200 µl PBS and observed in the white blood cell counting chamber of a Neubauer haemocytometer [ 8 , 23 ]. sperm motility = (no. of motile sperm/no. of immotile sperm)*100 sperm concentration = no. of sperm*10 6 *0.1(dilution factor) 2.5 Histological and immunohistochemical studies The testis, which was preserved in 70% alcohol, was dehydrated, and a tissue block was prepared using paraffin wax as per a previously described protocol [ 8 ]. The tissue block was cut using a microtome into a thin ribbon (5 µm), spread on a clean slide and incubated for 2 days. The prepared slide was used for histological study and immunohistochemical analysis. Testis histology was studied after processing the slide for haematoxylin and eosin staining [ 8 , 24 ]. Each tubule was observed carefully, and stages vii and viii of spermatogenesis was identified and their abundance recorded in each group. The Johnsen score was also evaluated in each seminiferous tubule, scored on a scale of 1 to 10 according to Johnsen (1970) [ 25 ]. In the Johnsen scoring system, all seminiferous tubules are carefully observed, and the presence, absence or abundance of spermatogonia, spermatocytes, spermatogonia and Sertoli cells are recorded. It evaluates the level of sperm maturation and degree of spermatogenesis on a 10-point scale, with 10 indicating complete spermatogenesis with a perfect tubule and 1 indicating no germ cells or an empty tubule. Immunohistochemical analysis of proliferating cell nuclear antigen PCNA and (Germ cell nuclear antigen) GCNA were performed as per the protocol of Nicy et al. (2024) [ 9 ]. First, the tissue sectioned was rehydrated and blocked using goat serum (1:100, diluted in PBS) for 1 h in a wet chamber for 1 h. The blocked section was incubated with primary antibody PCNA (cat# sc-7907, Santa Cruz Biotechnology, Dallas, Texas, United States) and GCNA (cat# 10D9G11, DSHB, University of Iowa, Dept of Biology, Iowa, United States) at a dilution of 1:100 each overnight in a wet chamber at 4℃. The excess antibody was washed off using PBS and was incubated for 4 h at room temperature with HRP-conjugated IgG secondary antibody (Goat anti-rabbit for PCNA, cat# E-AB-1102, Elabscience, Houston, Texas, United States and Goat anti-mouse for GCNA, cat# E-AB-1001, Elabscience, Houston, Texas, United States). The secondary antibody was rinsed in PBS to reduce nonspecific binding, and antibody bound to the desired antigen was detected using DAB (3′3′-diaminobenzidine tetrahydrochloride hydrate (0.6%) solution in 0.05M Tris buffer containing hydrogen peroxide). After the dot appeared at the periphery of the seminiferous tubules, it was dehydrated in a graded series of alcohol, cleared in xylene and mounted with DPX. 2.6 Hormonal assay The serum was used to analyse the circulating hormonal levels of testosterone, oestradiol, luteinising hormone and follicle stimulating hormone using a mouse ELISA kit (Testosterone, cat#KBH12112, Krishgen Biosystems, Mumbai, India; Oestradiol, cat#KBH11279, Krishgen Biosystems, Mumbai, India; Luteinizing Hormone Cat#E-EL-M3053, Elabscience, USA; Follicle Stimulating Hormone Cat# E-EL-M0511, Elabscience, USA) as per the manufacturer’s instructions. The sensitivity of testosterone was found to be 3.7 pmol/ml, and the intra-assay and inter-assay precision was found to be < 15% and < 18%, respectively. The sensitivity of oestradiol was found to be 4.45 pg/ml, and the intra-assay and inter-assay precision was found to be < 8% and < 10%, respectively. The sensitivity of follicle-stimulating hormone (FSH) was found to be 0.94 ng/ml, and the intra-assay and inter-assay precision was found to be < 4.87% and < 4.23%, respectively, with a coefficient variation of < 10%. The sensitivity of LH was found to be 0.19 ng/ml, and the intra-assay and inter-assay precision was found to be < 4.64%, with a coefficient variation of < 10%. 2.7 Oxidative stress and antioxidant analysis For the analysis of oxidative stress and antioxidants, a 10% tissue homogenate was prepared using PBS, and the supernatant was collected. Quantification of protein was also performed to determine the total protein concentration in the sample [ 26 ]. Lipid peroxidation analysis: as per the protocol given earlier [ 27 ], the testis supernatant was mixed in an equal ratio with 15% trichloroacetic acid (TCA) and 0.0375% thiobarbituric acid, and the mixture was boiled for 15 minutes for reactions to occur. The supernatant was collected by centrifugation, and the colour intensity was measured by spectrophotometry at 532 nm. The malondialdehyde concentration was expressed as Mol/mg protein. Superoxide dismutase analysis: in this enzyme assay, 10 µl tissue supernatant was mixed with nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH) and phenazine methosulphate (PMS) and incubated for 90 seconds at 30°C until a bluish colour appeared. Acetic acid and butanol were added and centrifuged for 30 seconds, and the absorbance of the coloured solution was read using a spectrophotometer at a wavelength of 560 nm. The SOD activity was expressed as U/mg protein [ 28 ]. Catalase analysis: in this assay, 25 µl tissue supernatant was incubated with H 2 O 2 for 2 minutes at 37°C [ 29 ]. The working solution containing cobalt (II), sodium hexametaphosphate, and sodium bicarbonate was added onto the solution and incubated for 10 more minutes in dark conditions at room temperature as per the previously described protocol. Using a spectrophotometer, the colour intensity was measured by reading the absorbance at 440 nm. The catalase activity was expressed as U/mg protein. Glutathione peroxidase analysis: this enzyme assay was performed by mixing the tissue supernatant with EDTA, sodium azide, reduced GSH, H 2 O 2 and phosphate buffer solution, incubating for 10 minutes at 37℃ and adding trichloroacetic acid. The solution was centrifuged, and 50 µl of the supernatant was pipetted out and mixed with 300 µl disodium hydrogen phosphate and 100 µl DTNB. Using a spectrophotometer, the colour intensity was measured by reading the absorbance at 412 nm. The GPx activity was expressed as µmol/min/mg protein [ 30 ]. 2.8 Western blot analysis Western blot analysis was performed based on a protocol described earlier [ 9 ]. The western blot analysis for testis homogenate was conducted to quantify our protein of interest. The testis was homogenized with PBS, and the supernatant was collected. The protein concentration was calculated using the Bradford method [ 26 ], and the testis supernatant was mixed with gel loading buffer in an equal ratio. A 50 µg protein sample from each group was separated by 10% SDS-PAGE. The protein in the gel was transferred onto a nitrocellulose membrane for 14 h at 4℃. The protein membrane was blocked in skimmed milk (cat#28582, SRL, Mumbai, India) for 30 minutes and then incubated with primary antibody (ERα, 1:500, cat# Bz1, DSHB, University of Iowa, Dept of Biology, Iowa, United States; ERβ, 1:500, cat# CWK-F12, DSHB, University of Iowa, Dept of Biology, Iowa, United States; BCL2, 1:1000, cat# sc-7382, Santa Cruz Biotechnology, Dallas, Texas, United States; Apelin receptor, 1:500, lot# ABD43, Millipore; Anti-caspase, 1:1000, cat#STJ97448-200, St John’s Laboratory, UK; Androgen receptor, 1:250, cat# PA5-16363 Invitrogen; β-Tubulin, 1;1000, cat#E7, DSHB, University of Iowa, Dept of Biology, Iowa, United States) overnight in a wet chamber at 4°C. The membrane was washed with PBST to removed unspecific antibody binding and probed with HRP-conjugated secondary antibody (Goat anti-rabbit for Apelin Receptor, Active-caspase 3, BCL2, cat# E-AB-1102, Elabscience, Houston, Texas, United States and Goat anti-mouse for ERα, ERβ, β-Tubulin, cat# E-AB-1001, Elabscience, Houston, Texas, United States) for 4 h and then washed with PBST to removed excess antibody binding. After washing, the signal was developed with enchanced chemiluminescence and detected by exposure to X-ray film. β-tubulin was used as the internal control. 2.9 Statistical analysis Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) and the mean ± SEM was used as a means to present the results. One way ANOVA followed by Tukey’s test was used to compare the means of the groups. Statistical significance was concluded when p < 0.05. 3. Results 3.1. Effects of paternal CuNP exposure on the body weight, testis weight and gonado-somatic index of male offspring The F1 generation from all the groups showed no significant change in body weight ( Fig. 1 A ) , testis weight ( Fig. 1 B ) and the gonado-somatic index ( Fig. 1 C ) although a slight decrease in the sperm concentration was found in males of the CuNP-treated groups. Sperm motility was significantly decreased (p < 0.05) in the F1 generation of 100 mg/kg and 200 mg/kg CuNP-treated parental mice ( Fig. 1 D ) . 3.2. Effects of paternal CuNP exposure on the testicular histology and Johnsen Score of male offspring Spermatogenesis stages vii & viii were examined in all the seminiferous tubules and found to be significantly (p < 0.05) decreased in males from the 100 mg/kg and 200 mg/kg groups compared to control (Fig. 2AB) . The Johnsen score ( Fig. 2 C ) was greater than 8 in most groups and highest in the control group. 3.3. Effects of paternal CuNP exposure on germ cell proliferation by PCNA in male offspring No significant differences in PCNA were found between groups, and no conspicuous changes was observed among the groups (Fig. 3AD) . 3.4. Effects of paternal CuNP exposure on germ cell proliferation by GCNA in male offspring A distinct difference in GCNA immunostaining ( Fig. 4 A D) was found between groups. Germ cell staining positivity was the strongest in the control group ( Fig. 4 A ) and the weakest in the treated group, with very few positive tubules detected in F1 generation males of 100 mg/kg and 200 mg/kg CuNP treated paternal mice (Fig. 4CD) . 3.5. Effects of paternal CuNP exposure on oxidative stress (malondialdehyde levels, MDA) and antioxidant enzymes (Gpx, SOD and catalase) in male offspring The MDA ( Fig. 5 A ) and catalase ( Fig. 5 D ) levels were significantly increased (p < 0.05) in the 100 mg/kg and 200 mg/kg groups. The SOD level ( Fig. 5 C ) , on the other hand, was significantly decreased (p < 0.05) in the 100 mg/kg and 200 mg/kg treatment groups. However, the GPx level ( Fig. 5 B ) did not show a significant difference between groups. 3.6. Effects of paternal CuNP exposure on circulating testosterone, FSH, oestradiol and luteinizing hormone levels in male offspring The FSH levels of the F1 generation from different levels of CuNP-induced paternal exposure was not affected; however, the circulating testosterone ( Fig. 6 . AC) and oestradiol levels were significantly decreased (p < 0.05) in the 100 mg/kg and 200 mg/kg groups. The luteinizing hormone (LH) levels ( Fig. 6 D ) were significantly elevated (p < 0.05) in all the F1 generation groups of CuNP-exposed male parents. 3.7. Effects of paternal CuNP exposure on the expression of AR, ERα, ERβ and AR by western blot analysis in male offspring The protein expression of ERα ( Fig. 7 A ) was unaffected; however, a decreasing trend of protein expression was found in ERβ ( Fig. 7 B ) in the F1 generation of the treatment group compared to the control, although it was not significant. The AR ( Fig. 7 C ) protein expression was found to be significantly up-regulated (p < 0.05) in all the F1 generation groups of CuNPs-exposed male parents. 3.8. Effects of paternal CuNP exposure on the expression of BCL2, anti-caspase and apelin receptor by western blot analysis in male offspring The protein expression of BCL2 ( Fig. 8 A ) and APJ ( Fig. 8 C ) were found to be significantly (p < 0.05) down-regulated in all the F1 generation of CuNP-treated groups compared to the control group. Although statistically not significant, an increasing trend in active-caspase 3 ( Fig. 8 B ) protein expression was found in all the F1 generation of paternal treatment groups compared to the control. 4. Discussion In the present study, we have shown that chronic exposure of males to CuNPs has a negative impact on the testicular activity of their male offspring, even if the mother had no exposure to CuNPs. Earlier, our laboratory also showed that chronic exposure of males to CuNPs severely impairs spermatogenesis and steroidogenesis [ 8 ]; moreover, discontinuation of CuNPs failed to restore testicular functions to a control level, and male mice showed compromised fertility [ 9 ]. A gross examination of testicular histology did not show observable pathological signs in the male offspring of CuNPs-treated male parents at all the doses; however, the number of tubules in spermatogenesis stages vii&viii and sperm motility showed a significant decline in the male offspring of male parents treated at higher doses of 100 and 200 mg/kg compared with control and 10 mg/kg CuNPs-treated paternal males. Furthermore, the sperm count showed a decline in the male offspring of all CuNPs-treated male parents; however, it was not significant. Despite the decrease in sperm count in male offspring of CuNPs-treated male parents, the absence of a significant difference vs the control could be due to individual variations, where some of the males could be resistant to CuNPs treatment. These findings clearly suggest that paternal CuNPs exposure impairs testicular functions, which could be due to genetic changes in the paternal gametes. It has been shown that parental ionic copper exposure to zebra fish can cause alterations in the sperm methylome and transcriptome, which can be passed down to their fertilized offspring [ 31 ]. Spermatogenesis encompasses continuous proliferation of germ cells, and slight impairment of sperm parameters and the testis prompted us to examine germ cell proliferation. The proliferation marker GCNA showed a lower abundance in the male offspring from paternal males treated at higher doses of CuNPs, while PCNA did not show changes among all the groups. Proliferation and germ cell apoptosis in the testis occurs simultaneously, and this maintains germ cell balance [ 32 ]. The pro-apoptotic marker active caspase3 showed elevated expression in all the groups; however, the anti-apoptotic marker Bcl2 showed down-regulation in the male offspring of male parents treated with a higher dose of CuNPs. These results showed evidence of compromised proliferation and elevated apoptosis in the male offspring of CuNPs-treated male parents. These findings also partially support the decrease in sperm parameters such as the sperm count and motility due to elevated apoptosis. Despite the toxic effects of CuNPs on embryo toxicity, such as reduction in blastocyst quality and the live birth rate in mice [ 33 ], the effects of paternal CuNPs exposure on the male offspring in relation to testicular functions have not been investigated. It has been shown that paternal exposure to endocrine disruptors could impair testicular functions in the male offspring, characterised by decreased sperm parameters and testicular histopathology [ 11 ]. Paternal exposure to another chemical toxicant, vinclozolin, elevates testicular germ cell apoptosis in the male offspring and leads to compromised sperm parameters [ 12 , 34 , 35 ]. It has also been shown that paternal exposure to bisphenol causes testicular sperm pathology via elevated oxidative stress in the offspring [ 36 ]. Our results also showed elevated oxidative stress (high MDA) and decreased antioxidant enzyme (SOD) in the male offspring of male parents treated at a higher dose of CuNPs (100 and 200 mg/kg). However, GPx enzyme did not show any significant changes, and only catalase was elevated in the male offspring of male parents treated at a higher dose of CuNPs (100 and 200 mg/kg). The increased catalase could be a mechanism to counteract elevated oxidative stress. To best of our knowledge, transgenerational toxicity in male offspring after CuNPs exposure in either parent is scant, thus, the findings of the present study are supported by other environmental or chemical contaminants and endocrine disruptors. Testicular functions are well regulated by testicular steroid hormones and gonadotropins from the pituitary [ 37 ].Thus, we have also evaluated circulating testosterone and estrogen levels in F1 males, and our results showed that both circulating testosterone and estrogen were elevated in the male offspring of male parents treated with higher doses (100 and 200 mg/kg) of CuNPs. Furthermore, circulating FSH levels did not show significant changes; however, the levels of LH were high in the male offspring of all CuNPs-treated male parents. The elevated LH levels in the male offspring of 100 and 200 mg/kg CuNPs-treated male parents explain the elevated testosterone levels, because LH stimulates testosterone production. However, the elevated LH and lack of change in circulating testosterone in the F1 male offspring of 10 mg/kg CuNPs-treated male parents remains unclear as a decrease in circulating testosterone levels was expected in the male offspring after paternal exposure to CuNPs, and our data showed elevated testosterone. A previous study showed that paternal acrylamide exposure decreased circulating testosterone in the male offspring [ 38 ]. However, maternal exposure to genistein during gestation increased circulating testosterone in the offspring [ 39 ]. The elevated testosterone and estrogen clearly showed hormonal imbalances in the male offspring; thus, we also analysed the expression of AR and ERs. Our data showed that the expression of AR and ERβ were down-regulated in the male offspring of all CuNPs-treated male parents. However, ERα did not show significant changes. These results clearly showed endocrine imbalances in the circulating steroids as well as their signalling in the testis of male offspring, when the male parent had been exposed to CuNPs. To the best of our knowledge, no study has been conducted to analyse AR and ER expression in the testes of male offspring mice from CuNPs-exposed male parents. Previous studies from our group have shown that continuous chronic CuNPs exposure for 70 days and even after termination of CuNPs, decreased expression of AR in the testis, and we have also shown that apelin receptor, APJ, was up-regulated in the CuNPs-treated testis, which coincides with decreased testosterone levels [ 8 , 9 ]. Thus, we also examined the expression of APJ in the testis of F1 males. The expression of APJ was down-regulated in the testis of male offspring from all the paternal CuNPs-treated groups. APJ is well known to be expressed in the testis and regulates various functions in normal and pathological conditions [ 40 , 41 ]. Elevated APJ expression in the diabetic testis has been shown to supress testosterone biosynthesis [ 40 ] and apelin signalling in the testis supresses testosterone biosynthesis [ 41 , 42 , 43 ]. However, how does the paternal sperm after CuNPs exposure transmit this information to the F1 males, not exactly known from the findings of the present study? It has been shown that in males, reproductive abnormalities caused by exposure to endocrine disrupting chemicals can be transmitted to future generations through epigenetic modifications of germ cells and exhibits wide heterogeneity in reproductive traits [ 44 ]. We did not perform an epigenetic study, which is a very important limitation of our study and requires further investigation; moreover, commenting on any aspect of epigenetic-meditated transmittance to male offspring after exposure of their male parent to CuNPs would be speculative. Various studies have shown that nanoparticles impair testicular function in a mouse model [ 45 , 46 , 47 ]. It has been suggested that toxic substances may have an unknown impact on offspring, since even minor and transient alterations to the testicular genome may increase the mortality and morbidity of offspring [ 47 ]. It has been shown that those nanoparticles can have transgenerational effects on living organisms [ 48 ]. Furthermore, epigenetic mechanisms are significant in mediating the responses to transgenerational toxicity resulting from environmental contaminants [ 49 ]. A previous study has also shown that parental CuNPs exposure causes transgenerational toxicity in C. elegans , which causes reproductive and general toxicity [ 14 ]. To the best of our knowledge, transgenerational reproductive toxicity of CuNPs has not been shown in rodents. 5. Conclusion In conclusion, the present study showed for the first time that paternal exposure to CuNPs at different doses impairs testicular functions and spermatogenesis in male offspring, and male offspring from higher-dose-treated male parents exhibited more dysfunctions. However, none of the studied parameters in the present study showed dose-dependent effects. The impairment of testicular functions in the male offspring could be mediated by elevated oxidative stress, endocrine imbalances, deregulated signalling of AR, ERs and APJ. Despite impaired or compromised spermatogenesis, sperm quality must be evaluated in male offspring. The transgenerational effects of CuNP exposure observed in the present study may be mediated by germline transmission. These results suggest the presence of heritable genetic alterations transmitted to male offspring, potentially explaining the observed reproductive abnormalities. Further studies are needed to better understand the effects of CuNPs in male offspring. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution VKR, GG, VN: Conceptualization, Experiment design, resources generation of study. VN, Performed the experiments. VKR, GG, VN: Experimental section analysis, Data analysis, Writing of manuscript. Acknowledgement Nicy Vanrohlu acknowledges the fellowship received at D ST/INSPIRES Fellowship (DST/INSPIRE/03/2021/001312) from DST New Delhi. The research infrastructure facility provided to the Department of Zoology, Mizoram University by the DST-FIST program, DST, New Delhi is greatly acknowledged. Data availability statement The data are available from the corresponding author upon reasonable request. 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14:11:39","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24556,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/5343fa98bf4fb3f755b817db.png"},{"id":96917168,"identity":"5c0a5f5c-744a-4143-bbbe-3346ad7bc5fb","added_by":"auto","created_at":"2025-11-27 14:09:19","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21164,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/a7b99b7247ead13e8d0020c7.png"},{"id":96803825,"identity":"b596b0e2-fbcc-45d6-b881-f38aab1812f4","added_by":"auto","created_at":"2025-11-26 09:03:35","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104241,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/0e3281f4ae88ae4d1ee6bea7.png"},{"id":96803827,"identity":"bc27a9b4-8217-4cc6-92d9-7278e8429e4f","added_by":"auto","created_at":"2025-11-26 09:03:35","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96708,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/e9a939e65d15c70cbaa66e66.png"},{"id":96917771,"identity":"0698a0c8-9eb3-468a-9e95-8ec13c6916cd","added_by":"auto","created_at":"2025-11-27 14:10:33","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129491,"visible":true,"origin":"","legend":"","description":"","filename":"59287520f5b048c191329d81614ee3eb1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/3f395bdef5e801c4ae7a81aa.xml"},{"id":96916576,"identity":"a9149634-bf2f-43a9-ad53-d68e202021e5","added_by":"auto","created_at":"2025-11-27 14:08:44","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139531,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/63031ad87759393103189fd2.html"},{"id":96803790,"identity":"f39ec564-1374-43ee-8620-95b789c4b101","added_by":"auto","created_at":"2025-11-26 09:03:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":246578,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on the body weight (A), testis weight (B), relative testis weight (C), sperm concentration (D) and sperm motility (E) of male offspring. A decreasing trend was found in all male offspring of CuNP-exposed male parents, but no significant change was observed in any parameters (ABCD) except for sperm motility (E), where a significant decline (p \u0026lt; 0.05) was found in the 100 mg/kg and 200 mg/kg groups. Data are represented as mean ± SEM (n = 5). Different letters on the graph show a significant difference.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/e0340e0de32e5458cb4f41ca.png"},{"id":96917524,"identity":"7cd03401-d455-46cb-906d-8ed2a73ffa2f","added_by":"auto","created_at":"2025-11-27 14:09:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3616172,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on testis histology (20x magnification) (A = control, B = 10 mg/kg, C = 100 mg/kg, D = 200 mg/kg), spermatogenesis (E) and Johnsen score (F) in the male offspring. The histological section showed many seminiferous tubules cross-sectioned undergoing stages vii\u0026amp;viii in the control group (A), with the least present in the 100 mg/kg (C) and 200 mg/kg groups (D). The graph (E) showed a significant decrease (p \u0026lt; 0.05) in stage vii\u0026amp;viii of spermatogenesis in the 100 mg/kg and 200 mg/kg groups. The Johnsen score (F) also showed a decreasing trend in a dose-dependent manner, but no significant change was observed. Data are represented as mean ± SEM (n = 5). Different letters on the graph show a significant difference.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/b023ffb43a18d511ae093686.png"},{"id":96803801,"identity":"0fc82c79-61b5-43b3-b0f7-5235dbffef19","added_by":"auto","created_at":"2025-11-26 09:03:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6039830,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on the localization of PCNA (40x magnification, A = control, B = 10 mg/kg, C = 100 mg/kg, D = 200 mg/kg) in male offspring. Immunostaining of PCNA showed equal staining in all the groups with no differences in immunolocalization. *arrow indicates the positive immunostaining area\u003c/p\u003e","description":"","filename":"fig3new.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/d39966e094666d3e29c9b99b.png"},{"id":96916485,"identity":"25c67574-bc15-4c0d-8067-6ae60e27af84","added_by":"auto","created_at":"2025-11-27 14:08:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5429355,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on the localization of GCNA (40x magnification, A = control, B = 10 mg/kg, C = 100 mg/kg, D = 200 mg/kg) in male offspring. Immunostaining of GCNA showed a distinct difference in localization in different groups. Immunolocalization of GCNA in the control (A) and 10 mg/kg (B) groups showed a similar pattern while the faint staining of GCNA was found in the 100 mg/kg (C) and 200 mg/kg (D) groups. *arrow indicates the positive immunostaining area\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/244fec8c825535e56b1b6bd9.png"},{"id":96803795,"identity":"120db24d-a9d7-4d22-9dc5-cabd0a0558a9","added_by":"auto","created_at":"2025-11-26 09:03:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":302138,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on lipid peroxidation (MDA) and antioxidant enzymes in male offspring. A significant increase (p \u0026lt; 0.05) in the MDA level (A) and catalase (D) was found in male offspring from CuNP-treated groups. However, a significant decline (p \u0026lt; 0.05) in superoxide dismutase (C) was found in the 100 mg/kg and 200 mg/kg groups. Data are represented as mean ± SEM (n = 5). Different letters on the graph show a significant difference.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/796d5f395222405e52c86921.png"},{"id":96803793,"identity":"4c15e678-bf76-491b-b8b6-7c0011b0419d","added_by":"auto","created_at":"2025-11-26 09:03:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":262218,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on circulating testosterone (A), FSH (B), oestradiol (C) and LH (D) levels in male offspring. The levels of circulating testosterone, oestradiol and LH were found to be significantly elevated (p \u0026lt; 0.05) in the male offspring from the CuNP-treated groups, while the FSH level remained unaffected. Data are represented as mean ± SEM (n = 5). Different letters on the graph show a significant difference.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/246a177d96873bf12531b9f8.png"},{"id":96803799,"identity":"ee040e9e-8dba-4a5e-bd5e-937bc9f33d4b","added_by":"auto","created_at":"2025-11-26 09:03:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":601540,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on the expression of ER-α (A), ER-β (B) and androgen receptor (C) in male offspring. The protein expression of ER-β was down-regulated in the treated group, although no significant change was found, and the ER-α protein expression was same in all the groups. However, a significant decrease (p \u0026lt; 0.05) in the protein expression of the androgen receptor was found in all the treated groups compared to the control group. Data are represented as mean ± SEM (n = 5). Different letters on the graph show a significant difference.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/e70aec2b029dc23b2908fc8a.png"},{"id":96917442,"identity":"f3930fb4-f66b-4cc3-8a1e-9902aa957b11","added_by":"auto","created_at":"2025-11-27 14:09:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":560876,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of paternal CuNP exposure on the expression of BCL2 (A), active-caspase 3 (B) and apelin receptor (C) in male offspring. The protein expression of BCL2 and APJ was found to be significantly (p \u0026lt; 0.05) down-regulated in the male offspring of CuNP-treated male parents, while active-caspase 3 was found to be up-regulated compared to the control group, although it was not significant. Data are represented as mean ± SEM (n = 5). Different letters on the graph show a significant difference.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/a66314f612cbb5369a36afb1.png"},{"id":105224491,"identity":"03ef3e01-ed3d-4464-b9c3-a8bb1e9598f8","added_by":"auto","created_at":"2026-03-23 16:14:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16525182,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8068308/v1/c807976e-a1d5-4ea2-b002-4fbb858fc837.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Paternal chronic exposure to copper nanoparticles (CuNPs) impairs testicular androgen and estrogen signalling in adult male offspring in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs human living standards rise quickly, thousands of chemical pollutants are released into the environment by industry, agriculture and pharmaceuticals, causing serious harm to the plants and animal health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Copper metals have been used in various human applications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and copper is also a trace metal that regulates various biological functions in humans and animals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Other forms of copper, such as the nano form and nano particles, are being used in water treatment, agriculture, livestock, wood preservation and textile manufacturing [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite being used in various applications, copper nanoparticles (CuNPs) have also been known to cause health hazards [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has been shown that different concentrations (50, 100 and 200 mg/kg) of CuNPs damages the kidneys and liver; moreover, a higher dose of CuNPs showed necrosis in the proximal kidney tubules [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies from our group and another laboratory have also shown that CuNPs exposure impairs testicular functions in males [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nanoparticles have been known to acts as endocrine disruptors, which alter the male reproductive system by affecting testicular structure, steroid hormone levels and spermatogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt has also been shown that the direct exposure of parental germ cells to toxic materials causes epigenetic changes and intergenerational inheritance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The inheritance of transgenerational toxic effects through parents affects the physiological functions of offspring [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We have shown that exposure of male mice to CuNPs causes endocrine imbalances such as suppression of testosterone levels and decreased androgen receptor (AR) expression [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, whether the F1 male offspring of treated male mice manifest reproductive dysfunction has not been investigated. It has been shown that parental exposure of \u003cem\u003eC. elegans\u003c/em\u003e to CuNPs at a dose of 150 mg/L causes developmental and reproductive toxicity in their progeny [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It has also been suggested that paternal endocrine disruptors affect the offspring [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Other endocrine disruptors, like bisphenol A exposure at the adult or embryonic stage in male mice, led to abnormal offspring with decreased sperm quality and spermatogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Only recently, it was shown that CuNPs exposure for one spermatogenic cycle followed by termination of CuNPs treatment for another 35 days, induces oxidative stress, impairs spermatogenesis and compromises epigenetic modification, poor sperm quality and developmental anomalies in male offspring [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe above- mentioned evidence, which showed that paternal exposure to chemical toxicants and endocrine disruptors causes anomalies in the offspring, still needs further investigation. As in our previous studies, we showed compromised testicular functions along with altered reproductive parameters in mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], therefore, we hypothesised that male offspring of CuNPs-treated male parents would show compromised testicular functions. The aim of the present work was to investigate the effects of paternal CuNPs exposure on the testes of male offspring by evaluating various parameters, such as circulating steroids levels, proliferation, apoptosis and oxidative stress.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals and experimental design\u003c/h2\u003e\u003cp\u003eThe animals used in this experiment were the male offspring of three-month-old Swiss albino (\u003cem\u003eMus musculus\u003c/em\u003e) male mice taken from an inbred colony of the Animal Housing Facility, Zoology Department, Mizoram University, Aizawl, Mizoram. Animals were housed under controlled conditions, on a 12 h light/dark cycle, with a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ and food and water ad libitum. All animal experiments were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. The animals experiment protocol was approved by the Institutional Ethical Committee (process number: MZU/IAEC/2020/10), Mizoram University, Aizawl, Mizoram, India and was compiled with ARRIVE guidelines.\u003c/p\u003e\u003cp\u003eFour different experimental groups with five animals in each group were formed. Group 1 (the control) was given phosphate buffered saline (PBS), and groups 2, 3 and 4 were administered, by oral gavage, 10, 100 and 200 mg/kg bw, respectively, CuNPs dissolved in PBS for 70 consecutive days, according to previous experiments [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, it has been documented that 10\u0026ndash;12 mg/person/day is an acceptable copper intake besides natural intake of 1\u0026ndash;2 mg/person/day [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, based on this data, an acceptable upper exposure limits 0.15 mg/kgbw/day was fixed by \u003cb\u003eEuropean Food Safety Authority\u003c/b\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and 0.2 mg/kgbw/day exposure has been permitted for adult. The dose in our study is 50, 500 and 1000 times higher than these permissible limits for 10, 100 and 200 mg/kg CuNPs respectively. After the treatment was over, the treated male mice were house with healthy female mice (1 male per 1 female mouse) and were allowed to breed. The number of pups was recorded, and the pups were separated from their parents at weaning and allowed to attain adulthood (three months old) to further investigate whether treatment of male parents with CuNPs showed a notable effect on the next generation. In the present study, we investigated the possible effects of CuNPs exposure of male parents on testicular activity in male offspring. Previously we have shown that 70 days of consecutive CuNPs treatment resulted in impaired spermatogenesis in male mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The male offspring of treated males were compared with offspring derived from untreated male parents.\u003c/p\u003e\u003cp\u003eThe male mouse offspring (n\u0026thinsp;=\u0026thinsp;5, each individual from different treated or control male parents) were euthanized at three months of age by decapitation under mild anaesthesia (90 mg/kg ketamine and 4.5 mg/kg xylazine, by intraperitoneal injection of 1 ml/kg bw). The collected blood was centrifuged to separate the serum and was used for hormonal analysis. The sperm parameters were analysed immediately, the testis from one side was frozen at -20℃ while the other one was fixed with Bouins fluid (picric acid 75% + formaldehyde 25% + glacial acetic acid 5%) for 24 h and then kept in 70% alcohol for 24 h and processed for histopathological and immunohistochemical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Nanoparticles used\u003c/h2\u003e\u003cp\u003eThe nanoparticles used in this experiment were Copper (II) oxide (Cat# 544868-5G), which was purchased from Sigma-Aldrich Chemicals Pvt Ltd (St. Louis, Missouri, United States). The nanoparticles were characterized by TEM analysis and were in nano-powder form; their details were previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Body weight and testis weight\u003c/h2\u003e\u003cp\u003eThe mice were weighed and the weights recorded just before sacrifice. The testis weight was also recorded, and the following index was calculated:\u003c/p\u003e\u003cp\u003eGonado-somatic index = (testis weight/body weight)*100\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Sperm parameter analysis\u003c/h2\u003e\u003cp\u003eThe cauda epididymis was dissected out and minced in 250 \u0026micro;l PBS maintained at 37℃, and sperm motility was observed in 10 different areas of sperm homogenate dropped onto a clean slide. Sperm homogenate (20 \u0026micro;l) was further diluted in 200 \u0026micro;l PBS and observed in the white blood cell counting chamber of a Neubauer haemocytometer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003esperm motility = (no. of motile sperm/no. of immotile sperm)*100\u003c/p\u003e\u003cp\u003esperm concentration\u0026thinsp;=\u0026thinsp;no. of sperm*10\u003csup\u003e6\u003c/sup\u003e *0.1(dilution factor)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Histological and immunohistochemical studies\u003c/h2\u003e\u003cp\u003eThe testis, which was preserved in 70% alcohol, was dehydrated, and a tissue block was prepared using paraffin wax as per a previously described protocol [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The tissue block was cut using a microtome into a thin ribbon (5 \u0026micro;m), spread on a clean slide and incubated for 2 days. The prepared slide was used for histological study and immunohistochemical analysis.\u003c/p\u003e\u003cp\u003eTestis histology was studied after processing the slide for haematoxylin and eosin staining [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Each tubule was observed carefully, and stages vii and viii of spermatogenesis was identified and their abundance recorded in each group. The Johnsen score was also evaluated in each seminiferous tubule, scored on a scale of 1 to 10 according to \u003cb\u003eJohnsen (1970)\u003c/b\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the Johnsen scoring system, all seminiferous tubules are carefully observed, and the presence, absence or abundance of spermatogonia, spermatocytes, spermatogonia and Sertoli cells are recorded. It evaluates the level of sperm maturation and degree of spermatogenesis on a 10-point scale, with 10 indicating complete spermatogenesis with a perfect tubule and 1 indicating no germ cells or an empty tubule.\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis of proliferating cell nuclear antigen PCNA and (Germ cell nuclear antigen) GCNA were performed as per the protocol of \u003cb\u003eNicy et al. (2024)\u003c/b\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. First, the tissue sectioned was rehydrated and blocked using goat serum (1:100, diluted in PBS) for 1 h in a wet chamber for 1 h. The blocked section was incubated with primary antibody PCNA (cat# sc-7907, Santa Cruz Biotechnology, Dallas, Texas, United States) and GCNA (cat# 10D9G11, DSHB, University of Iowa, Dept of Biology, Iowa, United States) at a dilution of 1:100 each overnight in a wet chamber at 4℃. The excess antibody was washed off using PBS and was incubated for 4 h at room temperature with HRP-conjugated IgG secondary antibody (Goat anti-rabbit for PCNA, cat# E-AB-1102, Elabscience, Houston, Texas, United States and Goat anti-mouse for GCNA, cat# E-AB-1001, Elabscience, Houston, Texas, United States). The secondary antibody was rinsed in PBS to reduce nonspecific binding, and antibody bound to the desired antigen was detected using DAB (3\u0026prime;3\u0026prime;-diaminobenzidine tetrahydrochloride hydrate (0.6%) solution in 0.05M Tris buffer containing hydrogen peroxide). After the dot appeared at the periphery of the seminiferous tubules, it was dehydrated in a graded series of alcohol, cleared in xylene and mounted with DPX.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Hormonal assay\u003c/h2\u003e\u003cp\u003eThe serum was used to analyse the circulating hormonal levels of testosterone, oestradiol, luteinising hormone and follicle stimulating hormone using a mouse ELISA kit (Testosterone, cat#KBH12112, Krishgen Biosystems, Mumbai, India; Oestradiol, cat#KBH11279, Krishgen Biosystems, Mumbai, India; Luteinizing Hormone Cat#E-EL-M3053, Elabscience, USA; Follicle Stimulating Hormone Cat# E-EL-M0511, Elabscience, USA) as per the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eThe sensitivity of testosterone was found to be 3.7 pmol/ml, and the intra-assay and inter-assay precision was found to be \u0026lt;\u0026thinsp;15% and \u0026lt;\u0026thinsp;18%, respectively.\u003c/p\u003e\u003cp\u003eThe sensitivity of oestradiol was found to be 4.45 pg/ml, and the intra-assay and inter-assay precision was found to be \u0026lt;\u0026thinsp;8% and \u0026lt;\u0026thinsp;10%, respectively.\u003c/p\u003e\u003cp\u003eThe sensitivity of follicle-stimulating hormone (FSH) was found to be 0.94 ng/ml, and the intra-assay and inter-assay precision was found to be \u0026lt;\u0026thinsp;4.87% and \u0026lt;\u0026thinsp;4.23%, respectively, with a coefficient variation of \u0026lt;\u0026thinsp;10%.\u003c/p\u003e\u003cp\u003eThe sensitivity of LH was found to be 0.19 ng/ml, and the intra-assay and inter-assay precision was found to be \u0026lt;\u0026thinsp;4.64%, with a coefficient variation of \u0026lt;\u0026thinsp;10%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Oxidative stress and antioxidant analysis\u003c/h2\u003e\u003cp\u003eFor the analysis of oxidative stress and antioxidants, a 10% tissue homogenate was prepared using PBS, and the supernatant was collected. Quantification of protein was also performed to determine the total protein concentration in the sample [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLipid peroxidation analysis: as per the protocol given earlier [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the testis supernatant was mixed in an equal ratio with 15% trichloroacetic acid (TCA) and 0.0375% thiobarbituric acid, and the mixture was boiled for 15 minutes for reactions to occur. The supernatant was collected by centrifugation, and the colour intensity was measured by spectrophotometry at 532 nm. The malondialdehyde concentration was expressed as Mol/mg protein.\u003c/p\u003e\u003cp\u003eSuperoxide dismutase analysis: in this enzyme assay, 10 \u0026micro;l tissue supernatant was mixed with nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH) and phenazine methosulphate (PMS) and incubated for 90 seconds at 30\u0026deg;C until a bluish colour appeared. Acetic acid and butanol were added and centrifuged for 30 seconds, and the absorbance of the coloured solution was read using a spectrophotometer at a wavelength of 560 nm. The SOD activity was expressed as U/mg protein [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCatalase analysis: in this assay, 25 \u0026micro;l tissue supernatant was incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 2 minutes at 37\u0026deg;C [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The working solution containing cobalt (II), sodium hexametaphosphate, and sodium bicarbonate was added onto the solution and incubated for 10 more minutes in dark conditions at room temperature as per the previously described protocol. Using a spectrophotometer, the colour intensity was measured by reading the absorbance at 440 nm. The catalase activity was expressed as U/mg protein.\u003c/p\u003e\u003cp\u003eGlutathione peroxidase analysis: this enzyme assay was performed by mixing the tissue supernatant with EDTA, sodium azide, reduced GSH, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and phosphate buffer solution, incubating for 10 minutes at 37℃ and adding trichloroacetic acid. The solution was centrifuged, and 50 \u0026micro;l of the supernatant was pipetted out and mixed with 300 \u0026micro;l disodium hydrogen phosphate and 100 \u0026micro;l DTNB. Using a spectrophotometer, the colour intensity was measured by reading the absorbance at 412 nm. The GPx activity was expressed as \u0026micro;mol/min/mg protein [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e\u003cp\u003eWestern blot analysis was performed based on a protocol described earlier [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The western blot analysis for testis homogenate was conducted to quantify our protein of interest. The testis was homogenized with PBS, and the supernatant was collected. The protein concentration was calculated using the Bradford method [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and the testis supernatant was mixed with gel loading buffer in an equal ratio. A 50 \u0026micro;g protein sample from each group was separated by 10% SDS-PAGE. The protein in the gel was transferred onto a nitrocellulose membrane for 14 h at 4℃. The protein membrane was blocked in skimmed milk (cat#28582, SRL, Mumbai, India) for 30 minutes and then incubated with primary antibody (ERα, 1:500, cat# Bz1, DSHB, University of Iowa, Dept of Biology, Iowa, United States; ERβ, 1:500, cat# CWK-F12, DSHB, University of Iowa, Dept of Biology, Iowa, United States; BCL2, 1:1000, cat# sc-7382, Santa Cruz Biotechnology, Dallas, Texas, United States; Apelin receptor, 1:500, lot# ABD43, Millipore; Anti-caspase, 1:1000, cat#STJ97448-200, St John\u0026rsquo;s Laboratory, UK; Androgen receptor, 1:250, cat# PA5-16363 Invitrogen; β-Tubulin, 1;1000, cat#E7, DSHB, University of Iowa, Dept of Biology, Iowa, United States) overnight in a wet chamber at 4\u0026deg;C. The membrane was washed with PBST to removed unspecific antibody binding and probed with HRP-conjugated secondary antibody (Goat anti-rabbit for Apelin Receptor, Active-caspase 3, BCL2, cat# E-AB-1102, Elabscience, Houston, Texas, United States and Goat anti-mouse for ERα, ERβ, β-Tubulin, cat# E-AB-1001, Elabscience, Houston, Texas, United States) for 4 h and then washed with PBST to removed excess antibody binding. After washing, the signal was developed with enchanced chemiluminescence and detected by exposure to X-ray film. β-tubulin was used as the internal control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) and the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM was used as a means to present the results. One way ANOVA followed by Tukey\u0026rsquo;s test was used to compare the means of the groups. Statistical significance was concluded when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003e3.1. Effects of paternal CuNP exposure on the body weight, testis weight and gonado-somatic index of male offspring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe F1 generation from all the groups showed no significant change in body weight \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, testis weight \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e and the gonado-somatic index \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e although a slight decrease in the sperm concentration was found in males of the CuNP-treated groups. Sperm motility was significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the F1 generation of 100 mg/kg and 200 mg/kg CuNP-treated parental mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effects of paternal CuNP exposure on the testicular histology and Johnsen Score of male offspring\u003c/h2\u003e\u003cp\u003eSpermatogenesis stages vii \u0026amp; viii were examined in all the seminiferous tubules and found to be significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decreased in males from the 100 mg/kg and 200 mg/kg groups compared to control \u003cb\u003e(Fig.\u0026nbsp;2AB)\u003c/b\u003e. The Johnsen score \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e was greater than 8 in most groups and highest in the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effects of paternal CuNP exposure on germ cell proliferation by PCNA in male offspring\u003c/h2\u003e\u003cp\u003eNo significant differences in PCNA were found between groups, and no conspicuous changes was observed among the groups \u003cb\u003e(Fig.\u0026nbsp;3AD)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effects of paternal CuNP exposure on germ cell proliferation by GCNA in male offspring\u003c/h2\u003e\u003cp\u003eA distinct difference in GCNA immunostaining \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003eD)\u003c/b\u003e was found between groups. Germ cell staining positivity was the strongest in the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and the weakest in the treated group, with very few positive tubules detected in F1 generation males of 100 mg/kg and 200 mg/kg CuNP treated paternal mice \u003cb\u003e(Fig.\u0026nbsp;4CD)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5. Effects of paternal CuNP exposure on oxidative stress (malondialdehyde levels, MDA) and antioxidant enzymes (Gpx, SOD and catalase) in male offspring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe MDA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and catalase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e levels were significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the 100 mg/kg and 200 mg/kg groups. The SOD level \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, on the other hand, was significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the 100 mg/kg and 200 mg/kg treatment groups. However, the GPx level \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e did not show a significant difference between groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6. Effects of paternal CuNP exposure on circulating testosterone, FSH, oestradiol and luteinizing hormone levels in male offspring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe FSH levels of the F1 generation from different levels of CuNP-induced paternal exposure was not affected; however, the circulating testosterone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003cb\u003eAC)\u003c/b\u003e and oestradiol levels were significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the 100 mg/kg and 200 mg/kg groups. The luteinizing hormone (LH) levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e were significantly elevated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in all the F1 generation groups of CuNP-exposed male parents.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.7. Effects of paternal CuNP exposure on the expression of AR, ERα, ERβ and AR by western blot analysis in male offspring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protein expression of ERα \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e was unaffected; however, a decreasing trend of protein expression was found in ERβ \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e in the F1 generation of the treatment group compared to the control, although it was not significant. The AR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e protein expression was found to be significantly up-regulated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in all the F1 generation groups of CuNPs-exposed male parents.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.8. Effects of paternal CuNP exposure on the expression of BCL2, anti-caspase and apelin receptor by western blot analysis in male offspring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protein expression of BCL2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and APJ \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e were found to be significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) down-regulated in all the F1 generation of CuNP-treated groups compared to the control group. Although statistically not significant, an increasing trend in active-caspase 3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e protein expression was found in all the F1 generation of paternal treatment groups compared to the control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present study, we have shown that chronic exposure of males to CuNPs has a negative impact on the testicular activity of their male offspring, even if the mother had no exposure to CuNPs. Earlier, our laboratory also showed that chronic exposure of males to CuNPs severely impairs spermatogenesis and steroidogenesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; moreover, discontinuation of CuNPs failed to restore testicular functions to a control level, and male mice showed compromised fertility [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A gross examination of testicular histology did not show observable pathological signs in the male offspring of CuNPs-treated male parents at all the doses; however, the number of tubules in spermatogenesis stages vii\u0026amp;viii and sperm motility showed a significant decline in the male offspring of male parents treated at higher doses of 100 and 200 mg/kg compared with control and 10 mg/kg CuNPs-treated paternal males. Furthermore, the sperm count showed a decline in the male offspring of all CuNPs-treated male parents; however, it was not significant. Despite the decrease in sperm count in male offspring of CuNPs-treated male parents, the absence of a significant difference vs the control could be due to individual variations, where some of the males could be resistant to CuNPs treatment. These findings clearly suggest that paternal CuNPs exposure impairs testicular functions, which could be due to genetic changes in the paternal gametes. It has been shown that parental ionic copper exposure to zebra fish can cause alterations in the sperm methylome and transcriptome, which can be passed down to their fertilized offspring [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSpermatogenesis encompasses continuous proliferation of germ cells, and slight impairment of sperm parameters and the testis prompted us to examine germ cell proliferation. The proliferation marker GCNA showed a lower abundance in the male offspring from paternal males treated at higher doses of CuNPs, while PCNA did not show changes among all the groups. Proliferation and germ cell apoptosis in the testis occurs simultaneously, and this maintains germ cell balance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The pro-apoptotic marker active caspase3 showed elevated expression in all the groups; however, the anti-apoptotic marker Bcl2 showed down-regulation in the male offspring of male parents treated with a higher dose of CuNPs. These results showed evidence of compromised proliferation and elevated apoptosis in the male offspring of CuNPs-treated male parents. These findings also partially support the decrease in sperm parameters such as the sperm count and motility due to elevated apoptosis. Despite the toxic effects of CuNPs on embryo toxicity, such as reduction in blastocyst quality and the live birth rate in mice [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], the effects of paternal CuNPs exposure on the male offspring in relation to testicular functions have not been investigated. It has been shown that paternal exposure to endocrine disruptors could impair testicular functions in the male offspring, characterised by decreased sperm parameters and testicular histopathology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Paternal exposure to another chemical toxicant, vinclozolin, elevates testicular germ cell apoptosis in the male offspring and leads to compromised sperm parameters [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It has also been shown that paternal exposure to bisphenol causes testicular sperm pathology via elevated oxidative stress in the offspring [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our results also showed elevated oxidative stress (high MDA) and decreased antioxidant enzyme (SOD) in the male offspring of male parents treated at a higher dose of CuNPs (100 and 200 mg/kg). However, GPx enzyme did not show any significant changes, and only catalase was elevated in the male offspring of male parents treated at a higher dose of CuNPs (100 and 200 mg/kg). The increased catalase could be a mechanism to counteract elevated oxidative stress.\u003c/p\u003e\u003cp\u003eTo best of our knowledge, transgenerational toxicity in male offspring after CuNPs exposure in either parent is scant, thus, the findings of the present study are supported by other environmental or chemical contaminants and endocrine disruptors. Testicular functions are well regulated by testicular steroid hormones and gonadotropins from the pituitary [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].Thus, we have also evaluated circulating testosterone and estrogen levels in F1 males, and our results showed that both circulating testosterone and estrogen were elevated in the male offspring of male parents treated with higher doses (100 and 200 mg/kg) of CuNPs. Furthermore, circulating FSH levels did not show significant changes; however, the levels of LH were high in the male offspring of all CuNPs-treated male parents. The elevated LH levels in the male offspring of 100 and 200 mg/kg CuNPs-treated male parents explain the elevated testosterone levels, because LH stimulates testosterone production. However, the elevated LH and lack of change in circulating testosterone in the F1 male offspring of 10 mg/kg CuNPs-treated male parents remains unclear as a decrease in circulating testosterone levels was expected in the male offspring after paternal exposure to CuNPs, and our data showed elevated testosterone. A previous study showed that paternal acrylamide exposure decreased circulating testosterone in the male offspring [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, maternal exposure to genistein during gestation increased circulating testosterone in the offspring [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe elevated testosterone and estrogen clearly showed hormonal imbalances in the male offspring; thus, we also analysed the expression of AR and ERs. Our data showed that the expression of AR and ERβ were down-regulated in the male offspring of all CuNPs-treated male parents. However, ERα did not show significant changes. These results clearly showed endocrine imbalances in the circulating steroids as well as their signalling in the testis of male offspring, when the male parent had been exposed to CuNPs. To the best of our knowledge, no study has been conducted to analyse AR and ER expression in the testes of male offspring mice from CuNPs-exposed male parents. Previous studies from our group have shown that continuous chronic CuNPs exposure for 70 days and even after termination of CuNPs, decreased expression of AR in the testis, and we have also shown that apelin receptor, APJ, was up-regulated in the CuNPs-treated testis, which coincides with decreased testosterone levels [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, we also examined the expression of APJ in the testis of F1 males. The expression of APJ was down-regulated in the testis of male offspring from all the paternal CuNPs-treated groups. APJ is well known to be expressed in the testis and regulates various functions in normal and pathological conditions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Elevated APJ expression in the diabetic testis has been shown to supress testosterone biosynthesis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and apelin signalling in the testis supresses testosterone biosynthesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, how does the paternal sperm after CuNPs exposure transmit this information to the F1 males, not exactly known from the findings of the present study? It has been shown that in males, reproductive abnormalities caused by exposure to endocrine disrupting chemicals can be transmitted to future generations through epigenetic modifications of germ cells and exhibits wide heterogeneity in reproductive traits [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We did not perform an epigenetic study, which is a very important limitation of our study and requires further investigation; moreover, commenting on any aspect of epigenetic-meditated transmittance to male offspring after exposure of their male parent to CuNPs would be speculative.\u003c/p\u003e\u003cp\u003eVarious studies have shown that nanoparticles impair testicular function in a mouse model [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. It has been suggested that toxic substances may have an unknown impact on offspring, since even minor and transient alterations to the testicular genome may increase the mortality and morbidity of offspring [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. It has been shown that those nanoparticles can have transgenerational effects on living organisms [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, epigenetic mechanisms are significant in mediating the responses to transgenerational toxicity resulting from environmental contaminants [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. A previous study has also shown that parental CuNPs exposure causes transgenerational toxicity in \u003cem\u003eC. elegans\u003c/em\u003e, which causes reproductive and general toxicity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To the best of our knowledge, transgenerational reproductive toxicity of CuNPs has not been shown in rodents.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, the present study showed for the first time that paternal exposure to CuNPs at different doses impairs testicular functions and spermatogenesis in male offspring, and male offspring from higher-dose-treated male parents exhibited more dysfunctions. However, none of the studied parameters in the present study showed dose-dependent effects. The impairment of testicular functions in the male offspring could be mediated by elevated oxidative stress, endocrine imbalances, deregulated signalling of AR, ERs and APJ. Despite impaired or compromised spermatogenesis, sperm quality must be evaluated in male offspring. The transgenerational effects of CuNP exposure observed in the present study may be mediated by germline transmission. These results suggest the presence of heritable genetic alterations transmitted to male offspring, potentially explaining the observed reproductive abnormalities. Further studies are needed to better understand the effects of CuNPs in male offspring.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eVKR, GG, VN: Conceptualization, Experiment design, resources generation of study. VN, Performed the experiments. VKR, GG, VN: Experimental section analysis, Data analysis, Writing of manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNicy Vanrohlu acknowledges the fellowship received at D ST/INSPIRES Fellowship (DST/INSPIRE/03/2021/001312) from DST New Delhi. The research infrastructure facility provided to the Department of Zoology, Mizoram University by the DST-FIST program, DST, New Delhi is greatly acknowledged.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eThe data are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDi Gregorio I, Busiello RA, Burgos Aceves MA, Lepretti M, Paolella G, Lionetti L (2019) Environmental pollutants effect on brown adipose tissue. 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B 281(1785):20140311.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2014.0311\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2014.0311\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Testis, CuNPs, F1 males, Spermatogenesis, Testosterone","lastPublishedDoi":"10.21203/rs.3.rs-8068308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8068308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAt present, the use of copper nanoparticles (CuNPs) is very common for various human applications. Despite its uses to improve human welfare, the toxicity of CuNPs is well documented. It can cause toxicity to different vital organs, including the male reproductive organs, or testes. Whether the offspring of CuNP-treated males show testicular impairment has not been well documented. The present study investigated the effects of paternal exposure to CuNPs on the testes of male offspring (Swiss Albino mice) divided into the following four groups: a control group and 10 mg/kg, 100 mg/kg and 200 mg/kg exposure groups, whose only male parent was exposed to CuNPs at 0, 10, 100 and 200 mg/kg, respectively, for 70 days, which covered two spermatogenic cycles. The findings reveal that exposure of male parents to CuNPs at higher doses, 100 and 200 mg/kg, compromises spermatogenesis in the testes of male offspring due to decreased germ cell proliferation. Our results showed that oxidative stress was also elevated in the male offspring of male parents treated with a higher dose of CuNPs. However, elevated apoptosis (increased caspase3) was noted in the male offspring of all treated male parental groups. Circulating testosterone and estrogen levels were elevated in the F1 males of higher dose CuNP-treated male paternal groups; however, the expression of androgen receptor (AR), apelin receptor (APJ) and estrogen receptor-β (Erβ) was decreased in the male offspring from all treated parental groups. In conclusion, paternal exposure to CuNPs was found to disrupt spermatogenesis and steroid signalling function in the offspring of F1 males.\u003c/p\u003e","manuscriptTitle":"Paternal chronic exposure to copper nanoparticles (CuNPs) impairs testicular androgen and estrogen signalling in adult male offspring in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 09:03:30","doi":"10.21203/rs.3.rs-8068308/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T20:57:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T10:54:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196659620344716138976745332372381487960","date":"2026-01-05T11:34:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75734363950676552308781592462730164017","date":"2026-01-05T10:30:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8535090866267526431024838988622132399","date":"2026-01-05T03:15:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T05:29:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183399600328555134457454819254351584663","date":"2025-11-22T16:30:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287864110264459960281250924190524248256","date":"2025-11-22T02:27:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-17T02:17:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-12T13:33:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T02:15:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2025-11-09T09:56:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bf9e75b2-d2bf-4f84-a5ff-5960540d6350","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:11:11+00:00","versionOfRecord":{"articleIdentity":"rs-8068308","link":"https://doi.org/10.1007/s12011-026-05077-7","journal":{"identity":"biological-trace-element-research","isVorOnly":false,"title":"Biological Trace Element Research"},"publishedOn":"2026-03-21 15:57:33","publishedOnDateReadable":"March 21st, 2026"},"versionCreatedAt":"2025-11-26 09:03:30","video":"","vorDoi":"10.1007/s12011-026-05077-7","vorDoiUrl":"https://doi.org/10.1007/s12011-026-05077-7","workflowStages":[]},"version":"v1","identity":"rs-8068308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8068308","identity":"rs-8068308","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}