Effects of acute or chronic administration gold nanospheres on testicular functions in male rats

Effects of acute or chronic administration gold nanospheres on testicular functions in male rats | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of acute or chronic administration gold nanospheres on testicular functions in male rats Doaa Mansour, Sahar Orabi, Said Fathalla, Shaaban Gadallah, Amina Gamal el Din, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1490709/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 4 You are reading this latest preprint version Show more versions Abstract This study investigated the effect of gold nanospheres (AuNPs) administration on the rat male reproductive function. Experiment 1: Fifty-four rats were assigned into three groups (n = 18/group). Group І (control), Group ІІ (50 nm AuNPs) and Group ІІІ (10 nm AuNPs), in which rats were intramuscular (i.m) injected with a single dose of 75µg AuNPs/kg/bwt. Experiment 2: Eighteen rats were randomly assigned to three groups (n = 6/group). Group І (control), Group ІІ (50nm AuNPs) and Group ІІІ (10 nm AuNPs): rats were injected repeated doses of 75µg AuNPs /kg/bwt for 5 weeks followed by 3 weeks washout period. Blood, testes and epididymis samples were collected on Days-3, 7, and 60 in experiment 1 and on Day-60 in the 2nd experiment. Results indicated that acute or chronic administration of 10nm AuNPs caused significantly increased serum testosterone level on day-60. Chronic administration of 50nm AuNPs has an antioxidant effect. Acute or chronic study induced no histopathological changes and no changes in Proliferating Cell Nuclear Antigen (PCNA) and caspase-3 in testicular tissues. Acute or chronic administration of 10nm AuNPs significantly increased the spermatozoa in epididymal ducts and testicular tissue on Day-60 post-treatment. In conclusion, the administration of AuNPs (10nm and 50nm size) is safe on the male reproductive function. Male rats Acute chronic study histopathology morphometric analysis Immunohistochemistry Oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Gold is a biologically inert metal that is found only at the macroscopic level, but gold at the nanoscale size has unique physical and chemical characteristics, such as stability, inertia, small size, simple penetration, biocompatibility, low cytotoxicity, and surface Plasmon resonance excitation characteristics ( 1 , 2 ). Therefore, gold nanoparticles (AuNPs) are utilized in various fields, including nanomedicine, diagnosis, drug and gene delivery, biological imaging, photothermal therapy, biosensing, contrast agents for cancer and pharmaceuticals ( 3 , 4 ). They are also used in several industrial applications such as cosmetics, hair tonics, conductive ink, lubricant oil ( 5 ), foodstuffs, textiles, electronic and sports instruments ( 6 ). Despite the benefits of AuNPs in many areas of medicine and industry, there are potentially damaging effects on living organisms, including human health particularly male reproductive function ( 7 ). The tiny size of nanoparticles (NPs) enables them to overcome the body defense barriers without being prevented ( 8 ). Bio-persistence of NPs in testes appears to rely on the size of NPs, the exposure route, and the agglomeration of particles ( 9 ). A previous study has reported that the testis is a minor distribution target for NPs compared to other organs where very limited quantities of nanoparticles can reach the testes irrespective of the exposure routes and the physicochemical characteristics of NPs ( 10 ). The process through which NPs may affect testicular functions involves reduced sperm production ( 11 ) and reduced testosterone synthesis ( 12 ). Furthermore, following intraperitoneal injection (IP) of AuNPs in male mice declined sperm motility and morphology and elevated the abnormal spermatozoa number ( 13 ). Administration of AuNPs to male rats for 2 months induced penetration of the seminiferous tubules without any effect on their basic functions, as well as having no influence on the proliferation of spermatogonia and on the apoptosis of mature germinal cells ( 14 ). Also, Polyethylene Glycol-coated gold nanoparticles (PEG-AuNPs) have the ability to cross the blood-testis barrier (BTB) and elevated plasma testosterone levels without any detrimental effects on fertility ( 15 ). Moreover, no testicular histopathological changes were detected following IP injection of PEG-AuNPs in mice ( 16 ). It was reported that the main mechanism of AuNPs toxicity is via oxidative stress causing damage to the lipids, carbohydrates, protein, and DNA ( 17 ). There are contradictory data about the oxidative stress induced by AuNPs. Some studies indicated that AuNPs cause significant oxidative stress and may lead to inflammation, DNA damage, and cell death ( 18 , 19 ). In contrast, other experimenters revealed that AuNPs are potent antioxidants and have no significant induction of oxidative stress or inflammatory response ( 20 ). Acute or chronic administration of GNPs alters the liver, kidney functions, and blood profile on day 7 post-treatment ( 21 ). Presently, data on the in vivo toxicity of gold nanospheres on the male reproductive system are scared. So, this study was carried out to investigate the effect of acute and chronic gold nanospheres administration on the male reproductive function via estimation of serum testosterone level, oxidative stress parameters, histopathological, morphometric, and immunohistochemical analyses of testicular tissue in male rats. Materials And Methods Spherical gold nanoparticles at the size 10nm were purchased from Sigma-Aldrich Com., Germany (PCode: 4100798324). while AuNPs (50nm size) were synthesized at the national research center, Dokki. Preparation of gold nanospheres (AuNPs at the size 50nm): Gold nanoparticles were synthesized according to the Turkevich method (22). Simply, a mixture of 1 ml of a solution of 5 mm Chloroauric Acid (HAuCl4) in deionized water (DI water) and 18 ml of DI water was heated until it started boiling, then add one milliliter of 0.5% trisodium citrate solution. It was heated up to an apparent color change, then taken out of the heater and stirred until cool down to room temperature. Characterization of AuNPs: The absorption spectra of AuNPs were collected by using V-630 UV-VIS Spectrophotometer (Jasco, Japan). Transmission electron microscopy (TEM) images were obtained using JEOL JEM 2010 TEM operated at 100- KV accelerating voltage. Animals and experimental design: Seventy-two adult male Albino Wister rats (110-150 g/bwt) were used in the current study. They were obtained from AL-Zyade Experimental Animals Production Center, Giza, Egypt. They were kept in well-ventilated metal cages at 23 ± 2 °C, 40–60% relative humidity and light/dark cycle of 12 h, Feed of balanced ration and water were added ad libitum and left 2 weeks for adaptation. They were randomly divided into 2 experiments. Experiment I: Effect of acute intramuscular administration of 10nm and 50nm gold nanospheres on reproductive function in male rats. Fifty-four adult male rats were randomly assigned to three equal groups (n=18/group). Group І (control group): rats were intramuscular (i.m.) injected of 1 ml of normal saline 0.9%.Group ІІ (50nm AuNPs) & Group ІІІ (10nm AuNPs): in which rats were i.m. injected with 75μg AuNPs/kg/bwt (23) 50nm and 10nmAuNPs, respectively as one shot. AuNPs were diluted using a physiological saline solution. Rats were sacrificed by head dislocation after 3, 7, and 60 days to obtain blood and tissue samples (testes and epididymis). Experiment II: Effect of chronic intramuscular administration of 10nm and 50nm gold nanospheres on reproductive function in male rats. Eighteen adult male rats were randomly assigned to three equal groups (n=6/group). Group І (control group): in which rats i.m injected with 1 ml normal saline 0.9%.Group ІІ (50nm AuNPs) & Group ІІІ (10nm AuNPs): in which rats were i.m injected with 75μg50nm or 10nm AuNPs/kg/bwt, respectively (23). AuNPs were diluted with physiological saline solution, injected i.m once weekly for five consecutive weeks, followed by a 21day for a washout period. On Day-60, they were sacrificed by head dislocation to obtain blood and tissue samples (testes and epididymis). Sampling: Blood samples were collected from the medial canthus of the eye using capillary tubes. The collected blood samples were left to clot, followed by centrifugation at 1500g for 15 minutes and serum samples were separated and kept at – 20°C for estimation of serum testosterone. Testes and epididymis were immediately picked up. Samples from testes were divided into 2 parts, one part was stored at -80 0 C for assaying lipid peroxidation (MDA) and antioxidant defense system (GSH and SOD) and the second part was fixed at 10% neutral buffered formalin for assaying immunohistochemical studies and also with epididymis for histopathological and morphometric analyses. Hormonal and oxidative stress analyzing: Serum testosterone level was determined by the quantitative method (24) by using the LIAISON® Testosterone assay method, DiaSorin, Italy, (Ref. Code: 310410). Tissue homogenate was prepared according to a previously published method (25). Commercial diagnostic kits (Biodiagnostic, Egypt) were used for the assay of malondialdehyde (MDA) (Cat. No. MD 25 29) were measured as previously described (26, 27), reduced glutathione (GSH) (Cat. No. GR 25 11) was determined according to a previously published method (28) and superoxide dismutase (SOD) (Cat. No. SD 25 21) was evaluated following the previously described method (29). Histopathological examination: Testicular and epididymal tissue samples were cut and fixed at 10% neutral buffered formalin. The tissues were dehydrated and embedded in paraffin blocks. Micro sections (4 μm) were prepared and stained with hematoxylin and eosin dye and were examined under a light microscope (30). Morphometric evaluation of seminiferous tubules and epididymis: The morphometric analysis was performed using a Leica Qwin 500 Image Analyzer (Leica Imaging Systems Ltd, Cambridge, UK). At every time point of the experiment, 10 sections of seminiferous tubules from control and AuNPs-treated rats were evaluated (round or almost round in form). At × 400 magnifications, the diameter of seminiferous tubules was calculated; the major and minor axes of the tubule and the epithelial thickness were measured from the spermatogenic cells on the basement membrane via the sidelines cells of the lumen of tubules (31). Also, various spermatogenic cells, such as spermatogonia, spermatocytes, and spermatids were recognized based upon their distinctive morphology and count (32). The morphometric analysis of the epididymis was performed using 20 sections of ducts difference. The major and minor axes diameter and epithelial height of the ducts difference were measured with sperm counts at × 400 magnifications. Immunohistochemical investigation: Another section of the testicular tissue block was deparaffinized, rehydrated, and immune-stained using primary mouse monoclonal antibody against PCNA Ab-1 (Clone PC10) Thermo Fisher Scientific; Fremont, CA 94538, USA; localized in the nucleus; for cellular proliferation evaluation (33). Also, another section of the same block was immune-stained using mouse monoclonal antibody: Caspase-3 (CPP32) Ab-4 primary rabbit polyclonal antibody (Thermo Scientific); localized mostly in the cytoplasm and some in the nucleus; for apoptosis assessment (34). Diaminobenzidine (DAB) was used to detect both immune stains (35). The sections were examined under an Olympus research microscope CX41 and the digital photomicrographs sections were photographed at different magnifying powers with Olympus SC100digital CCD camera. Spermatogenic cells lining the seminiferous tubules were regarded to be positive for activating caspase-3with brown cytoplasm and/or nuclei, while they were deemed positive for PCNA with dark brown nuclei. Statistical analysis: All statistical analysis was performed using SPSS software (Statistical Package for Social Sciences) Version 22 using one-way ANOVA with P < 0.05 regarded as statistically significant. All data were expressed as mean ± standard errors of the means (SEM). Results Preparation and characterization of gold nanospheres The morphology and size of AuNPs were shown by using transmission electron microscopy (TEM) in Figures 1a and b from this image, it is clear that a uniform sphere shape of AuNPs with regular distribution was formed. The small size particle averaged was 10nm in length. While in large size particles, the diameter was average 50nm. It was found that the surface Plasmon absorption of AuNPs was 525-530 nm. Effect of AuNPs on serum testosterone level (ng/ml) Data presented in Table 1 represent the acute and chronic effects of i.m. injection of 75μg AuNPs /kg/bwt on serum testosterone level. Results showed that i.m. injection of a single dose of 50nm or 10nm AuNPs had no significant effect on serum testosterone level compared to the control group on days 3 and 7. However, i.m injection of single or repeated doses of 10nm AuNPs significantly increased serum testosterone level in Day-60 post-treatment compared to 50nm AuNPs or the control group. Effect of AuNPs on testicular tissue MDA level, GSH content, and SOD activity Data illustrated in Table 2 represent the effect of acute and chronic i.m injection of 75μg 50nm or 10nm AuNPs /kg/bwt on the testicular tissue lipid peroxidation (MDA) and antioxidant defense system biomarkers as GSH content and SOD activity. Results demonstrated that i.m. injection of single or repeated doses of 50nm AuNPs (2 nd Group) or 10nm AuNPs (3 rd Group) had no significant effect on the testicular tissue MDA level and GSH content compared to the control group at any point of the experiment. But, they significantly (P < 0.05) increased testicular tissue SOD activity compared to the control group on Day-60. However, i.m. injection of repeated doses of 50nm AuNPs significantly (P < 0.05) decreased testicular tissue MDA level and (P < 0.05) increased GSH content and SOD activity. Histopathological examination of the testes and epididymis: Data for histopathological examination of testes and epididymis tissues in different groups of rats are shown in Figure 2 and Figure 3, respectively. Results indicated that acute or chronic i.m. injection of 75μg of 50nm or 10nm AuNPs /kg/bwt has no adverse effect on the histological evaluation of the testicular tissue. AuNPs treated male rats as in the control group showed normal, well preserved histological structure at all times of experiment, including normal seminiferous tubules of average diameter and normal basement membranes. Epithelial lining showed numerous Sertoli cells and cells at different stages of the spermatogenic series; including spermatogonia, spermatocytes, spermatids, and spermatozoa. For histological evaluation of the epididymal ducts in both control and AuNPs -treated rats; histopathological examination revealed normal well structurally preserved, without any histopathological abnormalities regardless of the time of analysis in both control and experimental groups. The epididymal ducts were lined by regular pseudostratified epithelial cells with rounded bland basophilic nuclei and cilia projecting into dilated lumina in which numerous spermatozoa were seen. Morphometric studies: The obtained data for morphometric analysis of testes and epididymis tissue in control and AuNPs treated rats are presented in Table 3, Table4, Table 5, and Table 6, respectively. Results revealed that i.m. injection of 75μg 50nm or 10nm AuNPs/kg/bwt significantly (P < 0.05) increased the mean number of spermatogonia on Day-7 post-treatment. While rats that received a single dose of 10nm AuNPs showed a significant (P < 0.05) increase in the mean number of spermatogonia and spermatozoa on Day-60 post-treatment compared to the control group. However, repeated doses of 10nm AuNPs significantly (P < 0.05) increased the mean number of spermatocytes, spermatids, and spermatozoa on Day-60 post-injection compared to the control group. With regard to the epididymis, i.m. injection of a single dose of 10 nm or 50 nm AuNPs significantly (P < 0.05) increased the average number of spermatozoa per epididymal ducts on Day-60 post-treatment compared with the control group. Also, a single dose of 10 nm AuNPs significantly (P < 0.05) decreased the minor axis of epididymal ducts on Day-7 post injection compared to the control rats. However, repeated injection of 10nm AuNPs significantly (P < 0.05) decreased the minor axis of epididymal ducts and significantly (P < 0.05) increased the average number of spermatozoa per epididymal ducts on Day-60 post-injection compared to the control rats. Immunohistochemical study: The obtained data for the immunohistochemical examination of proliferation biomarker Proliferating Cell Nuclear Antigen (PCNA) and apoptosis marker (Caspase-3) in control and AuNPs injected rats are illustrated in Table 7 and Figure 4 and Figure 5, respectively. The data showed no alteration in the expression of PCNA within the nuclei of spermatogenic cells lining the seminiferous tubules in testicular tissues for 10nm or 50nm AuNPs injected rats when compared to the control group at each point of the experiments. At the same time, the expression of caspase-3within the cytoplasm of spermatogenic cells lining the seminiferous tubules in testicular tissues was similar in control and 10nm or 50nm AuNPs injected rats at each time point of the experiments. Table 1 : Effects of acute or chronic I.m injection of 10nm or 50nm AuNPs on serum testosterone (ng/ml) level in male albino rats (mean ±SEM) Treatment Time Control group 50nm AuNPs 10nm AuNPs Acute Day-3 1.01±0.27 1.62±0.24 1.67±0.24 Day-7 1.21±0.26 1.08±0.16 1.12±0.14 Day-60 1.16±0.13 a 1.39±0.14 a 2.09±0.23 b Chronic Day-60 1.88±0.24 a 1.14±0.31 a 4.13±0.37 b a,b differ significantly within the same row at each time point at P< 0.05, number of rats=6/group Table 2: Effects of acute or chronic i.m injection of 10nm or 50nm AuNPson testicular MDA, GSH and SOD in male albino rats (mean ±SEM). Treatment Time Group MDA nmol/gm GSH mg/gm. SOD U/gm Acute Day-3 G1 48.6 ±2.96 5.02±0.29 573.1±24.4 G2 44.41±4.04 4.99±0.44 646.8±28.25 G3 45.08±3.04 4.58±0.67 596.6±31.67 Day-7 G1 50.38±6.98 4.94±0.07 612.9±23.3 G2 60.2±5.41 4.69±0.64 651.5±14 G3 61.32±7.73 4.13±0.21 667.2±19.8 Day-60 G1 57.2±3.43 4.77±0.69 584.3±20 a G2 61.79±2.42 5.08±0.48 732.8±25.1 b G3 66.69±3.34 5.68±0.82 714.1±16.8 b Chronic Day-60 G1 59.54±2.68 a 4.28±0.31 a 585.94±20.43 a G2 35.75±3.17 b 6.93±0.64 b 720.31±15.25 b G3 45.83±4.98 ab 5.97±0.68 ab 685.78±28.24 b a,b differ significantly within the same row at each time point at P< 0.05.number of rats=6/group Table 3: Morphometric analysis of testes in control and AuNPs -injected male rats at the different time points of the experiment (Single injection of AuNPs) Time Group Diameter of seminiferous tubules (µm) Cell count Major axis Minor axis Epithelial height Spermatogonia Spermatocytes Spermatid Spermatozoa Day-3 G1 314.51± 6.77 246.28 ±6.35 62.99±2.35 61.25 ± 1.83 58.21 ± 1.78 64.01±1.83 115.33 ± 2.91 G2 330.97± 7.61 236.78±4.66 58.58±2.64 66.21 ± 1.58 66.01 ± 3.35 67.33±3.01 110.81 ± 2.42 G3 331.88 ± 8.56 248.39 ±7.89 62.95±1.67 62.51 ± 2.13 60.57 ± 4.22 56.67±1.67 108.01 ± 2.24 Day-7 G1 309.55 ± 7.77 248.69 ±6.17 64.44±3.48 62.01 ± 1.93 a 61.51 ± 1.61 67.41±1.83 115.33 ± 4.91 G2 336.39 ± 9.79 254.41 ±6.53 57.26±4.07 73.41 ± 2.11 b 65 .75 ± 1.55 66.57±2.13 118.01 ± 7.25 G3 338.77 ±10.43 246.58 ±4.03 60.09±3.51 74.01 ± 1.01 b 66.17 ± 2.02 72.61±2.98 133.61 ± 7.53 Day-60 G1 400.66 ±12.89 287.54±12.18 65.55±2.82 59.25 ± 2.19 a 56.81 ± 1.39 a 56.01±2.12 103.61±1.93 a G2 369.86 ±13.38 247.17±14.11 64.38±2.68 60.11 ± 1.01 a 59.51 ± 2.85 a 61.25±2.71 109.11±2.25 a G3 376.32 ±13.09 269.27±9.98 61.35±3.91 64.67 ± 2.91 b 81.13± 2.13 b 64.67±2.19 194.83±1.88 b a,b differ significantly within the same column at each time point at P< 0.05.number of rats=6/group The number of spermatogonia was significantly (P< 0.05) higher in 10nm and 50nm AuNPs injected rats on Day-7 post-injection when compared with control. Also, i.m. injection of 10nm AuNPs produced significantly (P< 0.05) increase in the number of spermatogonia, spermatocytes, and spermatozoa on Day-60 post-injection in male rats. Table 4: Morphometric analysis of testes in control and AuNPs -injected male rats at the different time points of the experiment (Repeated injection of AuNPs) Time Group Diameter of seminiferous tubules (µm) Cell count Major axis Minor axis Epithelial height Spermatogonia Spermatocytes Spermatid Spermatozoa Day-60 G1 420.89 ±14.45 280.72 ±13.96 65.55±2.82 59.25 ± 2.19 59.25 ± 1.89 a 50.01±1.91 a 101.86 ± 1.75 a G2 397.64 ±16.04 254.98 ±10.17 60.85±3.69 56.51 ± 1.01 51.51 ± 1.85 a 49.71±1.86 a 105.51 ± 2.39 a G3 402.94 ±15.39 268.96 ± 8.84 69.17±3.83 62.81 ± 1.65 71.67± 1.26 b 67.75±3.95 b 188.81 ±3.99 b a,b differ significantly within the same column at each time point at P< 0.05. number of rats=6/group Repeated injection of 10nm AuNPs in male rats showed significantly (P< 0.05) higher number of spermatocyte, spermatids and spermatozoa on Day-60 post-injection compared with that in the control group. Table 5: Morphometric analysis of epididymis in control and AuNPs -injected male rats at the different time points of the experiment (Single injection of AuNPs) Time Group Diameter of epididymal ducts (µm) Epithelial height (µm) Spermatozoa Major axis Minor axis Day-3 G1 323.69±6.53 238.36±7.71 26.16±1.81 145.67±9.17 G2 317.22±9.62 207.96±10.42 28.86±3.13 172.21±8.07 G3 342.13±5.31 199.06±11.83 27.28±1.98 167.67±7.19 Day-7 G1 359.17±10.28 244.91±4.51 a 25.51±1.89 149.51±7.36 G2 338.56±9.95 229.48±3.78 a 25.79±1.79 144.25±8.61 G3 339.03±16.96 185.51±6.09 b 29.36±1.83 143.33±10.41 Day-60 G1 383.67±7.53 277.04±12.84 25.53±1.96 121.17±9.51 a G2 420.43±11.78 265.54±14.99 25.47±2.08 149.51±10.11 b G3 399.14±12.74 247.01±13.93 22.91±2.06 194.44±6.99 c a,b,c differ significantly within the same column at each time point at P< 0.05.number of rats=6/group Data revealed that i.m. injection of a single dose of 10nm AuNPs resulted in a significant (P< 0.05) decrease in minor axis diameter of the epididymis of injected male rats on Day- 7 post-treatment when compared with 50nm AuNPs or control animals. Also, 10nm AuNPs injected rats showed significantly (P< 0.05) higher number of spermatozoa on Day- 60 post-injection when compared with control or 50nm AuNPs group. Table 6: Morphometric analysis of epididymis in control and AuNPs -injected male rats at the different time points of the experiment (Repeated injection of AuNPs) Time Group Diameter of epididymal ducts (µm) Epithelial height (µm) Spermatozoa Major axis Minor axis Day-60 G1 384.67±10.63 277.04±12.8 a 25.53±1.96 122.83±10.92 a G2 397.12±12.29 269.06±10.61 a 22.49±2.47 117.41±8.71 a G3 407.11±14.45 206.79±14.83 b 26.62±1.63 178.75±10.15 b a,b differ significantly within the same column at each time point at P< 0.05.number of rats=6/group Repeated injection of 10 nm AuNPs in male rats showed significant (P< 0.05) decrease in minor axis diameter of the epididymis of injected male rats and also significant(P< 0.05) increase in number of spermatozoa on Day-60 post-injection when compared with control or 50nm AuNPs group. Table 7: Effects of acute or chronic i.m injection of 10nm or 50nm AuNPson testicular immunohistochemical changes in PCNA and Caspase-3 in male albino rats (mean ±SEM) Treatment Time Group PCNA/1mm Caspase-3/HPF. Acute Day-3 G1 91.33±6.31 3.25±0.45 G2 90.67±4.06 3.41±0.68 G3 90.33±4.91 3.01±0.58 Day-7 G1 91.51±5.17 3.21±0.86 G2 88.01±7.81 4.5±0.76 G3 88.66±5.24 2.67±0.61 Day-60 G1 89.61±4.33 3.33±0.81 G2 88.01±4.14 2.51±0.65 G3 93.25±4.61 4.51±0.69 Chronic Day-60 G1 90.21±4.31 3.33±0.81 G2 95.02±3.97 2.51±0.65 G3 92.01±4.04 4.51±0.59 Number of rats=6/group Discussion Our data revealed that i.m. injection of either single or repeated doses of 10nm AuNPs significantly (P < 0.05) increased serum testosterone level compared to control rats, this was detected on Day-60 after injection. In similar, some studies reported that IP injection of 75 µg AuNPs/kg/bwt of male rats significantly elevated serum total and free testosterone levels compared to the control group on days 1, 3, 7, and 14 post-gold nanorods (AuNRs) injection ( 20 ). Also, intravenous (IV)injection of ω-aminoethyl spherical PEG capped AuNPs (PEG-NH 2 @ AuNPs) in mice has elevated plasma testosterone levels, but had no effect on plasma follicle stimulating hormone (FSH) or luteinizing hormone (LH) levels that was accompanied by no any alterations on male fertility, fetal survival or fetal development ( 15 ). Conversely, other studies showed that treatment with AuNPs at different doses 100, 50, 25 ppm for 10 days reduced serum testosterone levels in AuNPs-treated rats compared to the controls especially at treatment with high doses ( 12 ). This discrepancy could be due to the AuNP size or dose used or at the time of exposure. Concerning oxidative stress, our results showed that AuNPs has an antioxidant effect. Repeated injection of 50nm AuNPs significantly decreased testicular tissue MDA level and significantly (P < 0.05) increased of testicular tissue GSH content and SOD activity on Day-60 post-injection. Meanwhile, the injection of 10nm or 50nm AuNPs significantly (P < 0.05) increased testicular tissue SOD activity, while the MDA level and GSH content were similar to the control group on Day-60 post-injection. However, i.m. injection of 10nm or 50nm AuNPs has no effect on that oxidative stress parameters on Day-3 or 7after injection. Similarly, IP injection of 75 µg/kg/bwt AuNRs (50nm size) significantly reduced serum MDA and increased the antioxidant markers including catalase, glutathione peroxidase (GPx) and SOD activities compared to control which indicated the antioxidant activity of AuNRs ( 20 ). Additionally, AuNPs are considered as antioxidants through reduction of the production of reactive oxygen species and nitrite species oxygen-free radicals, that is maybe owing to their interaction with the thioredoxin that acts over an endogenous inhibitor, thioredoxin-interacting protein (Txnip) which is related to the regulation of cellular redox ( 36 , 37 , 38 ). Conversely, other studies found that after IP injection of 10nm AuNRs induced a significant increase in MDA values in rat liver, lung, heart and kidney organs and significant decrease in SOD in rat kidney, liver and lung organs compared to the control after 3 and 7 days from treatment ( 39 ). This difference could be due to the Au nanoparticle shape, dose, and route of administration. For the histopathological study, the present work reported that there were no histopathological changes in the testes or epididymis of rats i.m. injected with either single or repeated doses of 10 nm or 50 nm AuNPs at each time point of the experiment when compared to control rats. These results are matched with other studies recorded that the testes and epididymis in male rat IP injected with a single dose of 50nm AuNRs at (75 µg/kg/bwt.) induced no any obvious histopathological alterations at each time of the experiment (Days-1, 3, 7 and 14) ( 20 ). Also, IP injection of male mice with different sizes of spherical PEG-coated AuNPs at a dose of 4000 µg/kg caused well preserved histological structure in the testes of AuNPs-treated mice compared with control ( 16 ). On the other hand, there are degenerative changes in the seminiferous tubules in Wister rats, which IP injected with AuNPs at a dose of 100 ppm ( 12 ). This contradictory, could be related to the dose of AuNPs used. For morphometric analysis, it is considered very delicate tools for assessing structural modifications by which histological examination could not be correctly proven. Our results are consistent with other findings declared that single IP injection of 75 µg/kg/bwt of AuNRs (50nm size) significantly increased the diameter of the minor axis of the seminiferous tubule, the height of the epithelial lining of the seminiferous and the average numbers of spermatocytes and spermatid after 7days of treatment ( 20 ). About the epididymis, the number of spermatozoa was significantly increased after 7 and 14 days compared to the control rats. Spermatogenesis is a complex process through which the spermatogonia in the testis evolve into mature sperm by continuous mitosis, meiosis and cell distinction in the testis ( 40 ). Proliferation of spermatogonia and apoptosis are responsible for maintaining balance in spermatogenesis. Apoptosis (Programmed cell death) occurs in the testes limiting the number of germinal cells in the epithelium of the testes ( 41 ). Our results showed no changes within PCNA or caspase-3 positive immunoreactivity within the nuclei or cytoplasm of spermatogenic cells lining the seminiferous tubules in testicular tissues of injected rats either with single or repeated doses of 10 nm or 50 nm AuNPs throughout the experiment when compared to control rats. These results are matched with other studies showed that orally administration of AuNPs to male rats for 8 weeks did not change the expression of proliferation proteins (KI-67 and D1 cyclin), or the activity of apoptosis (TUNEL method)in the spermatogenic epithelium ( 14 ). Conversely, other studies reported that Hesperetin conjugated AuNPs have anti-proliferative effects causing inhibition of PCNA expression in rat liver ( 42 ). Additionally, AuNPs have led to the release of free radicals and an elevation of apoptosis ( 43 ) which induced considerable up-regulation of mRNA expression of p53, bax, caspase-3, and caspase-9 which increased apoptosis ( 44 ). This difference could be attributed to variations in size, type, shape or dose of gold nanoparticles used. Conclusion Single or repeated i.m. injection of 10nm or 50nm AuNPs in male rats is safe on the male reproductive function; also, they have an antioxidant effects. Declarations Ethics approval and consent to participate All animal-handling procedures, as well as samples' collection and disposal, were according to the regulations of the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, University of Sadat City (Approval number: VUSC-006-1-19). Consent for publication All authors agree for publication Competing interests The authors declare no conflict of interest. Acknowledgements The authors thankfullyappreciatethe central laboratory, Faculty of Veterinary Medicine, University of Sadat City Authors’ contributions SO, AA, SF, SG contributed to the conception, design. Both DM, AA, SO and AG contributed to collection and/or assembly of data, data analysis and interpretation, and manuscript writing. SO, AA, SF, AG contributed to manuscript revision. All authors read and approved the final manuscript. Funding No Funding Availability of data and materials All data generated and analyzed during this research are included in this published article. References Drucker E, Krapfenbauer K. Pitfalls and limitations in translation from biomarker discovery to clinical utility in predictive and personalized medicine. EPMA J. 2013; 4: 7. https://doi.org/10.1186/1878-5085-4-7 Jenkins JT, Halaney DL, Sokolov KV, Ma LL, Shipley HJ, Mahajan S, Louden CL, Asmis R, Milner TE, Johnsons KP, Feldman MD. Excretion and toxicity of gold–iron nanoparticles. 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A study of the nucleation and growth process in the synthesis of colloidal gold. Discuss. Faraday Soc. 1951; 11:55–75. Abdoon AS, Al-Ashkar EA, Kandil OM, Shaban AM, Khaled HM, El Sayed MA, El Shaer MM, Shaalan AH, Eisa WH, Eldin AA, Hussein HA, El Ashkar MR, Ali MR, Shabaka AA. Efficacy and toxicity of plasmonic photothermal therapy (PPTT) using gold nanorods (GNRs) against mammary tumors in dogs and cats. Nanomed. 2016; 12:2291–2297. https://doi.org/10.1016/j.nano.2016.07.005 Feldman HA, Longcope C, Derby CA, Johannes CB, Araujo AB, Coviello AD, Bremner WJ, McKinlay JB. Age trends in the level of serum testosterone and other hormones in middle-aged men: longitudinal results from the Massachusetts male aging study. J Clin Endocrin Meta. 2002; 87:589-598.https://doi.org/10.1210/jcem.87.2.8201 Shawky SM, Ramadan SG, Orabi SH. Hemato-biochemical, Behavioral and Neurological effects of Vitamin C administration against lead exposure in mice.Inter J Advan Res. 2014; 2: 418-429. Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method.Clin Chim Acta. 1978; 15:37-43. https://doi.org/10.1016/0009-8981(78)90081-5 Ohkawa H, Ohishi W, Yagi k.Assay for lipid peroxide in animal tissue by thiobarbituric acid reaction.Anal Biochem. 1979; 95:351-358.https://doi.org/10.1016/0003-2697(79)90738-3 Beutler E,Duron O,Kelly BM. Improved method for the determination of blood glutathione.J Lab Clin Med. 1963; 61:882-888. Nishikimi M, Roa NA, Yogi K. The Occurrence of Superoxide Anion in the Reaction of Reduced Phenazine Methosulfate and Molecular Oxygen. Biochem Biophys Res Commun. 1972; 46:849-854. Drury RAB, Wallington EA, Cameron R. Carleton’s Histological Techniques: 4th ed., Oxford University Press NY. USA, 1976;279-280. Osinubi AA, Noronha CC, Okanlawon AO. Morphological and stereological assessment of the effect of long term administration of quinine on the morphology of rat testis. West Afr J Med. 2005; 24:200–205. Seethalakshmi L, Flores C, Carboni AA, Bala R, Diamond DA, Menon M. Cyclosporine: its effects on testicular function and fertility in the prepubertal rat. J Androl. 1990; 11: 17–24. https://doi.org/10.1002/j.1939-4640.1990.tb01568.x Hall PA, Levison DA, Woods AL, Yu CCW, Kellock DB, Watkins JA, Barnes DM, Gillett CE, Camplejohn R, Dover R, Waseem NH, Lane DP. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol. 1990; 162: 285-294. Virkajärvi N, Pääkkö P, Soini Y. Apoptotic index and apoptosis influencing proteins bcl-2, mcl-1, bax and caspases 3, 6 and 8 in pancreatic carcinoma. Histopath. 1998; 33: 432-439. https://doi.org/10.1046/j.1365-2559.1998.00553.x Shi ZR, Itzkowitz SH, Kim YS. A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues. J Histochem Cytochem. 1988; 36: 317-322. https://doi.org/10.1177/36.3.3278057 Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview. Langmuir. 2005; 21:10644-10654 . h ttps://doi.org/10.1021/la0513712 Yakimovich NO, Ezhevskii AA, Guseinov DV, Smirnova LA, Gracheva TA, Klychkov KS. Antioxidant properties of gold nanoparticles studied by ESR spectroscopy. Russ Chem Bull. 2008; 57: 520–523. https://doi.org/10.1007/s11172-008-0080-1 Barathmanikanth S, Kalishwaralal K, Sriram M, Pandian SR, Youn HS, Eom S, Gurunathan S. Anti-oxidant effect of gold nanoparticles restrains hyperglycemic conditions in diabetic mice. J Nanobiotech. 2010; 8:16-30 . https://doi.org/10.1186/1477-3155-8-16 Abdelhalim MA. Uptake of gold nanoparticles in several rat organs after intraperitoneal administration in vivo: a fluorescence study. Biomed Res Int. 2013; 2013: 353695. https://doi.org/10.1155/2013/353695 Griswold MD. Spermatogenesis: the commitment to meiosis.Physiological reviews. 2016; 96: 1-17.https://doi.org/10.1152/physrev.00013.2015 Shaha C, Tripathi R, Mishra DP. Male germ cell apoptosis: regulation and biology.Philosophical Transactions of the Royal Society B: Biol Sci 2010; 365: 15011501-1515. https://doi.org/10.1098/rstb.2009.0124 Krishnan G, Subramaniyan J, Subramani PC, Muralidharan B, Thiruvengadam D. Hesperetin conjugated PEGylated gold nanoparticles exploring the potential role in anti-inflammation and anti-proliferation during diethylnitrosamine-induced hepatocarcinogenesis in rats.Asian J PharmaSci. 2017; 12:442-455. https://doi.org/10.1016/j.ajps.2017.04.001 Ding F, Li Y, Liu J, Liu L, Yu W, Wang Z, Ni H, Liu B, Chen P. Over endocytosis of gold nanoparticles increases autophagy and apoptosis in hypoxic human renal proximal tubular cells. Int J Nanomed. 2014; 9:4317-4330. doi: 10.2147/IJN.S68685 Noël C, Simard JC, Girard D. Gold nanoparticles induce apoptosis, endoplasmic reticulum stress events and cleavage of cytoskeletal proteins in human neutrophils.Toxicol In Vitro. 2016; 31: 12-22. https://doi.org/10.1016/j.tiv.2015.11.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 2 posted Editorial decision: Reject after review 19 Apr, 2022 Submission checks completed at journal 05 Apr, 2022 Editor assigned by journal 05 Apr, 2022 First submitted to journal 04 Apr, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1490709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-04-04 15:28:00","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":99429042,"identity":"6b8ef966-400c-4f72-b4ea-52aa43480e61","order_by":0,"name":"Doaa Mansour","email":"","orcid":"","institution":"University of Sadat City","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Doaa","middleName":"","lastName":"Mansour","suffix":""},{"id":99429043,"identity":"62cf5b35-3c31-480f-8104-a6f54ceece6d","order_by":1,"name":"Sahar Orabi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie2RMUvDQBiGvxDodHrrZUn/wieBQrHgX6kIcQmKSwnocBJIFyFrios/JeHAKTbrlXZI9w4RFwUVL6Hocil1E7xnuY+Dh/e97wAMhj8J2Z7Muq1+bnv7KREC4K8U6LG9lP70KX8JYXRJ76P4moTvgKWooJ4IoPRBq2BxceYU4A/TVR4vSYGA0kcrnQtw0kqvQIAOB4EgT+PFLG4UAvZBLNSQ6YslG++tUfpKuZp9KqUswP5QykmHAjIYtCmoFHjmSskCsK0mhXJ9MbkZDDn6eLTKI1Y/esRRb8nv5ueEyY6NJYG34OEI3eV0XY9vXPewFOvqdXLs0qSj2HYJ37TflLUDG+9Q9NBdKQaDwfCP+AKal15cveec6wAAAABJRU5ErkJggg==","orcid":"","institution":"University of Sadat City","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sahar","middleName":"","lastName":"Orabi","suffix":""},{"id":99429044,"identity":"29f7daec-e96b-4877-a4a3-1b423baf9d07","order_by":2,"name":"Said Fathalla","email":"","orcid":"","institution":"University of Sadat City","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Said","middleName":"","lastName":"Fathalla","suffix":""},{"id":99429045,"identity":"4a4c0e63-f46c-4de5-a64b-3acb368ddd37","order_by":3,"name":"Shaaban Gadallah","email":"","orcid":"","institution":"University of Sadat City","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaaban","middleName":"","lastName":"Gadallah","suffix":""},{"id":99429046,"identity":"b30edfd7-18f8-4cf0-8095-b358b7a7a6a7","order_by":4,"name":"Amina Gamal el Din","email":"","orcid":"","institution":"National Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amina","middleName":"Gamal el","lastName":"Din","suffix":""},{"id":99429047,"identity":"7c3d79df-2198-4e4b-bb54-5590b02d7365","order_by":5,"name":"Ahmed Abdoon","email":"","orcid":"","institution":"National Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Abdoon","suffix":""}],"badges":[],"createdAt":"2022-03-25 23:29:11","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1490709/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1490709/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20507525,"identity":"994f37d4-df0b-4b53-bc52-085162c02701","added_by":"auto","created_at":"2022-04-19 15:10:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":139973,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of AuNPs using TEM at size \u003cstrong\u003e(a)-\u003c/strong\u003e 10nm, \u003cstrong\u003e(b)-\u003c/strong\u003e 50nm\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-1490709/v2/0ad37fef645509686d6f7f44.png"},{"id":20507528,"identity":"d953ee71-ae4e-40b1-b2be-501d824bb24a","added_by":"auto","created_at":"2022-04-19 15:10:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":904288,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of cross-sections of H\u0026amp;E-stained testicular sections showing the effect of i.m. injection of single or repeated doses of 50nm or 10nm AuNPs male rats and showing normal seminiferous tubules of average diameter and normal basement membranes. Epithelial lining showed numerous Sertoli cells and cells at different stages of spermatogenic series; including spermatogonia, spermatocytes, spermatids, and spermatozoa. A) Control, B-G representing AuNPs on Days-3, 7, 60 post-injection. H) For the control group of repeated injection, and I and J for repeated injection of 50nm or 10nm AuNPs\u003cstrong\u003e(x400).\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-1490709/v2/c0013036d5cdab2143f578b3.png"},{"id":20507527,"identity":"7fc2dd59-ac71-4d7e-93c4-e1d5b00d2f22","added_by":"auto","created_at":"2022-04-19 15:10:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":885913,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of cross-sections of H\u0026amp;E-stained epididymal sections showing the effect of i.m. injection of single or repeated doses of 50nm or 10nm AuNPs male rats and showing normal preserved structure of epithelial lining and the presence of spermatozoa in the lumen. A) Control, B-G representing single injection of 50nm or 10nm AuNPs on Days-3, 7, 60 post-injection, respectively. H) For the control group of repeated injection, and I and J for repeated injection of 50nm or 10nm AuNPs\u003cstrong\u003e(x400).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-1490709/v2/48fa184798a9fd09c4a3b9ba.png"},{"id":20507526,"identity":"e18171af-38cc-4891-815b-c800afe0af58","added_by":"auto","created_at":"2022-04-19 15:10:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":909761,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of cross-sections of Caspase immunoreactive stained testicular sections showing the effect of i.m. injection of single or repeated doses of 50nm or 10nm AuNPs male rats and showing similar expression patterns in control and AuNPs injected rats. A) Control, B-G representing AuNPs on Days-3, 7, 60 post-injection. H) For the control group of repeated injection, and I and J for repeated injection of 50nm or 10nm AuNPs\u003cstrong\u003e(x400).\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-1490709/v2/5c5ede19790cf1166e51f9cf.png"},{"id":20507529,"identity":"a5060cd1-3f67-41ad-8dcf-a5cb4dbb0ada","added_by":"auto","created_at":"2022-04-19 15:10:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":932268,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of cross-sections of PCNA immunoreactive stained testicular sections showing the effect of i.m. injection of single or repeated doses of 50nm or 10nm AuNPs male rats and showing similar expression patterns in control and AuNPs injected rats. A) Control, B-G representing AuNPs on Days-3, 7, 60 post-injection. H) For the control group of repeated injection, and I and J for repeated injection of 50nm or 10nm AuNPs\u003cstrong\u003e(x400).\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-1490709/v2/f99abd2358993bec3dc63e9d.png"},{"id":20507530,"identity":"0412b2ba-d3ef-46b1-947e-a340276edde5","added_by":"auto","created_at":"2022-04-19 15:10:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1478647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1490709/v2/f9396002-a927-446a-a202-90025e538608.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of acute or chronic administration gold nanospheres on testicular functions in male rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGold is a biologically inert metal that is found only at the macroscopic level, but gold at the nanoscale size has unique physical and chemical characteristics, such as stability, inertia, small size, simple penetration, biocompatibility, low cytotoxicity, and surface Plasmon resonance excitation characteristics (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Therefore, gold nanoparticles (AuNPs) are utilized in various fields, including nanomedicine, diagnosis, drug and gene delivery, biological imaging, photothermal therapy, biosensing, contrast agents for cancer and pharmaceuticals (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). They are also used in several industrial applications such as cosmetics, hair tonics, conductive ink, lubricant oil (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), foodstuffs, textiles, electronic and sports instruments (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the benefits of AuNPs in many areas of medicine and industry, there are potentially damaging effects on living organisms, including human health particularly male reproductive function (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The tiny size of nanoparticles (NPs) enables them to overcome the body defense barriers without being prevented (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Bio-persistence of NPs in testes appears to rely on the size of NPs, the exposure route, and the agglomeration of particles (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). A previous study has reported that the testis is a minor distribution target for NPs compared to other organs where very limited quantities of nanoparticles can reach the testes irrespective of the exposure routes and the physicochemical characteristics of NPs (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe process through which NPs may affect testicular functions involves reduced sperm production (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and reduced testosterone synthesis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Furthermore, following intraperitoneal injection (IP) of AuNPs in male mice declined sperm motility and morphology and elevated the abnormal spermatozoa number (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Administration of AuNPs to male rats for 2 months induced penetration of the seminiferous tubules without any effect on their basic functions, as well as having no influence on the proliferation of spermatogonia and on the apoptosis of mature germinal cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Also, Polyethylene Glycol-coated gold nanoparticles (PEG-AuNPs) have the ability to cross the blood-testis barrier (BTB) and elevated plasma testosterone levels without any detrimental effects on fertility (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Moreover, no testicular histopathological changes were detected following IP injection of PEG-AuNPs in mice (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt was reported that the main mechanism of AuNPs toxicity is via oxidative stress causing damage to the lipids, carbohydrates, protein, and DNA (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). There are contradictory data about the oxidative stress induced by AuNPs. Some studies indicated that AuNPs cause significant oxidative stress and may lead to inflammation, DNA damage, and cell death (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In contrast, other experimenters revealed that AuNPs are potent antioxidants and have no significant induction of oxidative stress or inflammatory response (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Acute or chronic administration of GNPs alters the liver, kidney functions, and blood profile on day 7 post-treatment (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePresently, data on the in vivo toxicity of gold nanospheres on the male reproductive system are scared. So, this study was carried out to investigate the effect of acute and chronic gold nanospheres administration on the male reproductive function via estimation of serum testosterone level, oxidative stress parameters, histopathological, morphometric, and immunohistochemical analyses of testicular tissue in male rats.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eSpherical gold nanoparticles at the size 10nm were purchased from Sigma-Aldrich Com., Germany (PCode: 4100798324). while AuNPs (50nm size) were synthesized at the national research center, Dokki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of gold nanospheres (AuNPs at the size 50nm):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGold nanoparticles were synthesized according to the Turkevich method (22). Simply, a mixture of 1 ml of a solution of 5 mm Chloroauric Acid (HAuCl4) in deionized water (DI water) and 18 ml of DI water was heated until it started boiling, then add one milliliter of 0.5% trisodium citrate solution. It was heated up to an apparent color change, then taken out of the heater and stirred until cool down to room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of AuNPs:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe absorption spectra of\u0026nbsp;AuNPs\u0026nbsp;were collected by using V-630 UV-VIS Spectrophotometer (Jasco, Japan). Transmission electron microscopy (TEM) images were obtained using JEOL JEM 2010 TEM operated at 100- KV accelerating voltage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals and experimental design:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeventy-two adult male Albino Wister rats (110-150 g/bwt) were used in the current study. They were obtained from AL-Zyade Experimental Animals Production Center, Giza, Egypt. They were kept in well-ventilated metal cages at 23 \u0026plusmn; 2 \u0026deg;C, 40\u0026ndash;60% relative humidity and light/dark cycle of 12 h, Feed of balanced ration and water were added ad libitum and left 2 weeks for adaptation. They were randomly divided into 2 experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment I: Effect of acute intramuscular administration of 10nm and 50nm gold nanospheres on reproductive function in male rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFifty-four adult male rats were randomly assigned to three equal groups (n=18/group). Group І (control group): rats were intramuscular (i.m.) injected of 1 ml of normal saline 0.9%.Group ІІ (50nm\u0026nbsp;AuNPs) \u0026amp; Group ІІІ (10nm\u0026nbsp;AuNPs): in which rats were i.m. injected with 75\u0026mu;g\u0026nbsp;AuNPs/kg/bwt\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(23) 50nm and 10nmAuNPs, respectively as one shot.\u0026nbsp;AuNPs\u0026nbsp;were diluted using a physiological saline solution. Rats were sacrificed\u0026nbsp;by head dislocation\u0026nbsp;after 3, 7, and 60 days to obtain blood and tissue samples (testes and epididymis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment II: Effect of chronic intramuscular administration of 10nm and 50nm gold nanospheres on reproductive function in male rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEighteen adult male rats were randomly assigned to three equal groups (n=6/group). Group І (control group): in which rats i.m injected with 1 ml normal saline 0.9%.Group ІІ (50nm\u0026nbsp;AuNPs) \u0026amp; Group ІІІ (10nm\u0026nbsp;AuNPs): in which rats were i.m injected with\u0026nbsp;75\u0026mu;g50nm or 10nm AuNPs/kg/bwt, respectively (23).\u0026nbsp;AuNPs were diluted with physiological saline solution,\u0026nbsp;injected i.m once weekly for five consecutive weeks, followed by a 21day for a washout period. On\u0026nbsp;Day-60, they were\u0026nbsp;sacrificed\u0026nbsp;by head dislocation\u0026nbsp;to obtain blood and tissue samples (testes and epididymis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSampling:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from the medial canthus of the eye using capillary tubes. The collected blood samples were\u0026nbsp;left to clot, followed by centrifugation at 1500g for 15 minutes\u0026nbsp;and serum samples were separated and kept at \u0026ndash; 20\u0026deg;C for estimation of serum testosterone. Testes and epididymis were\u0026nbsp;immediately picked up. Samples from testes\u0026nbsp;were divided into 2 parts, one part was stored at -80\u003csup\u003e0\u003c/sup\u003eC for assaying lipid peroxidation (MDA) and antioxidant defense system (GSH and SOD) and the second part was fixed at 10% neutral buffered formalin for assaying immunohistochemical studies and also with epididymis for histopathological and morphometric analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHormonal and oxidative stress analyzing:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum testosterone level was determined by the quantitative method\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(24) by using\u0026nbsp;the LIAISON\u0026reg;\u0026nbsp;Testosterone assay method, DiaSorin, Italy, (Ref. Code: 310410). Tissue homogenate was prepared according to a previously published method (25). Commercial diagnostic kits (Biodiagnostic, Egypt) were used for the assay of\u0026nbsp;malondialdehyde\u0026nbsp;(MDA) (Cat. No. MD 25 29) were measured as previously described (26, 27), reduced glutathione (GSH) (Cat. No. GR 25 11) was determined according to a previously published method (28)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand superoxide dismutase (SOD) (Cat. No. SD 25 21) was evaluated following the previously described method (29).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological examination:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesticular and epididymal tissue samples were cut and fixed at 10% neutral buffered formalin. The tissues were dehydrated and embedded in paraffin blocks.\u0026nbsp;Micro sections (4 \u0026mu;m) were prepared and stained with hematoxylin and eosin dye and were examined under a light microscope (30).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphometric evaluation of seminiferous tubules and epididymis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphometric analysis was performed using a Leica Qwin 500 Image Analyzer (Leica Imaging Systems Ltd, Cambridge, UK). At every time point of the experiment, 10 sections of seminiferous tubules from control and\u0026nbsp;AuNPs-treated rats were evaluated (round or almost round in form). At \u0026times; 400 magnifications, the diameter of seminiferous tubules was calculated; the major and minor axes of the tubule and the epithelial thickness were measured from the spermatogenic cells on the basement membrane via the sidelines cells of the lumen of tubules (31). Also, various spermatogenic cells, such as spermatogonia, spermatocytes, and spermatids were recognized based upon their distinctive morphology and count (32).\u003c/p\u003e\n\u003cp\u003eThe morphometric analysis of the epididymis was performed using 20 sections of ducts difference. The major and minor axes diameter and epithelial height of the ducts difference were measured with sperm counts at \u0026times; 400 magnifications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical investigation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnother section of the testicular tissue block was deparaffinized, rehydrated, and immune-stained using primary mouse monoclonal antibody against PCNA Ab-1 (Clone PC10) Thermo Fisher Scientific; Fremont, CA 94538, USA; localized in the nucleus; for cellular proliferation evaluation (33). Also, another section of the same block was immune-stained using mouse monoclonal antibody: \u0026nbsp;Caspase-3 (CPP32) Ab-4 primary rabbit polyclonal antibody (Thermo Scientific); localized mostly in the cytoplasm and some in the nucleus; for apoptosis assessment (34). Diaminobenzidine (DAB) was used to detect both immune stains (35). The sections were examined under an Olympus research microscope CX41 and the digital photomicrographs sections were photographed at different magnifying powers with Olympus SC100digital CCD camera. Spermatogenic cells lining the seminiferous tubules were regarded to be positive for activating caspase-3with brown cytoplasm and/or nuclei, while they were deemed positive for PCNA with dark brown nuclei.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analysis was performed using SPSS software (Statistical Package for Social Sciences) Version 22 using one-way ANOVA with \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 regarded as statistically significant. All data were expressed as mean \u0026plusmn; standard errors of the means (SEM).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of gold nanospheres\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology and size of\u0026nbsp;AuNPs\u0026nbsp;were shown by using transmission electron microscopy (TEM) in Figures 1a and b\u0026nbsp;from this image, it is clear that a uniform sphere shape of\u0026nbsp;AuNPs\u0026nbsp;with regular distribution was formed. The small size particle averaged was 10nm in length. While in large size particles, the diameter was average 50nm. It was found that the surface Plasmon absorption of\u0026nbsp;AuNPs\u0026nbsp;was 525-530 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of AuNPs on serum testosterone level (ng/ml)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData presented in Table 1 represent the acute and chronic effects of\u0026nbsp;i.m.\u0026nbsp;injection of 75\u0026mu;g AuNPs /kg/bwt on serum testosterone level. Results showed that\u0026nbsp;i.m.\u0026nbsp;injection of a single dose of 50nm or 10nm AuNPs had no significant effect on serum testosterone level compared to the control group on days 3 and 7. However,\u0026nbsp;i.m\u0026nbsp;injection of single or repeated doses of 10nm AuNPs significantly increased serum testosterone level in Day-60 post-treatment compared to 50nm AuNPs or the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of AuNPs on testicular tissue MDA level, GSH content, and SOD activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData illustrated in Table 2 represent the effect of acute and chronic\u0026nbsp;i.m\u0026nbsp;injection of 75\u0026mu;g 50nm or 10nm AuNPs /kg/bwt on the testicular tissue lipid peroxidation (MDA) and antioxidant defense system biomarkers as GSH content and SOD activity. Results demonstrated that\u0026nbsp;i.m.\u0026nbsp;injection of single or repeated doses of 50nm AuNPs (2\u003csup\u003end\u003c/sup\u003eGroup) or 10nm AuNPs (3\u003csup\u003erd\u003c/sup\u003eGroup) had no significant effect on the testicular tissue MDA level and GSH content compared to the \u0026nbsp;control group at any point of the experiment. But, they significantly (P \u0026lt; 0.05) increased testicular tissue SOD activity compared to the control group on Day-60. However, i.m. injection of repeated doses of 50nm AuNPs significantly (P \u0026lt; 0.05) decreased testicular tissue MDA level and (P \u0026lt; 0.05) increased GSH content and SOD activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological examination of the testes and epididymis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData for histopathological examination of testes and epididymis tissues in different groups of rats are shown in Figure 2 and Figure 3, respectively. Results indicated that acute or chronic\u0026nbsp;i.m.\u0026nbsp;injection of 75\u0026mu;g of 50nm or 10nm AuNPs /kg/bwt has no adverse effect on the histological evaluation of the testicular tissue. AuNPs treated male rats as in the control group showed normal, well preserved histological structure at all times of experiment, including normal seminiferous tubules of average diameter and normal basement membranes. Epithelial lining showed numerous Sertoli cells and cells at different stages of the spermatogenic series; including spermatogonia, spermatocytes, spermatids, and spermatozoa.\u003c/p\u003e\n\u003cp\u003eFor histological evaluation of the epididymal ducts in both control and AuNPs -treated rats; histopathological examination revealed normal well structurally preserved, without any histopathological abnormalities regardless of the time of analysis in both control and experimental groups. The epididymal ducts were lined by regular pseudostratified epithelial cells with rounded bland basophilic nuclei and cilia projecting into dilated lumina in which numerous spermatozoa were seen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphometric studies:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe obtained data for morphometric analysis of testes and epididymis tissue in control and AuNPs treated rats are presented in Table 3, Table4, Table 5, and Table 6, respectively. Results revealed that i.m. injection of 75\u0026mu;g 50nm or 10nm AuNPs/kg/bwt significantly (P \u0026lt; 0.05) increased the mean number of spermatogonia on Day-7 post-treatment. While rats that received a single dose of 10nm AuNPs showed a significant (P \u0026lt; 0.05) increase in the mean number of spermatogonia and spermatozoa on Day-60 post-treatment compared to the control group. However, repeated doses of 10nm AuNPs significantly (P \u0026lt; 0.05) increased the mean number of spermatocytes, spermatids, and spermatozoa on Day-60 post-injection compared to the control group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith regard to the epididymis,\u0026nbsp;i.m.\u0026nbsp;injection of a\u0026nbsp;single dose of 10 nm or 50 nm\u0026nbsp;AuNPs significantly (P \u0026lt; 0.05) increased the average number of spermatozoa per epididymal ducts on Day-60 post-treatment compared with the control group. Also, a\u0026nbsp;single dose of 10 nm\u0026nbsp;AuNPs significantly (P \u0026lt; 0.05) decreased the minor axis of epididymal ducts on Day-7 post injection compared to the control rats. However, repeated injection of 10nm AuNPs significantly (P \u0026lt; 0.05) decreased the minor axis of epididymal ducts and significantly (P \u0026lt; 0.05) increased the average number of spermatozoa per epididymal ducts on Day-60 post-injection compared to the control rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical study:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe obtained data for the immunohistochemical examination of proliferation biomarker Proliferating Cell Nuclear Antigen (PCNA) and apoptosis marker (Caspase-3) in control and AuNPs injected rats are illustrated in Table 7 and Figure 4 and Figure 5, respectively.\u003c/p\u003e\n\u003cp\u003eThe data showed no alteration in the expression of PCNA within the nuclei of spermatogenic cells lining the seminiferous tubules in testicular tissues for 10nm or 50nm AuNPs injected rats when compared to the control group at each point of the experiments. At the same time, the expression of caspase-3within the cytoplasm of spermatogenic cells lining the seminiferous tubules in testicular tissues was similar in control and 10nm or 50nm AuNPs injected rats at each time point of the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e:\u0026nbsp;Effects of acute\u0026nbsp;or chronic I.m injection of 10nm or 50nm AuNPs on serum testosterone (ng/ml) level\u0026nbsp;in male albino rats (mean \u0026plusmn;SEM)\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.124293785310735%\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.994350282485875%\"\u003e\n \u003cp\u003eTime\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.305084745762713%\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.847457627118644%\"\u003e\n \u003cp\u003e50nm AuNPs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.728813559322035%\"\u003e\n \u003cp\u003e10nm AuNPs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"14.124293785310735%\"\u003e\n \u003cp\u003eAcute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.994350282485875%\"\u003e\n \u003cp\u003eDay-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.305084745762713%\"\u003e\n \u003cp\u003e1.01\u0026plusmn;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.847457627118644%\"\u003e\n \u003cp\u003e1.62\u0026plusmn;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.728813559322035%\"\u003e\n \u003cp\u003e1.67\u0026plusmn;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.131578947368421%\"\u003e\n \u003cp\u003eDay-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.138157894736842%\"\u003e\n \u003cp\u003e1.21\u0026plusmn;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.098684210526315%\"\u003e\n \u003cp\u003e1.08\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.63157894736842%\"\u003e\n \u003cp\u003e1.12\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.131578947368421%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.138157894736842%\"\u003e\n \u003cp\u003e1.16\u0026plusmn;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.098684210526315%\"\u003e\n \u003cp\u003e1.39\u0026plusmn;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.63157894736842%\"\u003e\n \u003cp\u003e2.09\u0026plusmn;0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.124293785310735%\"\u003e\n \u003cp\u003eChronic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.994350282485875%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.305084745762713%\"\u003e\n \u003cp\u003e1.88\u0026plusmn;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.847457627118644%\"\u003e\n \u003cp\u003e1.14\u0026plusmn;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.728813559322035%\"\u003e\n \u003cp\u003e4.13\u0026plusmn;0.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea,b differ significantly within the same row at each time point at P\u0026lt; 0.05,\u0026nbsp;number of rats=6/group\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Effects of acute or chronic i.m injection of 10nm or 50nm AuNPson testicular MDA, GSH and SOD in male albino rats (mean \u0026plusmn;SEM).\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.288135593220339%\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.757062146892656%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.59322033898305%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.88135593220339%\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003cp\u003enmol/gm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"21.89265536723164%\"\u003e\n \u003cp\u003eGSH\u003c/p\u003e\n \u003cp\u003emg/gm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"23.587570621468927%\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003cp\u003eU/gm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" valign=\"top\" width=\"12.288135593220339%\"\u003e\n \u003cp\u003eAcute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"8.757062146892656%\"\u003e\n \u003cp\u003eDay-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.59322033898305%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.88135593220339%\"\u003e\n \u003cp\u003e48.6 \u0026plusmn;2.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"21.89265536723164%\"\u003e\n \u003cp\u003e5.02\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"23.587570621468927%\"\u003e\n \u003cp\u003e573.1\u0026plusmn;24.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e44.41\u0026plusmn;4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e4.99\u0026plusmn;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e646.8\u0026plusmn;28.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e45.08\u0026plusmn;3.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e4.58\u0026plusmn;0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e596.6\u0026plusmn;31.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"9.98389694041868%\"\u003e\n \u003cp\u003eDay-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.077294685990339%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.08695652173913%\"\u003e\n \u003cp\u003e50.38\u0026plusmn;6.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"24.9597423510467%\"\u003e\n \u003cp\u003e4.94\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"26.89210950080515%\"\u003e\n \u003cp\u003e612.9\u0026plusmn;23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e60.2\u0026plusmn;5.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e4.69\u0026plusmn;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e651.5\u0026plusmn;14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e61.32\u0026plusmn;7.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e4.13\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e667.2\u0026plusmn;19.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"9.98389694041868%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.077294685990339%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.08695652173913%\"\u003e\n \u003cp\u003e57.2\u0026plusmn;3.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"24.9597423510467%\"\u003e\n \u003cp\u003e4.77\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"26.89210950080515%\"\u003e\n \u003cp\u003e584.3\u0026plusmn;20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e61.79\u0026plusmn;2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e5.08\u0026plusmn;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e732.8\u0026plusmn;25.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e66.69\u0026plusmn;3.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e5.68\u0026plusmn;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e714.1\u0026plusmn;16.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"12.288135593220339%\"\u003e\n \u003cp\u003eChronic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"8.757062146892656%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.59322033898305%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.88135593220339%\"\u003e\n \u003cp\u003e59.54\u0026plusmn;2.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"21.89265536723164%\"\u003e\n \u003cp\u003e4.28\u0026plusmn;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"23.587570621468927%\"\u003e\n \u003cp\u003e585.94\u0026plusmn;20.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e35.75\u0026plusmn;3.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e6.93\u0026plusmn;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e720.31\u0026plusmn;15.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"13.416815742397137%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.980322003577818%\"\u003e\n \u003cp\u003e45.83\u0026plusmn;4.98\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23%;\" valign=\"top\" width=\"27.728085867620752%\"\u003e\n \u003cp\u003e5.97\u0026plusmn;0.68\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.375%;\" valign=\"top\" width=\"29.874776386404292%\"\u003e\n \u003cp\u003e685.78\u0026plusmn;28.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea,b differ significantly within the same row at each time point at P\u0026lt; 0.05.number of rats=6/group\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e Morphometric analysis of testes in control and AuNPs -injected male rats at the different time points of the experiment (Single injection of AuNPs)\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"7.578947368421052%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"8.210526315789474%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"38.10526315789474%\"\u003e\n \u003cp\u003eDiameter of seminiferous tubules (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"46.10526315789474%\"\u003e\n \u003cp\u003eCell count\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.355805243445692%\"\u003e\n \u003cp\u003eMajor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.481897627965044%\"\u003e\n \u003cp\u003eMinor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.480649188514358%\"\u003e\n \u003cp\u003eEpithelial height\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.9812734082397%\"\u003e\n \u003cp\u003eSpermatogonia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.9812734082397%\"\u003e\n \u003cp\u003eSpermatocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.235955056179776%\"\u003e\n \u003cp\u003eSpermatid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.48314606741573%\"\u003e\n \u003cp\u003eSpermatozoa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"7.570977917981073%\"\u003e\n \u003cp\u003eDay-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.201892744479496%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.933753943217665%\"\u003e\n \u003cp\u003e314.51\u0026plusmn; 6.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.197686645636173%\"\u003e\n \u003cp\u003e246.28 \u0026plusmn;6.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.038906414300737%\"\u003e\n \u003cp\u003e62.99\u0026plusmn;2.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.618296529968454%\"\u003e\n \u003cp\u003e61.25 \u0026plusmn; 1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.618296529968454%\"\u003e\n \u003cp\u003e58.21 \u0026plusmn; 1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.46372239747634%\"\u003e\n \u003cp\u003e64.01\u0026plusmn;1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.35646687697161%\"\u003e\n \u003cp\u003e115.33 \u0026plusmn; 2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.873720136518772%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.993174061433447%\"\u003e\n \u003cp\u003e330.97\u0026plusmn; 7.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.196814562002276%\"\u003e\n \u003cp\u003e236.78\u0026plusmn;4.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.10693970420933%\"\u003e\n \u003cp\u003e58.58\u0026plusmn;2.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e66.21 \u0026plusmn; 1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e66.01 \u0026plusmn; 3.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.238907849829351%\"\u003e\n \u003cp\u003e67.33\u0026plusmn;3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.286689419795222%\"\u003e\n \u003cp\u003e110.81 \u0026plusmn; 2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.873720136518772%\"\u003e\n \u003cp\u003eG3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.993174061433447%\"\u003e\n \u003cp\u003e331.88 \u0026plusmn; 8.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.196814562002276%\"\u003e\n \u003cp\u003e248.39 \u0026plusmn;7.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.10693970420933%\"\u003e\n \u003cp\u003e62.95\u0026plusmn;1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e62.51 \u0026plusmn; 2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e60.57 \u0026plusmn; 4.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.238907849829351%\"\u003e\n \u003cp\u003e56.67\u0026plusmn;1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.286689419795222%\"\u003e\n \u003cp\u003e108.01 \u0026plusmn; 2.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"7.570977917981073%\"\u003e\n \u003cp\u003eDay-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.201892744479496%\"\u003e\n \u003cp\u003eG1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.933753943217665%\"\u003e\n \u003cp\u003e309.55 \u0026plusmn; 7.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.197686645636173%\"\u003e\n \u003cp\u003e248.69 \u0026plusmn;6.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.038906414300737%\"\u003e\n \u003cp\u003e64.44\u0026plusmn;3.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.618296529968454%\"\u003e\n \u003cp\u003e62.01 \u0026plusmn; 1.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.618296529968454%\"\u003e\n \u003cp\u003e61.51 \u0026plusmn; 1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.46372239747634%\"\u003e\n \u003cp\u003e67.41\u0026plusmn;1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.35646687697161%\"\u003e\n \u003cp\u003e115.33 \u0026plusmn; 4.91\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.873720136518772%\"\u003e\n \u003cp\u003eG2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.993174061433447%\"\u003e\n \u003cp\u003e336.39 \u0026plusmn; 9.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.196814562002276%\"\u003e\n \u003cp\u003e254.41 \u0026plusmn;6.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.10693970420933%\"\u003e\n \u003cp\u003e57.26\u0026plusmn;4.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e73.41 \u0026plusmn; 2.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e65 .75 \u0026plusmn; 1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.238907849829351%\"\u003e\n \u003cp\u003e66.57\u0026plusmn;2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.286689419795222%\"\u003e\n \u003cp\u003e118.01 \u0026plusmn; 7.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.873720136518772%\"\u003e\n \u003cp\u003eG3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.993174061433447%\"\u003e\n \u003cp\u003e338.77 \u0026plusmn;10.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.196814562002276%\"\u003e\n \u003cp\u003e246.58 \u0026plusmn;4.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.10693970420933%\"\u003e\n \u003cp\u003e60.09\u0026plusmn;3.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e74.01 \u0026plusmn; 1.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e66.17 \u0026plusmn; 2.02\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.238907849829351%\"\u003e\n \u003cp\u003e72.61\u0026plusmn;2.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.286689419795222%\"\u003e\n \u003cp\u003e133.61 \u0026plusmn; 7.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"7.570977917981073%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.201892744479496%\"\u003e\n \u003cp\u003eG1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.933753943217665%\"\u003e\n \u003cp\u003e400.66 \u0026plusmn;12.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.197686645636173%\"\u003e\n \u003cp\u003e287.54\u0026plusmn;12.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.038906414300737%\"\u003e\n \u003cp\u003e65.55\u0026plusmn;2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.618296529968454%\"\u003e\n \u003cp\u003e59.25 \u0026plusmn; 2.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.618296529968454%\"\u003e\n \u003cp\u003e56.81 \u0026plusmn; 1.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.46372239747634%\"\u003e\n \u003cp\u003e56.01\u0026plusmn;2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.35646687697161%\"\u003e\n \u003cp\u003e103.61\u0026plusmn;1.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.873720136518772%\"\u003e\n \u003cp\u003eG2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.993174061433447%\"\u003e\n \u003cp\u003e369.86 \u0026plusmn;13.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.196814562002276%\"\u003e\n \u003cp\u003e247.17\u0026plusmn;14.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.10693970420933%\"\u003e\n \u003cp\u003e64.38\u0026plusmn;2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e60.11 \u0026plusmn; 1.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e59.51 \u0026plusmn; 2.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.238907849829351%\"\u003e\n \u003cp\u003e61.25\u0026plusmn;2.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.286689419795222%\"\u003e\n \u003cp\u003e109.11\u0026plusmn;2.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"8.873720136518772%\"\u003e\n \u003cp\u003eG3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.993174061433447%\"\u003e\n \u003cp\u003e376.32 \u0026plusmn;13.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.196814562002276%\"\u003e\n \u003cp\u003e269.27\u0026plusmn;9.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.10693970420933%\"\u003e\n \u003cp\u003e61.35\u0026plusmn;3.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e64.67 \u0026plusmn; 2.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.651877133105803%\"\u003e\n \u003cp\u003e81.13\u0026plusmn; 2.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.238907849829351%\"\u003e\n \u003cp\u003e64.67\u0026plusmn;2.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.286689419795222%\"\u003e\n \u003cp\u003e194.83\u0026plusmn;1.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea,b differ significantly within the same column at each time point at P\u0026lt; 0.05.number of rats=6/group\u003c/p\u003e\n\u003cp\u003eThe number of spermatogonia was significantly (P\u0026lt; 0.05) higher in 10nm and 50nm AuNPs injected rats on Day-7 post-injection when compared with control. Also, i.m. injection of 10nm AuNPs produced significantly (P\u0026lt; 0.05) increase in the number of spermatogonia, spermatocytes, and spermatozoa on Day-60 post-injection in male rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u003c/strong\u003e Morphometric analysis of testes in control and AuNPs -injected male rats at the different time points of the experiment (Repeated injection of AuNPs)\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"6.081081081081081%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"9.45945945945946%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"34.45945945945946%\"\u003e\n \u003cp\u003eDiameter of seminiferous tubules (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eCell count\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.4%\"\u003e\n \u003cp\u003eMajor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.4%\"\u003e\n \u003cp\u003eMinor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12%\"\u003e\n \u003cp\u003eEpithelial height\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16%\"\u003e\n \u003cp\u003eSpermatogonia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16%\"\u003e\n \u003cp\u003eSpermatocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.8%\"\u003e\n \u003cp\u003eSpermatid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.4%\"\u003e\n \u003cp\u003eSpermatozoa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"6.081081081081081%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.45945945945946%\"\u003e\n \u003cp\u003eG1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.162162162162161%\"\u003e\n \u003cp\u003e420.89 \u0026plusmn;14.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.162162162162161%\"\u003e\n \u003cp\u003e280.72 \u0026plusmn;13.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.135135135135135%\"\u003e\n \u003cp\u003e65.55\u0026plusmn;2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.513513513513514%\"\u003e\n \u003cp\u003e59.25 \u0026plusmn; 2.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.513513513513514%\"\u003e\n \u003cp\u003e59.25 \u0026plusmn; 1.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.81081081081081%\"\u003e\n \u003cp\u003e50.01\u0026plusmn;1.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.162162162162161%\"\u003e\n \u003cp\u003e101.86 \u0026plusmn; 1.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"10.071942446043165%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.949640287769784%\"\u003e\n \u003cp\u003e397.64 \u0026plusmn;16.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.949640287769784%\"\u003e\n \u003cp\u003e254.98 \u0026plusmn;10.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.79136690647482%\"\u003e\n \u003cp\u003e60.85\u0026plusmn;3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.388489208633093%\"\u003e\n \u003cp\u003e56.51 \u0026plusmn; 1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.388489208633093%\"\u003e\n \u003cp\u003e51.51 \u0026plusmn; 1.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.510791366906474%\"\u003e\n \u003cp\u003e49.71\u0026plusmn;1.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.949640287769784%\"\u003e\n \u003cp\u003e105.51 \u0026plusmn; 2.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"10.071942446043165%\"\u003e\n \u003cp\u003eG3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.949640287769784%\"\u003e\n \u003cp\u003e402.94 \u0026plusmn;15.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.949640287769784%\"\u003e\n \u003cp\u003e268.96 \u0026plusmn; 8.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.79136690647482%\"\u003e\n \u003cp\u003e69.17\u0026plusmn;3.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.388489208633093%\"\u003e\n \u003cp\u003e62.81 \u0026plusmn; 1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.388489208633093%\"\u003e\n \u003cp\u003e71.67\u0026plusmn; 1.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.510791366906474%\"\u003e\n \u003cp\u003e67.75\u0026plusmn;3.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.949640287769784%\"\u003e\n \u003cp\u003e188.81 \u0026plusmn;3.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea,b differ significantly within the same column at each time point at P\u0026lt; 0.05.\u0026nbsp;number of rats=6/group\u003c/p\u003e\n\u003cp\u003eRepeated injection of 10nm AuNPs in male rats showed significantly (P\u0026lt; 0.05) higher number of spermatocyte, spermatids and spermatozoa on Day-60 post-injection compared with that in the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5:\u003c/strong\u003e\u0026nbsp; \u0026nbsp;Morphometric analysis of epididymis in control and AuNPs -injected male rats at the different time points of the experiment (Single injection of AuNPs)\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"9.655172413793103%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"15.172413793103448%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 39.168%;\" valign=\"top\" width=\"34.48275862068966%\"\u003e\n \u003cp\u003eDiameter of epididymal ducts (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 17.4043%;\" valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003eEpithelial height (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 18.5957%;\" valign=\"top\" width=\"20.689655172413794%\"\u003e\n \u003cp\u003eSpermatozoa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"48%\"\u003e\n \u003cp\u003eMajor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"52%\"\u003e\n \u003cp\u003eMinor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"9.655172413793103%\"\u003e\n \u003cp\u003eDay-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.172413793103448%\"\u003e\n \u003cp\u003eG1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"16.551724137931036%\"\u003e\n \u003cp\u003e323.69\u0026plusmn;6.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"17.93103448275862%\"\u003e\n \u003cp\u003e238.36\u0026plusmn;7.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e26.16\u0026plusmn;1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"20.689655172413794%\"\u003e\n \u003cp\u003e145.67\u0026plusmn;9.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.793893129770993%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"18.3206106870229%\"\u003e\n \u003cp\u003e317.22\u0026plusmn;9.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"19.84732824427481%\"\u003e\n \u003cp\u003e207.96\u0026plusmn;10.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"22.137404580152673%\"\u003e\n \u003cp\u003e28.86\u0026plusmn;3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"22.900763358778626%\"\u003e\n \u003cp\u003e172.21\u0026plusmn;8.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.793893129770993%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"18.3206106870229%\"\u003e\n \u003cp\u003e342.13\u0026plusmn;5.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"19.84732824427481%\"\u003e\n \u003cp\u003e199.06\u0026plusmn;11.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"22.137404580152673%\"\u003e\n \u003cp\u003e27.28\u0026plusmn;1.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"22.900763358778626%\"\u003e\n \u003cp\u003e167.67\u0026plusmn;7.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"9.655172413793103%\"\u003e\n \u003cp\u003eDay-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.172413793103448%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"16.551724137931036%\"\u003e\n \u003cp\u003e359.17\u0026plusmn;10.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"17.93103448275862%\"\u003e\n \u003cp\u003e244.91\u0026plusmn;4.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e25.51\u0026plusmn;1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"20.689655172413794%\"\u003e\n \u003cp\u003e149.51\u0026plusmn;7.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.793893129770993%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"18.3206106870229%\"\u003e\n \u003cp\u003e338.56\u0026plusmn;9.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"19.84732824427481%\"\u003e\n \u003cp\u003e229.48\u0026plusmn;3.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"22.137404580152673%\"\u003e\n \u003cp\u003e25.79\u0026plusmn;1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"22.900763358778626%\"\u003e\n \u003cp\u003e144.25\u0026plusmn;8.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.793893129770993%\"\u003e\n \u003cp\u003eG3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"18.3206106870229%\"\u003e\n \u003cp\u003e339.03\u0026plusmn;16.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"19.84732824427481%\"\u003e\n \u003cp\u003e185.51\u0026plusmn;6.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"22.137404580152673%\"\u003e\n \u003cp\u003e29.36\u0026plusmn;1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"22.900763358778626%\"\u003e\n \u003cp\u003e143.33\u0026plusmn;10.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"9.655172413793103%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.172413793103448%\"\u003e\n \u003cp\u003eG1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"16.551724137931036%\"\u003e\n \u003cp\u003e383.67\u0026plusmn;7.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"17.93103448275862%\"\u003e\n \u003cp\u003e277.04\u0026plusmn;12.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e25.53\u0026plusmn;1.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"20.689655172413794%\"\u003e\n \u003cp\u003e121.17\u0026plusmn;9.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.793893129770993%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"18.3206106870229%\"\u003e\n \u003cp\u003e420.43\u0026plusmn;11.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"19.84732824427481%\"\u003e\n \u003cp\u003e265.54\u0026plusmn;14.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"22.137404580152673%\"\u003e\n \u003cp\u003e25.47\u0026plusmn;2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"22.900763358778626%\"\u003e\n \u003cp\u003e149.51\u0026plusmn;10.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.793893129770993%\"\u003e\n \u003cp\u003eG3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.8242%;\" valign=\"top\" width=\"18.3206106870229%\"\u003e\n \u003cp\u003e399.14\u0026plusmn;12.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.668%;\" valign=\"top\" width=\"19.84732824427481%\"\u003e\n \u003cp\u003e247.01\u0026plusmn;13.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4043%;\" valign=\"top\" width=\"22.137404580152673%\"\u003e\n \u003cp\u003e22.91\u0026plusmn;2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.5957%;\" valign=\"top\" width=\"22.900763358778626%\"\u003e\n \u003cp\u003e194.44\u0026plusmn;6.99\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea,b,c differ significantly within the same column at each time point at P\u0026lt; 0.05.number of rats=6/group\u003c/p\u003e\n\u003cp\u003eData revealed that i.m. injection of a single dose of 10nm AuNPs resulted in a significant (P\u0026lt; 0.05) decrease in minor axis diameter of the epididymis of injected male rats on Day- 7 post-treatment when compared with 50nm AuNPs or control animals. Also, 10nm AuNPs injected rats showed significantly (P\u0026lt; 0.05) higher number of spermatozoa on Day- 60 post-injection when compared with control or 50nm AuNPs group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6:\u003c/strong\u003e Morphometric analysis of epididymis in control and AuNPs -injected male rats at the different time points of the experiment (Repeated injection of AuNPs)\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"10.839160839160838%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"14.335664335664335%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 32.9297%;\" valign=\"top\" width=\"34.26573426573427%\"\u003e\n \u003cp\u003eDiameter of epididymal ducts (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 18.375%;\" valign=\"top\" width=\"20.27972027972028%\"\u003e\n \u003cp\u003eEpithelial height (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 23.125%;\" valign=\"top\" width=\"20.27972027972028%\"\u003e\n \u003cp\u003eSpermatozoa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0664%;\" valign=\"top\" width=\"40.816326530612244%\"\u003e\n \u003cp\u003eMajor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3047%;\" valign=\"top\" width=\"59.183673469387756%\"\u003e\n \u003cp\u003eMinor axis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"10.839160839160838%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.335664335664335%\"\u003e\n \u003cp\u003eG1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.0664%;\" valign=\"top\" width=\"13.986013986013987%\"\u003e\n \u003cp\u003e384.67\u0026plusmn;10.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3047%;\" valign=\"top\" width=\"20.27972027972028%\"\u003e\n \u003cp\u003e277.04\u0026plusmn;12.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.375%;\" valign=\"top\" width=\"20.27972027972028%\"\u003e\n \u003cp\u003e25.53\u0026plusmn;1.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.125%;\" valign=\"top\" width=\"20.27972027972028%\"\u003e\n \u003cp\u003e122.83\u0026plusmn;10.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.07843137254902%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.0664%;\" valign=\"top\" width=\"15.686274509803921%\"\u003e\n \u003cp\u003e397.12\u0026plusmn;12.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3047%;\" valign=\"top\" width=\"22.745098039215687%\"\u003e\n \u003cp\u003e269.06\u0026plusmn;10.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.375%;\" valign=\"top\" width=\"22.745098039215687%\"\u003e\n \u003cp\u003e22.49\u0026plusmn;2.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.125%;\" valign=\"top\" width=\"22.745098039215687%\"\u003e\n \u003cp\u003e117.41\u0026plusmn;8.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.07843137254902%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.0664%;\" valign=\"top\" width=\"15.686274509803921%\"\u003e\n \u003cp\u003e407.11\u0026plusmn;14.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3047%;\" valign=\"top\" width=\"22.745098039215687%\"\u003e\n \u003cp\u003e206.79\u0026plusmn;14.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.375%;\" valign=\"top\" width=\"22.745098039215687%\"\u003e\n \u003cp\u003e26.62\u0026plusmn;1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.125%;\" valign=\"top\" width=\"22.745098039215687%\"\u003e\n \u003cp\u003e178.75\u0026plusmn;10.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ea,b differ significantly within the same column at each time point at P\u0026lt; 0.05.number of rats=6/group\u003c/p\u003e\n\u003cp\u003eRepeated injection of 10 nm AuNPs in male rats showed significant (P\u0026lt; 0.05) decrease in minor axis diameter of the epididymis of injected male rats and also significant(P\u0026lt; 0.05) increase in number of spermatozoa on Day-60 post-injection when compared with control or 50nm AuNPs group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7:\u003c/strong\u003e Effects of acute or chronic i.m injection of 10nm or 50nm AuNPson testicular immunohistochemical changes in PCNA and Caspase-3 in male albino rats (mean \u0026plusmn;SEM)\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.6923%;\" valign=\"top\" width=\"12.141882673942701%\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7692%;\" valign=\"top\" width=\"9.140518417462483%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"11.186903137789905%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"25.10231923601637%\"\u003e\n \u003cp\u003ePCNA/1mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"24.010914051841745%\"\u003e\n \u003cp\u003eCaspase-3/HPF.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" style=\"width: 12.6923%;\" valign=\"top\" width=\"12.552891396332862%\"\u003e\n \u003cp\u003eAcute\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 10.7692%;\" valign=\"top\" width=\"9.449929478138223%\"\u003e\n \u003cp\u003eDay-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"11.56558533145275%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"25.952045133991536%\"\u003e\n \u003cp\u003e91.33\u0026plusmn;6.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"24.823695345557123%\"\u003e\n \u003cp\u003e3.25\u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e90.67\u0026plusmn;4.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e3.41\u0026plusmn;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e90.33\u0026plusmn;4.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e3.01\u0026plusmn;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 10.7692%;\" valign=\"top\" width=\"10.806451612903226%\"\u003e\n \u003cp\u003eDay-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"13.225806451612904%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"29.677419354838708%\"\u003e\n \u003cp\u003e91.51\u0026plusmn;5.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"28.387096774193548%\"\u003e\n \u003cp\u003e3.21\u0026plusmn;0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e88.01\u0026plusmn;7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e4.5\u0026plusmn;0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e88.66\u0026plusmn;5.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e2.67\u0026plusmn;0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 10.7692%;\" valign=\"top\" width=\"10.806451612903226%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"13.225806451612904%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"29.677419354838708%\"\u003e\n \u003cp\u003e89.61\u0026plusmn;4.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"28.387096774193548%\"\u003e\n \u003cp\u003e3.33\u0026plusmn;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e88.01\u0026plusmn;4.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e2.51\u0026plusmn;0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e93.25\u0026plusmn;4.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e4.51\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 12.6923%;\" valign=\"top\" width=\"12.552891396332862%\"\u003e\n \u003cp\u003eChronic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 10.7692%;\" valign=\"top\" width=\"9.449929478138223%\"\u003e\n \u003cp\u003eDay-60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"11.56558533145275%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"25.952045133991536%\"\u003e\n \u003cp\u003e90.21\u0026plusmn;4.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"24.823695345557123%\"\u003e\n \u003cp\u003e3.33\u0026plusmn;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e95.02\u0026plusmn;3.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e2.51\u0026plusmn;0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.2308%;\" valign=\"top\" width=\"14.211438474870018%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31.9231%;\" valign=\"top\" width=\"31.889081455805893%\"\u003e\n \u003cp\u003e92.01\u0026plusmn;4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.3846%;\" valign=\"top\" width=\"30.502599653379548%\"\u003e\n \u003cp\u003e4.51\u0026plusmn;0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNumber of rats=6/group\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur data revealed that i.m. injection of either single or repeated doses of 10nm AuNPs significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased serum testosterone level compared to control rats, this was detected on Day-60 after injection. In similar, some studies reported that IP injection of 75 \u0026micro;g AuNPs/kg/bwt of male rats significantly elevated serum total and free testosterone levels compared to the control group on days 1, 3, 7, and 14 post-gold nanorods (AuNRs) injection (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Also, intravenous (IV)injection of ω-aminoethyl spherical PEG capped AuNPs (PEG-NH\u003csub\u003e2\u003c/sub\u003e @ AuNPs) in mice has elevated plasma testosterone levels, but had no effect on plasma follicle stimulating hormone (FSH) or luteinizing hormone (LH) levels that was accompanied by no any alterations on male fertility, fetal survival or fetal development (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Conversely, other studies showed that treatment with AuNPs at different doses 100, 50, 25 ppm for 10 days reduced serum testosterone levels in AuNPs-treated rats compared to the controls especially at treatment with high doses (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This discrepancy could be due to the AuNP size or dose used or at the time of exposure.\u003c/p\u003e \u003cp\u003eConcerning oxidative stress, our results showed that AuNPs has an antioxidant effect. Repeated injection of 50nm AuNPs significantly decreased testicular tissue MDA level and significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased of testicular tissue GSH content and SOD activity on Day-60 post-injection. Meanwhile, the injection of 10nm or 50nm AuNPs significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased testicular tissue SOD activity, while the MDA level and GSH content were similar to the control group on Day-60 post-injection. However, i.m. injection of 10nm or 50nm AuNPs has no effect on that oxidative stress parameters on Day-3 or 7after injection. Similarly, IP injection of 75 \u0026micro;g/kg/bwt AuNRs (50nm size) significantly reduced serum MDA and increased the antioxidant markers including catalase, glutathione peroxidase (GPx) and SOD activities compared to control which indicated the antioxidant activity of AuNRs (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Additionally, AuNPs are considered as antioxidants through reduction of the production of reactive oxygen species and nitrite species oxygen-free radicals, that is maybe owing to their interaction with the thioredoxin that acts over an endogenous inhibitor, thioredoxin-interacting protein (Txnip) which is related to the regulation of cellular redox (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Conversely, other studies found that after IP injection of 10nm AuNRs induced a significant increase in MDA values in rat liver, lung, heart and kidney organs and significant decrease in SOD in rat kidney, liver and lung organs compared to the control after 3 and 7 days from treatment (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). This difference could be due to the Au nanoparticle shape, dose, and route of administration.\u003c/p\u003e \u003cp\u003eFor the histopathological study, the present work reported that there were no histopathological changes in the testes or epididymis of rats i.m. injected with either single or repeated doses of 10 nm or 50 nm AuNPs at each time point of the experiment when compared to control rats. These results are matched with other studies recorded that the testes and epididymis in male rat IP injected with a single dose of 50nm AuNRs at (75 \u0026micro;g/kg/bwt.) induced no any obvious histopathological alterations at each time of the experiment (Days-1, 3, 7 and 14) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Also, IP injection of male mice with different sizes of spherical PEG-coated AuNPs at a dose of 4000 \u0026micro;g/kg caused well preserved histological structure in the testes of AuNPs-treated mice compared with control (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). On the other hand, there are degenerative changes in the seminiferous tubules in Wister rats, which IP injected with AuNPs at a dose of 100 ppm (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This contradictory, could be related to the dose of AuNPs used.\u003c/p\u003e \u003cp\u003eFor morphometric analysis, it is considered very delicate tools for assessing structural modifications by which histological examination could not be correctly proven. Our results are consistent with other findings declared that single IP injection of 75 \u0026micro;g/kg/bwt of AuNRs (50nm size) significantly increased the diameter of the minor axis of the seminiferous tubule, the height of the epithelial lining of the seminiferous and the average numbers of spermatocytes and spermatid after 7days of treatment (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). About the epididymis, the number of spermatozoa was significantly increased after 7 and 14 days compared to the control rats.\u003c/p\u003e \u003cp\u003eSpermatogenesis is a complex process through which the spermatogonia in the testis evolve into mature sperm by continuous mitosis, meiosis and cell distinction in the testis (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Proliferation of spermatogonia and apoptosis are responsible for maintaining balance in spermatogenesis. Apoptosis (Programmed cell death) occurs in the testes limiting the number of germinal cells in the epithelium of the testes (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Our results showed no changes within PCNA or caspase-3 positive immunoreactivity within the nuclei or cytoplasm of spermatogenic cells lining the seminiferous tubules in testicular tissues of injected rats either with single or repeated doses of 10 nm or 50 nm AuNPs throughout the experiment when compared to control rats. These results are matched with other studies showed that orally administration of AuNPs to male rats for 8 weeks did not change the expression of proliferation proteins (KI-67 and D1 cyclin), or the activity of apoptosis (TUNEL method)in the spermatogenic epithelium (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Conversely, other studies reported that Hesperetin conjugated AuNPs have anti-proliferative effects causing inhibition of PCNA expression in rat liver (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Additionally, AuNPs have led to the release of free radicals and an elevation of apoptosis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) which induced considerable up-regulation of mRNA expression of p53, bax, caspase-3, and caspase-9 which increased apoptosis (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). This difference could be attributed to variations in size, type, shape or dose of gold nanoparticles used.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSingle or repeated i.m. injection of 10nm or 50nm AuNPs in male rats is safe on the male reproductive function; also, they have an antioxidant effects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal-handling procedures, as well as samples' collection and disposal, were according to the regulations of the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, University of Sadat City (Approval number: VUSC-006-1-19).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree for publication\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thankfullyappreciatethe central laboratory, Faculty of Veterinary Medicine, University of Sadat City\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSO, AA, SF, SG contributed to the conception, design. Both DM, AA, SO and AG contributed to collection and/or assembly of data, data analysis and interpretation, and manuscript writing. SO, AA, SF, AG contributed to manuscript revision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo Funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this research are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDrucker E, Krapfenbauer K. Pitfalls and limitations in translation from biomarker discovery to clinical utility in predictive and personalized medicine. 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Gold nanoparticles induce apoptosis, endoplasmic reticulum stress events and cleavage of cytoskeletal proteins in human neutrophils.Toxicol In Vitro. 2016; 31: 12-22. https://doi.org/10.1016/j.tiv.2015.11.003\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Male rats, Acute, chronic study, histopathology, morphometric analysis, Immunohistochemistry, Oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-1490709/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1490709/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the effect of gold nanospheres (AuNPs) administration on the rat male reproductive function. Experiment 1: Fifty-four rats were assigned into three groups (n\u0026thinsp;=\u0026thinsp;18/group). Group І (control), Group ІІ (50 nm AuNPs) and Group ІІІ (10 nm AuNPs), in which rats were intramuscular (i.m) injected with a single dose of 75\u0026micro;g AuNPs/kg/bwt. Experiment 2: Eighteen rats were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;6/group). Group І (control), Group ІІ (50nm AuNPs) and Group ІІІ (10 nm AuNPs): rats were injected repeated doses of 75\u0026micro;g AuNPs /kg/bwt for 5 weeks followed by 3 weeks washout period. Blood, testes and epididymis samples were collected on Days-3, 7, and 60 in experiment 1 and on Day-60 in the 2nd experiment. Results indicated that acute or chronic administration of 10nm AuNPs caused significantly increased serum testosterone level on day-60. Chronic administration of 50nm AuNPs has an antioxidant effect. Acute or chronic study induced no histopathological changes and no changes in Proliferating Cell Nuclear Antigen (PCNA) and caspase-3 in testicular tissues. Acute or chronic administration of 10nm AuNPs significantly increased the spermatozoa in epididymal ducts and testicular tissue on Day-60 post-treatment. In conclusion, the administration of AuNPs (10nm and 50nm size) is safe on the male reproductive function.\u003c/p\u003e","manuscriptTitle":"Effects of acute or chronic administration gold nanospheres on testicular functions in male rats","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-04-19 15:10:31","doi":"10.21203/rs.3.rs-1490709/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Reject after review","date":"2022-04-19T08:55:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-04-05T07:16:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-05T07:16:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nanoscale Research Letters","date":"2022-04-04T19:23:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"782315cf-3af0-4e1d-b35b-8f5b7d671e84","owner":[],"postedDate":"April 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-04-19T15:10:32+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-19 15:10:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1490709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1490709","identity":"rs-1490709","version":["v2"]},"buildId":"wLkW0s4AflPzk-lpfg-fK","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}