Impact of chitosan administration on titanium dioxide nanoparticles induced testicular dysfunction

The potential reproductive toxic effects of oral TiO2 NPs in adult male rats as well as the possible alleviation of chitosan administration was investigated. Animals were allocated to four groups; the first group received deionized water and was assigned as a control group. In the second group, rats received chitosan at a dose of 5 mg/kg BW/day. The third group was designed for administration of TiO2 NPs at a dose of 150 mg/kg BW/day (1/80 LD50). Rats in the fourth group received both TiO2 NPs and chitosan. After 14 days, TiO2 NPs induced testicular lipid peroxidation as well as oxidative stress. Nano-titanium significantly upregulated genes that encode apoptosis and inflammation in testicular tissue. Moreover, it induced histological alteration in the testicular structure with impairment in spermatogenesis via reduction of PCNA immune-staining. Chitosan administration significantly improved the activities of testicular GPx, SOD, and CAT enzymes. In addition, it significantly down-regulated the relative expressions of pro-apoptotic and pro-inflammatory testicular genes. Chitosan was able to improve the testicular architecture as well as spermatogenesis. The current study revealed the capability of chitosan to ameliorate nano-titanium induced testicular toxicity. Thus, attention should be given to the extensive consumption of nano-titanium particles.

Index weight of testis. Rats that received TiO 2 NPs showed no alteration in the index weight of their testes (p > 0.05) ( Table 1). Serum testosterone level. Nano titanium particles significantly reduced serum levels of testosterone hormone compared to the control group (p < 0.05). Chitosan showed no obvious effect on testosterone hormone either alone or combined with TiO 2 NPs (Fig. 4). (Fig. 5). After 14 days, nano titanium particles significantly increased testicular MDA levels compared to the control group. Chitosan treatment significantly decreased MDA levels compared to the TiO 2 NPs group (p < 0.05).

Testicular oxidative/antioxidant biomarkers
Glutathione (GSH) levels were markedly reduced in the TiO 2 NPs group compared to the control group. Chitosan significantly alleviated the TiO 2 NPs-induced reduction of GSH. The activity of GST enzyme was seriously diminished in TiO 2 NPs intoxicated rats compared with the control group with a significant improvement in testicular activity of GST in TiO 2 NPs + Ch group compared to single administration of TiO 2 NPs. Compared to the control group, the activity of testicular GPx enzyme was markedly reduced in TiO 2 NPs-intoxicated rats in both TiO 2 NPs and TiO 2 NPs + Ch groups without significant variation between them.
Nano-titanium particles severely reduced the activity of testicular SOD enzyme compared to the control group (p < 0.05). Chitosan significantly mitigated the TiO 2 NPs-induced reduction in SOD activity in the TiO 2 NPs + Ch group. The activity of CAT enzyme was enormously declined in the TiO 2 NPs group compared to the control one. Chitosan significantly increased the testicular activity of CAT in both, chitosan and TiO 2 NPs + Ch groups compared to the control and the TiO 2 NPs groups, respectively (p < 0.05).
Relative expression of pro-apoptotic genes (Fig. 6A). The relative expressions of caspase 3 and BAX were immensely up-regulated in the TiO 2 NPs and TiO 2 NPs + Ch groups compared to the control group (p < 0.05) with a significant alleviating effect of chitosan in the TiO 2 NPs + Ch group.
Relative expression of pro-inflammatory genes (Fig. 6B). Chitosan showed significant downregulation of testicular genes encode inflammation, TNFα and IL-1β, compared to the control group (p < 0.05). Nanotitanium particles extremely upregulated the testicular mRNA expression of both TNFα and IL-1β in the TiO 2  Fig. 5, histological examination of the control group ( Fig. 7a1-a3) showed that the testicular parenchyma was studded with numerous normal seminiferous tubules that were separated from the interstitial tissues (Leydig cells and blood capillaries) by a well-defined basal lamina and flat myoid cells. The tubules are lined with 4-8 layers of closely and orderly arranged germinal epithelium (spermatogonia, primary spermatocytes, and spermatids) and Sertoli cells. The histological structure of the tes-   Fig. 7b1-b3) was almost similar to that of the control group ( Fig. 7a1-a3). However, in the chitosan group, the seminiferous tubules appeared overcrowded with the germinal epithelium and the interstitial tissues were densely packed with blood capillaries. In the TiO 2 NPs group, a significant reduction was observed in the number of normal seminiferous tubules with intact germinal epithelium (p < 0.05) compared to the control group ( Fig. 7c1-c3). Various degrees of germinal epithelium degeneration were detected, ranging from germinal epithelium disorganization, sloughing, detachment, and vacuolization. Moreover, some tubules are irregular, atrophied, and empty of their lining germinal epithelium. Although the interstitial blood vessels were markedly congested and large areas of interstitial tissues appeared vacuolated and covered with pale inflammatory exudates, the nuclei of Leydig cells appeared normal. In the TiO 2 NPs + Ch group (Fig. 7d1-d3), the mean percentage of preserved seminiferous tubules was significantly increased (p < 0.05) compared to the TiO 2 NPs group (Fig. 7A).

Histological analysis (H & E stain). As shown in
Spermatogenesis assessment (Fig. 8). Normal spermatogenesis was observed in seminiferous tubules in the control ( Fig. 8a1-a4,A) and chitosan ( Fig. 8b1-b4,A) groups, without a significant difference in the mean Johnsen's score between them. In the TiO 2 NPs group, the mean Johnsen's score was significantly decreased than in the control group, and seminiferous tubules showed maturation arrest ( Fig. 8c1-c4,A). In TiO 2 NPs + Ch group, the majority of tubules achieved a normalized Johnsen's score, with the exception of a small number  Caspase-3 Immunohistochemical determination (Fig. 9A). The density of caspase-3 staining in both the control (Fig. 9a) and chitosan groups (Fig. 9b) showed negligible variation. In the TiO 2 NPs group, the caspase-3 staining density was significantly increased compared to the control group (Fig. 7c). In the TiO 2 NPs + Ch group, caspase-3 expression was significantly decreased (Fig. 9d) compared to the TiO 2 NPs group.
PCNA Immunohistochemical determination (Fig. 9B). Expression of PCNA in the control group was quite evident in the nuclei of the spermatogonia (Fig. 9e). Chitosan administration significantly increased the expression of PCNA and was apparently detected in the spermatogonia and primary spermatocytes (Fig. 9f). The expression of PCNA was significantly decreased after TiO 2 NPs administration (Fig. 9g). In the TiO 2 NPs + Ch group, the expression of PCNA was significantly increased (Fig. 9h) compared to the TiO 2 NPs group and approached the PCNA expression in the control group (Fig. 9B).

Discussion
The aim of the present work was to investigate the potential ability of nano-titanium to induce testicular damage as well as the possible mitigation by chitosan administration.
In the current study, neither nano-titanium nor chitosan affected the index weight of the testes. Likewise, no significance was reported in either body weights or testicular weights in male mice exposed to TiO 2 NPs at doses Figure 5. Effects of TiO 2 NPs and/or chitosan on testicular oxidative/antioxidant markers. data are presented as means ± SEM. data are presented as means ± SEM. Data are expressed as mean ± standard error of the mean (n = 5). Means in the same row with different superscripts are significantly different (p < 0.05). MDA (malondialdehyde); GSH (Reduced glutathione); GST (Glutathione S transferase); GPx (Glutathione Peroxidase) SOD (superoxide dismutase) and CAT (catalase). www.nature.com/scientificreports/ up to 100 mg/kg BW for 28 days 13,14 . Mice exposed orally to TiO 2 NPs at doses of 10, 50 and 250 mg/kg BW from 28 to 70th postnatal day suffered reductions in the body weight gain solely at the highest dose without significant difference in absolute or relative weights of testes 15 . A significant reduction in testicular weight was observed in mice exposed to 300 mg/kg BW of TiO 2 NPs for 35 days without alteration in body weights 13,14 . Meena et al. 2015 9 observed a significant decrease in the average coefficients of testis at the higher dose of TiO 2 NPs (50 mg/ kg BW for 30 days), whereas, lower doses of TiO 2 NPs (5 and 25 mg/kg BW) did not show a significant effect. The reduction in body weight and testicular mass with rising doses of nano-titanium might be attributed to excess accumulation of TiO 2 NPs in testes 3 . This phenomenon showed that the accumulation of titanium in the organs was closely related to the organ-to-body-weight ratio 9 . Thus, the dose and the exposure time were among the factors that influenced body and organ weights. After 14 days of exposure time, TiO 2 NPs significantly reduced serum levels of testosterone hormone without obvious action of chitosan. In consistency, TiO 2 NPs at doses of 50 or 300 mg/kg BW for 30-35 days significantly decreased serum testosterone levels in rats and mice 9,16,17 . Serum levels of testosterone were significantly reduced after postnatal exposure of mice to 50 and 250 mg/kg/day nano-TiO 2 15 . Intragastric TiO 2 NPs for 90 consecutive days passed through the blood-testis barrier and accumulated in the testis, with subsequent testicular lesions, sperm malformations, and alterations in serum sex hormone levels 3 . Testosterone was produced in Leydig cells and in supported Sertoli cells and was required for the attachment of round spermatids to Sertoli cells 18 . Low levels of testosterone might suppress spermatogenesis and cause dysfunction of Sertoli cells 9,19,20 . This reduction may contribute to changes in testis histology and spermatogenesis.
Chitosan nanoparticles at a dose of 280 mg/kg BW for 45 consecutive days attenuated the reduction in testosterone induced by hydroxyapatite nanoparticles 21 . Pretreatment with chitosan nanoparticles (60 mg/kg BW) ameliorated the testicular level of testosterone significantly as compared to the potassium dichromate exposed group 21 . Following 30 successive days, chitosan nanoparticles at a dose of 280 mg/kg in combination with selenium significantly increased serum levels of testosterone in diclofenac-sodium intoxicated rats 22 . In the present work, chitosan might require more exposure time and/or a higher dose to achieve a significant attenuation of the hormonal reduction induced by nano-titanium particles.
In the present study, the testicular level of MDA was obviously increased in the TiO 2 NPs group, which is compatible with previous studies that were previously established 23 . MDA content serves as an indicator of the extent of lipid peroxidation and is an indirect reflection of the extent of cell damage 14 . MDA is the decomposition product of polyunsaturated fatty acids, and its increase is a result of significant accumulation under high antioxidant stress 14 . Carboxy methylated chitosan significantly reduced testicular lipid peroxidation in the TiO 2 NPs + Ch group compared to the TiO 2 NPs group, suggesting the capability of chitosan as a natural biomaterial to alleviate the harm induced by nano titanium particles.
Testicular antioxidant system comprises enzymatic and non-enzymatic constituents. The enzymatic components include SOD, GST and GPx enzymes. The defense mechanism involves conversion of superoxide anion  www.nature.com/scientificreports/ to hydrogen peroxide that is eliminated by CAT or GPx. While GST facilitates conjugation of glutathione with xenobiotic agents for their excretions as a detoxification process 24 . Nano-titanium particles adversely affected the testicular antioxidant system through reductions in GST, GPx, SOD and CAT activities. Similarly, testicular SOD activity was decreased in mice exposed to 100 mg/kg BW TiO 2 NPs for 28 days 14 . Intravenous TiO 2 NPs for 30 days significantly decreased the SOD and GPx activities at 25 and 50 mg/kg BW with an increase in CAT activity in Wister rats 9 . At a higher dose (300 mg/kg), TiO 2 NPs enhanced oxidative stress via increasing testicular levels of MDA and reducing testicular levels of SOD, CAT, and GSH 16 . Similar outcomes were reported at the cellular level using rat, human, or prepubertal porcine primary cultured Sertoli cells 25,26 . Nano-titanium particles stimulated production of reactive oxygen species in mouse testes after a 60-day exposure period with a subsequent alteration in the activities of spermatogenesis-dependent enzymes 27 . Thus, oxidative stress and lipid peroxidation might be attributed to the production of free radicals in the testicular tissues and are vital mechanistic paradigms to explain the toxic effects of TiO 2 NPs. Chitosan was able to ameliorate testicular oxidative stress induced by TiO 2 NPs, with a significant increase in CAT activity compared to the control group, confirming its antioxidant properties.
Chitosan increased GSH activity and exhibited antioxidant properties by preventing the decrease in CAT and GSH levels caused by sodium fluoride 28 . In addition, nano-chitosan with hydroxyapatite nanoparticles increased the activities of GPx, GST, CAT, SOD, TAC, and GSH in male rats 21 . Chitosan-NPs reversed the diclofenac-sodium mediated decrease in testicular antioxidant markers, SOD and CAT 22 . In accordance, Chitosan significantly lowered lead-induced testicular oxidative stress 29 . The pretreatment with nano chitosan (60 mg/kg) significantly decreased MDA levels and increased the activity of GPx levels 23 . Chitosan has a potent www.nature.com/scientificreports/ antioxidant activity and free radical scavenger potential, which can decrease the lipid peroxidation (MDA level) and increase the antioxidant defense system, which protects against free radical attack with subsequent nullifying of TiO 2 NPs-induced testicular damage.
Regarding the relationship between antioxidant defense enzymes and apoptotic factors. Glutathione and glutathione-related enzymes are decreased in both their levels and activities after the incidence of apoptosis 30 , as the depletion of GSH is critical for cellular death 31 . Moreover, GST is an enzyme that is required for the protection against DNA damage induced by apoptosis, where exhaustion of GST activity can be found due to the incidence of apoptosis. Superoxide dismutase is an enzyme that is associated with a defense mechanism against free radicals, found to decrease in the case of prostate carcinoma 32 , and found to be effective when supplemented to decrease apoptosis. The downregulation of catalase as well as the decrease in its activity might inhibit the process of apoptosis 33 ; due to overproduction of hydrogen peroxide. The overexpression of both catalase and Cu/ Zn superoxide dismutase decreased the incidence of apoptosis by 40% due to the decrease in caspase 9 34 . In the current study, the administration of TiO2 can result in the propagation of reactive oxygen species that induce the incidence of apoptosis in testicular tissue as a result of the increase of the degree of lipid peroxidation 35 . Moreover, proinflammatory cytokines such as TNF-alpha can contribute to the induction of apoptosis through stimulation of both c-ABL and p73 pathways during the degradation of retinoblastoma protein 36 .
In the present work, short term oral exposure to TiO 2 NPs extremely induced testicular inflammation via upregulation of mRNA expressions of TNFα and IL-1β genes with a significant anti-inflammatory action of chitosan. In primary cultured rat Sertoli cells, TiO 2 NPs induced an inflammatory response via increasing the expression of IL-1β, TNF-α, IFN-γ, and IL-10 in a concentration-dependent manner 37 . Inflammatory mediators present in the normal testis, such as interleukins and TNFα participate in the regulation of spermatogenesis; 38 , therefore the disruption in their expression could perturb normal spermatogenesis. In the current study, TiO 2 NPs significantly impaired spermatogenesis, evidenced by the reduction that was reported in Johnsen's score.
Our present work showed that the relative expression of both BAX and Caspase 3 was enormously upregulated in the TiO 2 NPs exposed group. Likewise, the expression levels of testicular caspase-3, Nrbp2, and cytochrome c and their proteins were significantly increased in mice exposed to oral TiO 2 NPs (2.5, 5 and 10 mg/ kg BW) for 90 days in a dose-dependent manner 39 . Significant upregulation of testicular BAX gene expression was detected in rats receiving oral 300 mg/kg BW/day of TiO 2 NPs for 30 days 16 . Intravenous administration of 50 mg/kg BW/week for 30 days of TiO 2 NPs significantly activated testicular caspase 3 in exposed rats with a significant reduction in total sperm count suggesting that apoptosis is considered to be involved in the impairment of spermatogenesis and the seminiferous tubules 9 . The pro-apoptotic member of Bcl-2 protein family, BAX, stimulates the mitochondrial pathway, whereas caspase-3 is activated in the apoptotic cell by extrinsic (death ligand) and intrinsic (mitochondrial) pathways 40 . Up-regulation of both BAX and caspase 3 suggests induction of testicular apoptosis via the mitochondrial pathway. A positive correlation was reported between ROS levels and apoptosis in testicular cells. Therefore, oxidative stress encourages apoptotic processes 9,24 . Additionally, the upregulation of the proinflammatory cytokines, IL-1β and TNFα, might have a crucial role in inducing testicular apoptosis via regulating Bcl2 family protein expression 41 .
In the current study, chitosan displayed a testicular anti-apoptotic effect in TiO 2 NPs + Ch group with respect to the TiO 2 NPs-intoxicated group. Pretreatment of mice with chitosan nanoparticles significantly decreased potassium dichromate-induced elevation in testicular contents of caspase 3 42 . Oral chitosan nanoparticles at a dose of 600 mg/kg BW for 11 days could protect rat testis from oxidative damage and apoptosis prompted by lead acetate through reducing MDA levels, caspase 3 mRNA expression and, in addition, increasing levels of SOD and GPx 43 .
The present results were confirmed by the histological and morphometric outcomes, including a significant reduction in the number of seminiferous tubules with irregular and atrophied tubules and marked germinal epithelium degeneration. In consistency, in rats exposed to 50 mg/kg TiO 2 NPs, disorganization and disruption in some seminiferous tubules were observed 9 . Numerous histological alterations were reported in rats exposed to 10 mg/kg of TiO 2 NPs for 90 days, including irregular arrangement of Sertoli cells in the seminiferous tubules, Sertoli cell apoptosis, necrosis of the seminiferous tubules, decreased thickness of the germinative layer and vacuolation 9 . No histological changes were observed in the testes of mice exposed to 10 mg/kg/day nano-TiO 2 . Whereas, at higher doses (50 and 250 mg/kg/day), seminiferous tubules showed vacuoles with decreased layers of spermatogenic cells in mice that received the high-dose (250 mg/kg/day) of nano-TiO 2 15 . Nano TiO 2 particles (50 mg/kg) significant histological alterations in seminiferous tubules, including reduction in their diameter, epithelial vacuolization, sloughing, detachment, and atrophy with abnormal spermatogenesis and significant decline in the Johnsen score 17 . Significant modifications in the testicular morphology of TiO 2 NP-treated rats might be the result of free reactive radicals and subsequent lipid peroxidation 16 .
Proliferating cell nuclear antigen (PCNA) is a cell cycle regulatory protein marker that is involved in DNA synthesis and has been linked to cell proliferation 44 . PCNA immune-expression in the testis is used as a proliferative marker for spermatogenesis estimation 45,46 . It is considered a rapid, reliable, sensitive, and quantitative approach for determining and detecting early testicular toxicity 47,48 . Spermatogenesis is a dynamic and synchronized process of maturation of stem spermatogonia into mature spermatozoa, which takes place in the seminiferous tubules 49 . In the current study, the testicular PCNA expression was mapped using the immunohistochemistry technique. The significant decrease in the PCNA immune-staining with TiO 2 NPs highlights the ability of nano-titanium particles to impair spermatogenesis, which is further supported by the histological findings and the significant decline in Johnsen's score. The toxic impact of TiO 2 NPs on the male spermatogonia has been previously demonstrated 50,51 . The reduction in Johnsen's score in the TiO 2 NPs group may be attributed to the induction of apoptosis and reduction of the active DNA content in the dividing spermatogonia 52 . The spermatogenic damage in the TiO 2 NPs group was attributed as well to the induction of lipid peroxidation and oxidative stress that had harmful effects on spermatogenesis 52 . www.nature.com/scientificreports/ It is noteworthy that chitosan significantly induced testicular immune-expression of PCNA suggesting its capability to improve spermatogenesis under basal conditions. At the same time, chitosan exerted a potential ameliorative effect counter to that of TiO 2 NPs through elevations in the regenerated seminiferous tubules, PCNA immune-staining, and a reduction in Caspase-3 expression in respect to the control group. The constructive effect of chitosan on spermatogenesis was obvious as well in raising Johnsen's score compared to the non-treated TiO 2 NPs group.
This work was the first that combined nano-titanium particles with chitosan to study their effects on various reproductive parameters. Briefly, we should highlight the ability of TiO 2 NPs to induce testicular dysfunction after short-term of exposure via promotion of inflammation, apoptosis and oxidative stress as well as the reduction in testosterone hormone, the issue that necessitate rising attention for the daily and excessive exposure to oral and occupational nano-titanium particles, and considering its potential toxic impact on reproduction. On the other hand, chitosan was the key to mitigating the adverse effect of TiO 2 NPs on testicular functions. In the current work, chitosan achieved its role as an antioxidant, anti-inflammatory, and anti-apoptotic biomaterial. In addition, it improved spermatogenesis as well as CAT activity under normal conditions (without TiO 2 NPs exposure), indicating its ability to upgrade testicular function, an issue that needs further investigation.

Materials and methods
This study was conducted at Mansoura University with an approved animal use protocol (R/50) in accordance with the Guiding Principles for the Care and Use of Research Animals, Faculty of Veterinary Medicine, Mansoura University, Egypt.
Chemicals. Titanium dioxide (TiO 2 ) was purchased from Sigma-Aldrich Chemical Co., USA. The titanium dioxide nanoparticles, TiO2 NPs, were prepared at the Nanotechnology Unit, Faculty of Postgraduate Studies in Advanced Sciences, Beni-Suef University, according to the methods described by Farghali et al. 46 . The size range of TiO 2 NPs is less than 60 nm. Solutions of dispersed TiO 2 NPs were freshly prepared via ultrasonication for 15 min just before oral administration. Carboxymethyl chitosan (CMC, 10%) was purchased from Xin Luk Biotech, China. A field emission scanning electron microscope, FESEM was used to examine the morphologies of the prepared materials (FEI-Quanta FEG-250 SEM). X-ray diffraction, XRD (PANalytical Empyrean, Netherlands) was used to determine phase identification and crystallinity using CuKa radiation (wavelength 1.54045), accelerating voltage of 40 kV, and current of 35 mA. Raman spectroscopy was performed with a Bruker Senterra Raman Microscope (Bruker Optics Inc., Germany). The modified TiO 2 NPs were successfully disseminated into the chitosan matrix, as determined by analysis using scanning electron microscopy (SEM) and atomic force microscopy (AFM), and the roughness of the chitosan-TiO 2 nanocomposites was greatly reduced. Additionally, thermogravimetric analysis (TGA) of the thermal characteristics revealed that the chitosan-TiO2 nanocomposites with 0.05 percent TiO 2 NPs concentration had the best thermal stability. These analyses also revealed that the chitosan interacted with TiO 2 NPs and displayed good compatibility 53 .

Animals and treatment.
Adult male Albino rats (3-4 months old), purchased from MERC lab (Mansoura University) were kept for acclimatization under standard laboratory conditions (temperature of 22-25 °C, 50-60% relative humidity, and 12 h dark/light cycle) for 7 days. Food and water were available ad libitum.
Animals were randomly distributed into four groups (5 rats each). The first group received deionized water and was assigned as a control group. In the second group, animals received chitosan at a dose of 5 mg/kg BW/ day according to Wang et al. 54 . The third group was designed for administration of TiO 2 NPs at a dose of 150 mg/ kg BW/day (1/80 LD 50 ) according to Azim et al. 55 . Rats in the fourth group received both TiO 2 NPs and chitosan (TiO 2 NPs + Ch) (150 and 5 mg/kg BW/day, respectively). Both TiO 2 NPs and chitosan were freshly prepared just before administration, following the manufacturers' instructions. All treatments were given via oral route for 14 consecutive days. On the 15th day, rats were weighed and killed by cervical dislocation. Blood and testes were collected from all groups. Relative testicular weights were calculated using the following equation according to Bearden and Fuquay 56 . Oxidative/antioxidant parameters. The lipid peroxidation marker, malondialdehyde (MDA), was measured according to Draper and Hadley 57 . Antioxidant defense markers were determined using colorimetric commercial kits [Bio-diagnostic Co, Giza, Egypt]. Reduced glutathione (GSH) concentration was measured colorimetrically using dithionitrobenzoate reagent according to Beutler et al. 58 . Glutathione S transferase (GST) activity was measured according to Habig et al. 59 . The activity of superoxide dismutase (SOD) was measured spectrophotometrically according to Nishikimi et al. 60 .
Relative testicular weight = testicular weight g /Body weight g * 100 .  Table 2, including their sequences and accession numbers in Genbank. The application of real-time PCR for amplification and relatively quantifying the specified genes in the current study was conducted on an Applied Biosystem Step One (Thermo Fisher Scientific, UK). Real-time PCR was performed using TOPreal qPCR 2 × premix (enzynomics, South Korea) with the following cycling conditions: Initial denaturation at 95 °C for 8 min, followed by 40 cycles of 95 °C for 40 s, 56 °C for 30 s, and 72 °C for 40 s, then the reaction was terminated by a final elongation cycle at 72 °C for 7 min. The expression analysis was done using the 2ΔΔ ct method adopted by Livak and Schmittgen 62 .
Histomorphometric and immunohistochemical studies. Hematoxylin and eosin staining. Testicular samples were fixed in 10% neutral buffered formalin solution for 24 h. The tissues were then gradually dehydrated with ascending ethanol concentrations, cleaned in xylene, and imbedded in liquid paraffin wax. Using a rotatory microtome, paraffinized blocks were sectioned at a thickness of 5 microns and mounted on either coated glass slides for H & E staining or positive glass slides for immunohistochemical examination 63 .
Immune-staining of caspase-3 and proliferating cell nuclear antigen (PCNA). The technique was applied according to Karen Petrosyan et al. (2002) 64 . Briefly, testicular sections were dewaxed, rehydrated, incubated with 3% hydrogen peroxide at room temperature for 30 min to inhibit endogenous peroxidase activity, and blocked for 15 min with 5% normal goat serum. The sections were then incubated overnight at 4 °C with primary antibodies against either caspase-3 (1:100, 56,046; Santa Cruz Biotechnology, CA, USA) or PCNA (1:500, ab18197; Abcam). Afterwards, the sections were washed, incubated with secondary antibodies, stained with diaminobenzidine, and counterstained with hematoxylin. The mean density of caspase-3 and PCNA expressions was evaluated and expressed as percent using the image analyzer program (version 1.36, NIH, USA).
Histomorphometric analyses. Using the light microscope (40 × magnification), morphometric analyses were performed on randomly selected five stained slides for each group (5 fields/slide). The mean percentage of normal seminiferous tubules of the testes 65,66 was determined using the image analyzer program (version 1.36, NIH, USA). Germinal epithelium maturity was graded according to a modified Johnsen's scoring method 67,68 . The score, ranging from 1 to 10, was calculated for each animal based on the stage of spermatogenesis. The image analyzer program was used to calculate the mean density of caspase-3 and PCNA immune expression in all groups.
Statistical analysis. The normality of quantitative parameters (apoptotic and inflammatory gene mRNA expression) was visually examined using normal probability plots and the Kolmogorov-Smirnov test. All data are presented as mean standard deviation of the mean (SEM). Duncan's multiple comparison test was used to perform post hoc multiple pairwise comparisons. The effect of TiO 2 NPs/chitosan on oxidative stress-antioxidant parameters, expression of apoptotic proteins, and changes in serum testosterone was studied using a mixed model one-way analysis of variance. SAS® was used for statistical analysis (version 9.2, SAS Institute, Cary, NC). For all analyses, the values were considered statistically significant when p < 0.05.
Ethics approval and consent to participate. All used protocols were approved by the Committee on the Ethics of Animal Experiments of the Faculty of Veterinary Medicine, Mansoura University Code No. R/51. Table 2. Sequences of forward and reverse primers used for qRT-PCR quantitation.