Article

Melatonin controlled apoptosis and protected the testes and sperm quality against bisphenol A-induced oxidative toxicity

Toxicology and Industrial Health 1–13 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714561286 tih.sagepub.com

Azza I Othman1, Gamal M Edrees1, Mohamed A El-Missiry1, Doaa A Ali1, Mohamed Aboel-Nour1 and Banan R Dabdoub2 Abstract Epidemiological reports have indicated a correlation between the increasing bisphenol A (BPA) levels in the environment and the incidence of male infertility. In this study, the protective effects of melatonin on BPAinduced oxidative stress and apoptosis were investigated in the rat testes and epididymal sperm. Melatonin (10 mg/kg body weight (bw)) was injected concurrently with BPA (50 mg/kg bw) for 3 and 6 weeks. The administration of BPA significantly increased oxidative stress in the testes and epididymal sperm. This was associated with a decrease in the serum testosterone level as well as sperm quality, chromatin condensation/decondensation level, and the percentage of haploid germ cells in the semen. BPA administration caused a significant increase in apoptosis accompanied by a decrease in the expression of the antiapoptotic proteins Bcl-2 in the testes and epididymal sperm. The concurrent administration of melatonin decreased oxidative stress by modulating the levels of glutathione, superoxide dismutase, and catalase as well as the malondialdehyde and hydrogen peroxide concentrations in the testes and sperm. Melatonin sustained Bcl-2 expression and controlled apoptosis. Furthermore, melatonin maintained the testosterone levels, ameliorated histopathological changes, increased the percentages of seminal haploid germ cells, and protected sperm chromatin condensation process, indicating appropriate spermatogenesis with production of functional sperm. In conclusion, melatonin protected against BPA-induced apoptosis by controlling Bcl-2 expression and ameliorating oxidative stress in the testes and sperm. Thus, melatonin is a promising pharmacological agent for preventing the potential reproductive toxicity of BPA following occupational or environmental exposures. Keywords Pollutants, endocrine disrupting chemical, oxidative stress, reproduction, apoptosis, testosterone, antioxidants

Introduction Bisphenol A (BPA) has received great attention due to its widespread presence in several consumer products and is detected in various human tissues and body fluids. BPA is used for manufacturing polycarbonate plastic and is a constituent of epoxy and the polystyrene resin (Doerge et al., 2011). BPA exhibits hormone-like properties that raise concern about its suitability in consumer products and food containers. It is present in several products, including the interior coatings of food cans, milk containers, and baby formula bottles, as well as in dental sealants (Welshons et al., 2006). It can be detected in water, food, indoor

dust, and other related media (Kleywegt et al., 2011; Noonan et al., 2011). Humans are mostly and innocently exposed to BPA, and it can be detected in the majority of 1

Department of Zoology, Faculty of Science, Mansoura University, Mansoura, Egypt 2 Department of Biology, Faculty of Education, Mosul University, Mosul, Iraq Corresponding author: Mohamed A El-Missiry, Department of Zoology, Faculty of Science, Mansoura University, Mansoura 35516, Egypt. Email: [email protected]

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individuals in many countries worldwide (Vandenberg et al., 2007). When BPA is consumed through food, it can be quickly absorbed from the digestive tract and can be detected in various body fluids, organs, and tissues (Dekant and Volkel, 2008; Fisher et al., 2011; Vandenberg et al., 2007). The reported concentrations of BPA in blood and urine of occupationally and environmentally exposed humans are reviewed elsewhere (Dekant and Volkel, 2008; Li et al., 2010; Vandenberg et al., 2010). The minimum toxic dose of BPA is estimated about 200 mg/kg/day in rats and mice (Takahashi and Oishi, 2003). Median lethal dose of BPA was 841 and 35.26 mg/kg body weight (bw) for intraperitoneal and intravenous route, respectively, in rats (Pant and Deshpande 2012). Reproductive and endocrine disruptions are the major kinds of toxicity induced by BPA (Erler and Novak, 2010). Testicular toxicity induced by BPA has been reported and may account for the increasing frequency of infertility (Takahashi and Oishi, 2001; Tohei et al., 2001). The reproductive toxicity of BPA is caused through multiple signaling pathways (D’Cruz et al., 2012) and may contribute to spermatogenesis failure in rats (Qiu et al., 2013). Moreover, BPA reversibly perturbs the integrity of the blood– testis barrier in Sertoli cells in vitro (Li et al., 2009a). Melatonin (N-acetyl-5-methoxytryptamine, MLT) is a neurohormone derived from tryptophan and is mainly released from the pineal gland. MLT participated in various homeostatic functions, such as reproduction regulation and circadian rhythms (Reiter et al., 2000), by its action at various levels of the hypothalamic– pituitary–gonadal axis (Pandi-Perumal et al., 2006). In addition, it has antioxidant and prophylatic properties against oxidative stress in several experimental and clinical conditions with a large safety margin upon its administration (El-Missiry, 2000; Reiter et al., 2004). MLT readily scavenges the most toxic reactive oxygen and nitrogen species, including the hydroxyl radical, the peroxynitrite anion, and hydrogen peroxide (H2O2). It stimulates the messenger RNA (mRNA) levels of antioxidant enzymes including superoxide dismutase (SOD) (Kotler et al., 1998). In addition to its use as an antistress, antiaging, and immunomodulatory agent, MLT has been used for sexual dysfunctions, gallbladder stones, obesity, and even cancer (Altun and Ugur-Altun, 2007; El-Missiry and Abd El-Aziz, 2000).These properties have suggested the potential use of MLT as a therapeutic agent. Therefore, the aim of this study was to investigate the protective effect of melatonin on BPA-induced oxidative

stress and apoptosis in rat testis and epididymal sperm quality.

Materials and methods Chemicals and reagents All reagents were of the highest purity available. BPA (CAS #80-05-7) and melatonin (CAS 73-31-4) were obtained from Sigma-Aldrich Co., (St Louis, Missouri, USA)

Animal exposure Adult male Sprague–Dawley rats (8-week old, 200– 220 g) were obtained from the Laboratory Animal Center, Mansoura University, Egypt. The animals were maintained under standard laboratory conditions (temperature 22 + 2 C; humidity 50%–70%; and 12-h light/12-h dark cycle). Food and water were provided ad libitum. All the experiments were performed in accordance with protocols and international guidelines for care and use of laboratory animals and approved by the local experimental ethics committee. After 2 weeks of acclimatization, the rats were randomly divided into six groups. A normal control group (n ¼ 6) did not receive any treatment. Groups 2 and 3 (n ¼ 6 each) were given either corn oil (0.2 mL) or saline orally and served as the experimental control groups. Group 4 (n ¼ 16) was given BPA dissolved in corn oil at 50 mg/kg body weight 3 days a week for 3 and 6 weeks. Group 5 (n ¼ 6) received 10 mg/kg body weight of melatonin dissolved in normal saline 3 days a week for 3 weeks. Group 6 (n ¼ 16) received 10 mg/kg of melatonin followed by 50 mg/kg of BPA 3 days a week for 6 weeks. The doses of melatonin and BPA were based on earlier studies (El-Missiry et al., 2007; Nakamura et al., 2010). Our study aimed to clearly identify the protective challenge of the pharmacological dose of melatonin, so one relatively high dose of BPA was used.

Sperm analysis Sperms were collected as previously described (Gupta et al., 2004). Briefly, the epididymides of both testes were removed and trimmed free of fat and placed in physiological saline at 37 C. Three deep cuts were made along the proximal and distal cauda of each epididymis. After 5 min incubation at 37 C, the tissue was removed, and the sperm suspensions were gently mixed and maintained at 37 C.

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Sperm counts

Germ cell preparation

Aliquots of sperm suspension were diluted 100 times with fresh medium, and sperm numbers were counted using a Neubauer chamber (Aydogan and Barlas, 2006).

The testicular tissue preparation for flow cytometry was performed as described (Dey et al., 2000). Briefly, the testicular tissue was obtained immediately after dissecting rats from the same region of the testis. The testicular tissue was immediately and finely minced in PBS, passed gently through the hub of a syringe and then vortexed briefly to maximize the release of germ cells from the seminiferous tubule minces. The cell suspension was then filtered using a nylon filter, and a drop of the filtrate was observed under the microscope to check the number and integrity of the released germ cells before fixing in 80% chilled ethanol and storing at 4 C until flow cytometric analysis.

Sperm motility Aliquots of this suspension were microscopically examined, and the percentage of motile sperm was determined by counting the number exhibiting forward motion against the total sperm count (Aydogan and Barlas, 2006).

Abnormality of epididymal sperm For sperm abnormalities, smears were fixed with alcohol, stained with eosin Y, and then examined for morphological abnormalities. The percentages of abnormal sperm in three fields were calculated according to a previous protocol (Aydogan and Barlas, 2006).

Biochemical determinations The left testes and epididymal sperms were homogenized in ice-cold (20 mM) phosphate-buffered saline (PBS; pH 7.4) for all of the following biochemical assays. The lipid peroxidation product, malondialdehyde (MDA), was measured by the thiobarbituric acid (TBA) assay, which is based on the MDA reaction with TBA to give thiobarbituric acid reactive substances (TBARS), a red species that absorbs at 535 nm (Ohkawa et al., 1979). The levels of reduced glutathione (GSH) were determined at 412 nm as previously described (Sedlak and Lindsay, 1968). The catalase (CAT) activity was spectrophotometrically determined by measuring the decomposition of H2O2 at 240 nm (Johansson and Borg, 1988). The SOD activity was spectrophotometrically measured using phenazine methosulfate to generate superoxide radicals that react with nitroblue tetrazolium (Nishikimi et al., 1972). All protein concentrations were determined as previously mentioned (Lowry et al., 1951). The plasma testosterone level was measured using enzyme-linked immunosorbent assay (ELISA) method using DRG ELISA testosterone kit (ELISA EIA-1559, 96 Wells kit, DRG Instruments, GmbH, Marburg, Germany) according to the kit manufacturer’s instructions. The H2O2 concentration was measured in seminal plasma using a colorimetric assay kit according to the kit manufacturer’s instructions (Biovision, California, USA).

Flow cytometric determination of epididymal sperm ploidy and condensation/de-condensation The epidydimal sperm samples were diluted and washed twice with PBS before fixation. The samples were fixed immediately with 1 mL ice-cold absolute alcohol drop by drop with gently shaking and preserved at 4 C until staining. Staining with propidium iodide (PI) for DNA ploidy was carried out according to the procedure described by Vindelov (1977). For measuring the de-condensation, another sample was treated with sodium dodecyl sulfate and processed according to the method described by Hacker-Klom et al. (1999). Flow cytometric analysis was aimed at measuring the parameters, such as (a) mature haploid spermatozoa percentage at 1 mL peak, (b) haploid round spermatids percentage at 1 mL peak, and (c) diploid spermatozoa percentage in 2 mL peak. Chromatin condensation percentage and chromatin decondensation percentage were analyzed using FACSCalibur™ flow cytometer (Becton Dickinson, Sunnyvale, California, USA).

Flow cytometric analysis of apoptosis with Annexin V–fluorescein isothiocyanate /PI For the annexin V assay, testicular cells and epididymal sperm were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V using the ApoAlert kit from Clontech (Palo Alto, California, USA) according to the manufacturer’s instructions. In this study, the testicular cells and epididymal sperm samples were fixed in 100 mL of cold 1% paraformaldehyde in PBS for 1 h at 4 C in a 96well plate. To prevent cell clumping, the cells were

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Table 1. Effect of MLT and BPA on sperm count and percentage of motility, abnormality, DNA ploidy levels, chromatin condensation, and de-condensation, as well as serum testosterone level in the epididymal sperm of the control and vehicle-treated rat groups.a Control Sperm count (106/mL) 437.1 Sperm motility 61.03 Sperm abnormality 7.90 Haploid sperm 97.12 Spermatid 2.28 Diploid sperm 2.88 Condensation 90.23 De-condensation 109.77

MLT

+ 13.9 483.1 + + 3.4 92.50 + + 1.90 9.19 + + 2.25 98.76 + + 3.11 1.77 + + 2.26 1.24 + + 3.6 93.62 + + 4.9 115.50 +

BPA (3 weeks) BPA (6 weeks) 9.3 2.2b 1.87 1.60 2.09 1.60 3.6 8.7

275.9 + 59.2b 28.67 + 1.4b 27.71 + 5.90b 91.63 + 1.67b 21.32 + 2.74 8.37 + 1.66b 93.42 + 16.6 99.21 + 4.5b

BPA þ MLT (3 weeks)

BPA þ MLT (6 weeks)

220.4 + 25.9b 464.50 + 18.9c 430.5 + 15.53 + 1.7b 63.86 + 11.4c 91.86 + b 43.71 + 6.91 7.63 + 1.10c 7.17 + b 75.91 + 2.98 94.51 + 4.73 96.66 + 60.72 + 3.59b 16.39 + 3.33c 17.79 + 24.09 + 2.98b 5.49 + 1.73b 3.34 + 54.30 + 5.8b 77.83 + 12.4c 79.00 + 62.98 + 8.8b 104.49 + 10.2c 109.48 +

38.7d 5.9bd 1.41d 1.32d 3.80d 1.32d 8.7d 5.5d

MLT: melatonin; BPA; bisphenol A. a Values are expressed as mean + SD, n ¼ 6. b p < 0.05: significant as compared to control group. c p < 0.05: significant as compared to BPA 3-week group. d p < 0.05: significant as compared to BPA 6-week group.

pipetted several times while adding paraformaldehyde and were agitated on a shaker during the fixation process. The fixed cells were centrifuged for 5 min and washed twice in 200 mL of PBS. The cells were then resuspended in 200 mL of PBS containing 50 mg/mL of ribonuclease A (RNase A) and 50 mg/mL of PI and incubated for 30 min at 37 C. The flow cytometric analyses were performed on a FACSCalibur cytometer using CellQuest Pro software (Becton Dickinson) for data acquisition and analysis (Juan et al., 2012).

Flow cytometric analysis of Bcl-2 The flow cytometric analysis of Bcl-2 was performed using methods similar to those previously reported (Aiello et al., 1992). A cell suspension of was prepared with PBS/ bovine serum albumin (BSA) buffer and then incubated with Mouse Anti-Bcl-2 FITC (Clone: 10C4, eBioscience, San Diego, California, USA) for 15 min at room temperature. Cells finally was resuspended in 0.5% paraformaldehyde in PBS/ BSA and analyzed by flow cytometry.

Testicular histology Testicular histological examination was conducted to study the effects of BPA on testis. Briefly, the testis was immersed in neutral-buffered formalin fixation fluid for 10 h. After washing with 70% alcohol, the testis sample was processed for dehydration, paraffin embedding, 5-m thick paraffin sections, and then stained with hematoxylin–eosin (Kumar et al., 2006).

Statistical analysis Data were presented as mean + SD. In all cases, n refers to the number of animals in each treatment group. Statistical analyses were performed by oneway analysis of variance followed by Students–Newman–Keul’s post hoc test. Statistical significance was considered as p < 0.05.

Results No mortality or morbidity was observed in any of the experimental animal groups during the study period. The treatment with either saline solution or corn oil three times per week for 3 and 6 weeks did not show statistical differences in all measured parameters when compared with each other and with the normal control group. The treatment with BPA caused a significant decrease in the mature sperm concentration and motility, while abnormal sperm concentration increased compared with the control and vehicletreated groups (Table 1). The treatment with MLT showed a significant increase in mature sperm motility compared with the control groups. The concurrent treatment with MLT and BPA caused a significant protection against BPA-induced changes and showed normal sperm count, mortality, and morphology compared with the BPA-treated group. The flow cytometry analysis of ploidy and condensation of epididymal sperm in relation to the effect of MLT and BPA are summarized in Table 1. The percentage of haploid sperm in BPA-treated group was significantly lower than that of the control and

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Figure 1. Plasma testosterone levels (ng/mL) in control and different animal groups. Each column represents mean + SD, n ¼ 6, ***p < 0.05: significant.

vehicle-treated groups. The percentage of diploid sperm and spermatids in rats treated with BPA was significantly higher than that in the control rats. Moreover, the mean sperm chromatin condensation/decondensation values reduced significantly in the BPA-treated rats compared with control groups. The effect of BPA was higher in BPA-treated groups for 6 weeks than for 3 weeks. In contrast, when MLT was administered concurrently with BPA, it significantly ameliorated the effects of BPA on the percentages of ploidy levels in the sperm. This combined treatment showed significant protection of sperm chromatin condensation/de-condensation ratio compared with the BPA-treated groups and appeared close to the control levels (Table 1). Furthermore, there was a significant decrease in the testosterone levels in the serum of the BPA-treated rats compared with the serum of the control groups (Figure 1). MLT treatment along with BPA significantly protected testosterone levels compared with the BPA-treated group and showed serum testosterone levels to be within the control range. The treatment with BPA three times per week for 3 and 6 weeks resulted in significant elevations in the MDA and H2O2 levels in the testes and epididymal sperm of the BPA-administered group compared with the control and vehicle-treated groups (Table 2). The treatment with MLT and BPA concurrently resulted in a significant decrease in the MDA and H2O2 levels in the testes and sperm compared with the BPA-

treated rats. In contrast, the GSH content in testes and epididymal sperm significantly decreased in the BPAtreated rats compared with the control groups (Table 2). The SOD and CAT activities significantly decreased in the testes and epididymal sperm in the BPA-treated groups. The decrease in the level of GSH and antioxidant enzymes was ameliorated in the MLT þ BPA-treated groups compared with the BPAtreated group. Interestingly, MLT treatment alone resulted in a significant increase in SOD activity in the testes but not in the epididymal sperm compared with the control rats (Table 2). Meanwhile, MLT treatment caused significant increase in the CAT activity in the epididymal sperm compared with the control group (Table 2). To elucidate the effect of MLT and BPA on numbers of apoptotic germ cells in the testes and epididymal sperm, annexin V-FITC/PI staining was used. The percentages of viable, apoptotic, and necrotic cells were scored and are summarized in Table 3. The number of apoptotic and necrotic cells showed a significant increase in response to BPA treatment compared with the control groups. Meanwhile, the viable cells in the testes and epididymal sperm significantly decreased after BPA treatment. Concurrent treatment with MLT and BPA protected germ cell viability, evidenced by a significant increase in the viable cells associated with a significant decrease in the apoptotic and necrotic cells compared with the BPA-treated rats. To investigate the involvement of Bcl-2 in BPAinduced apoptosis, we evaluated the expression of Bcl-2 by flow cytometry, and the results are summarized in Table 4. The expression of Bcl-2 in the testes and epididymal sperm significantly decreased compared with the control groups. MLT treatment produced significant increase in the levels of Bcl-2 compared with the controls. When MLT was concurrently administered with BPA, Bcl-2 significantly showed higher values than BPA-treated rats at 3 and 6 weeks of treatment. The testis sections of the control and MLT-treated rats exhibited normal architecture with active spermatogenesis and prominent interstitial cellularity (Figure 2(a) and (b)). The testes of rats treated with BPA for 3 weeks displayed vacuolization, sloughing, and reduction of spermatogenic cells (Figure 2(c)). In addition, the testes of 6-week BPA-treated rats displayed extensive histopathological alterations including atrophy with significant loss of spermatogenesis in most of the seminiferous tubules, marked vacuolization,

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MDA (nmol/g tissue) GSH (mg/g tissue) SOD (U/g tissue) CAT (KU/g tissue) H2O2 (mM/g tissue) MDA nmol/108 cell GSH (mg/108 cell) SOD (U/108 cell) CAT (KU/108 cell) H2O2 (mM/108 cell)

56.22 + 0.46 + 41.16 + 8.92 + 1.047 + 9.26 + 0.114 + 9.417 + 0.931 + 0.260 + 3.7 0.030 2.27 0.31 0.05 0.12 0.006 0.52 0.045 0.004

58.57 0.48 52.79 8.66 1.019 9.28 0.116 9.486 1.160 0.264

+ 4.65 + 0.032 + 4.52b + 0.44 + 0.02 + 0.70 + 0.015 + 0.49 + 0.031b + 0.035

MLT + 20.86b + 0.036b + 4.88b + 0.50b + 0.02b + 3.12b + 0.001b + 0.34b + 0.023b + 0.203b

153.73 0.32 27.32 2.87 1.443 40.85 0.082 4.891 0.613 1.070

3.33b 0.038 2.38b 0.90b 0.04b 4.84b 0.008b 0.52b 0.017b 0.015b

168.69 + 0.41 + 35.97 + 4.77 + 1.13 + 23.86 + 0.087 + 6.941 + 0.653 + 0.421 +

BPA (6 weeks)

BPA (3 weeks) 120.16 0.54 44.24 8.34 1.079 8.80 0.116 9.11 1.077 0.264

+ 6.06b,c + 0.032bc + 4.39 + 0.66c + 0.14c + 0.28c + 0.005c + 0.60c + 0.131c + 0.008c

BPA þ MLT (3 week) 71.17 0.47 40.70 7.22 1.084 12.41 0.114 9.564 1.059 0.265

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Viable Early apoptotic Late apoptotic Necrotic Viable Early apoptotic Late apoptotic Necrotic

76.3 17.1 3.8 2.8 70.2 23.4 3.3 3.2

+ 3.9 + 0.46 + 0.65 + 0.21 + 2.6 + 3.41 + 0.97 + 0.32

80.7 + 13.7 + 3.0 + 2.6 + 74.7 + 18.3 + 4.18 + 2.8 + 2.7 2.14 0.26 0.37 3.5 3.65 0.83 0.11

MLT

2.9b 0.69 0.96b 1.51b .84b 1.84 1.25b 0.9b 21.5 + 22.7 + 38.6 + 17.2 + 15.5 + 23.4 + 41.6 + 19.5 +

+ 2.7b + 1.73 + 1.29b + 0.38b + 5.5b + 1.24 + 1.19b + 0.60b 31.4 20.7 33.8 14.1 21.7 28.4 32.6 17.3

BPA (6 weeks)

BPA (3 weeks)

MLT: melatonin; BPA: bisphenol A. a Data are summarized and presented as the means + SD of two independent experiments. b p < 0.05: significant as compared to control group. c p < 0.05: significant as compared to BPA 3-week group. d p < 0.05: significant as compared to BPA 6-week group.

Sperm

Testis

Control

60.7 18.6 16.7 4.0 68.4 20.1 6.8 4.7

+ 4.4c + 2.12 + 2.62b,c + 0.53c + 2.3c + 1.55c + 0.95c + 0.41c

BPA þ MLT (3 weeks)

56.2 21.7 16.8 5.3 67.6 19.8 6.6 4.7

+ + + + + + + +

6.1b,d 2.07 1.52b,d 0.28d 6.0d 1.37 0.06d 0.26d

BPA þ MLT (6 weeks)

Table 3. The flow cytomertic data of the effect of MLT and BPA on the percentage of apoptosis in testis and epididymal sperm in adult male rats.a

+ 6.17b,d + 0.035d + 5.14d + 0.69d + 0.04d + 0.46b,d + 0.009d + 0.41d + 0.157d + 0.011d

BPA þ MLT (6 weeks)

MLT: melatonin; BPA: bisphenol A; MDA: malondialdehyde; GSH: reduced glutathione; SOD: superoxide dismutase; CAT: catalase; H2O2: hydrogen peroxide. a Values are expressed as mean + SD, n ¼ 6. b p < 0.05: significant as compared to control group. c p < 0.05: significant as compared to BPA 3-week group. d p < 0.05: significant as compared to BPA 6-week group.

Sperm

Testis

Control

Table 2. Effect of MLT and BPA on the levels of oxidative stress and antioxidants in testis and epididymal sperm in adult male rats.a

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Table 4. The flow cytomertic data of the effect of MLT and BPA on the percentage of Bcl-2 analyzed by flow cytometry in testis and epididymal sperm in adult male rats.a Control Testis Sperm

BPA (3 week) BPA (6 week) BPA þ MLT (3 weeks) BPA þ MLT (6 weeks)

MLT

16.3 + 1.36 16.7 + 1.4 78.1 + 4.69 80.5 + 5.56

12.0 + 2.6b 35.6 + 2.54b

9.3 + 1.3b 16.4 + 3.82b

14.3 + 2.1 52.7 + 3.53b,c

12.5 + 1.5b,d 43.6 + 5.41b,d

MLT: melatonin; BPA: bisphenol A. a Data are summarized and presented as the means + SD of two independent experiments. b p < 0.05: significant as compared to control group. c p < 0.05: significant as compared to BPA 3-week group. d p < 0.05: significant as compared to BPA 6-week group.

degeneration, sloughing, and reduction of spermatogenic cells. Moreover, interstitial hemorrhage, vacuolated, degenerated, and poorly developed Leydig cells were noticed (Figure 2(d)). The administration of MLT concurrently with BPA for 3 and 6 weeks markedly ameliorated BPA-induced histopathological effects (Figure 2(e) and (f)). The seminiferous tubules showed increases in the germinal cell population with active spermatogenesis and normal arrangement of spermatogenic cell compared with BPA-treated rats. Also the Leydig cells population and other structures were apparently normal.

Discussion There is an increasing concern regarding BPAinduced health effects among the general population. Meanwhile, oxidative stress and apoptosis are pathophysiological factors linking infertility to an exposure to environmental pollutants. In this study, BPA brought about significant increases in the levels of MDA and H2O2 and decreases in the levels of GSH, SOD, and CAT in the testes and epididymal sperm. This is associated with a significant reduction in the sperm quality parameters, suggesting an association between oxidative stress and reproductive perturbation due to BPA exposure. These findings agree with other studies (Kabuto et al., 2003; Rashid et al., 2009) and further indicate that the BPA-treated rats were under higher levels of oxidative stress than the control animals. This might be attributed to a direct action of BPA on the antioxidant synthesis pathways and/or an indirect function of the overwhelming response of BPA-stimulated generation of reactive oxygen species (ROS) leading to oxidative damage of cellular biomolecules. Testes are considered to be highly sensitive to oxidative stress because of the presence of abundant polyunsaturated fatty acids (Aitken and Roman, 2008). BPA can cause oxidative stress in

tissues and cells by increasing hydroxyl radical formation through disturbing the redox status between quinone and hydroquinone forms of BPA (Kabuto et al., 2003; Obata and Kubota, 2000). It is hypothesized that if oxidative stress is involved in the origin of BPA-induced reproductive toxicity, then successful antioxidant treatment should prevent that toxicity. In this study, MLT significantly ameliorated oxidative stress and reproductive indicators. This is verified by marked decreases in the levels of MDA and H2O2, normalization of the antioxidants levels with protection of testicular histological structure and semen quality in the MLT þ BPA-treated rats. MLT is attracting increased attention in recent years due to its known ability to reduce oxidative stress (El-Missiry et al., 2007; Othman et al., 2008), with negligible toxicity even in very high doses (El-Sokkary et al., 2005; Gurpinar et al., 2012; Reiter et al., 2013). MLT is not only an effective hydroxyl radical scavenger (Reiter et al., 2004) but also has the capacity to detoxify other ROS and reactive nitrogen species as well as their metabolites, peroxynitrous acid, and intermediates H2O2 (Tan et al., 2002). Moreover, MLT enhances the antioxidant potential of the cell by the upregulation of several antioxidant enzymes (Pandi-Perumal et al., 2013; Fischer et al., 2013). Since it was shown to stimulate -glutamylcysteine synthetase (Urata et al., 1999), it can be stated that MLT is vital for GSH homeostasis within the cell and it can also determine the total amount of cellular GSH. In addition, one vital role of MLT may be complementary in function to CAT and GPx in keeping intracellular H2O2 concentrations at steady-state levels (Tan et al., 2000).Thus, MLT is capable of ameliorating BPAinduced oxidative stress and perturbation of redox status in the testes and sperm. This study showed that melatonin treatment is able to increase the activity of SOD in testes but not in epididymal sperm. Depending on the tissue, the ability to

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Figure 2. Testis histopathology of rats treated with MLT and/or BPA. (a) Testis section of normal control rat showing well-developed seminiferous tubules with active spermatogenesis and prominent interstitial cellularity. (b) Testis section of MLT-treated rat showing no pathological patterns in morphology of seminiferous tubules was observed compared to that of the control. In addition, the process of spermatogenesis was not interrupted. (c) Testis section of 3-week BPAinjected rats illustrating vacuolization (V) and sloughing of SSC. (d) Testis section of 6-week BPA-injected rats showing atrophy of the seminiferous tubules (A); marked vacuolization (V); sloughing ofSSC; interstitial hemorrhage (H); and vacuolated, degenerated, and poorly developed LC. (e and f) Testes sections of 3- and 6-week MLT þ BPA group are displaying protection of seminiferous tubules and spermatogenesis in most of the seminiferous tubules by MLT. MLT: melatonin; BPA: bisphenol A; SSC: spermatogenic cells; LC: Leydig cells.

recruit exogenous melatonin varied, so that tissues and cells that accumulated more melatonin presented higher SOD activity and expression. This also is probably involving differences in melatonin receptors in terms of type and number in different tissues and cells, although this suggestion remains to be discovered.

Concomitant with oxidotoxicity in the testis and epididymal sperm, there were significant decreases in the sperm chromatin condensation in BPA-treated rats, designating that altered redox state is involved in this effect. It is established that the oxidation and reduction of sulfhydryl groups is critical to sperm chromatin condensation/de-condensation process,

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and GSH is an intermediary in sperm condensation (Aitken and Vernet, 1998). It has been proposed that BPA may either affect DNA synthesis in germinal cells or interfere with the normal replacement of nuclear histones in germ cells for transition proteins and cysteine-rich protamines in spermatids during sperm chromatin condensation (Chapman and Michael, 2003). In addition, semen samples obtained from epididymis of BPA-treated rats characterized by increased percentage of diploid germ cells. Tubulin and microtubules are targets of BPA, potentially leading to disturbance of chromosome segregation and aneuploidy (George et al., 2008). Several studies have indicated that BPA exerted transforming and genotoxic effects, such as aneuploidy and DNA adduct formation (Nakamura et al., 2010; Tsutsui et al., 1998; Wu et al., 2013). BPA exposure resulted in impaired chromosome synapsis and altered meiotic DNA double-strand break repair progression, (Allard and Colaiacovo, 2010). Thus, this data suggest a link between aneuploidy occurrence and elevated oxidative stress in germ cells due to BPA exposure. In this study, MLT markedly protected sperm chromatin condensation process, and the percentage of haploid cells increased with marked normalization of the antioxidant levels’ cellular oxidative stress. This indicates that controlled redox state is essential for proper sperm production. Since fertility in mammals involves interplay between MLT and the antioxidant defense system (Casao et al., 2010), it is suggested that MLT has a protective role in the process of sperm chromatin condensation/de-condensation during sperm maturity. A significant decrease in the serum testosterone level with marked testicular histological abnormalities and lesions was demonstrated in the BPAtreated rats. The reduced level of the testosterone has been suggested as being caused by the oxidative damage to the Leydig cell population (Aguilar-Mahecha et al., 2002) and might be related to the decreased numbers of Leydig cells (Nakamura et al., 2010). Furthermore, the exposure to BPA or its fluorinated derivative resulted in a dramatic decline in genes and protein involved in cholesterol biosynthesis, transport, and steroid biosynthesis (Feng et al., 2012) and caused a failure of spermatogenesis (Qiu et al., 2013). The concurrent treatment with MLT and BPA ameliorated the reduction in the testosterone level and sperm parameters. The amelioration of histopathological signs in the testes of MLT þ BPA-treated rats lends credence to MLT’s protection against the

primary causes of damage of the testis. Because testosterone plays a key role in the process of spermatocyte meiosis (Jin et al., 2013) and the relationship between MLT and testosterone has been well documented (Casao et al., 2010), it is suggested that modulation of the testosterone level by MLT potentially protected the meiosis of spermatocytes and production of normal sperm quality, and this effect might be attributed to its capacity to control cellular redox state. This study demonstrated a significant increase in the numbers of apoptotic and necrotic cells in the testes and semen of BPA-treated rats than controls. These results are supported by significant reductions in the antiapoptotic protein Bcl-2 levels in the same cells. It is suggested that in response to BPAinduced oxidative stress, germ cells were sent into apoptosis. When DNA subjected to damage cells turned to cell cycle arrest to give sufficient time for DNA repair mechanisms to function and to trigger apoptotic mechanisms to eliminate cells with damaged DNA. The present findings are consistent with the recent studies that reported an increase in the expression of Bax and concomitantly decreased the expression of Bcl-2 levels at both protein and mRNA levels following BPA exposure (Liu et al., 2013; Xu et al., 2002; Zatecka et al., 2013). In addition, BPAinduced germ and sertoli cell apoptosis through not only the mitochondrial apoptotic pathway but also the Fas/FasL signaling pathway (Iida et al., 2003; Li et al., 2009b; Wang et al., 2010). MLT concurrently administered with BPA normalized apoptosis and redox balance in the testis and sperm. The effect of MLT on apoptosis was significant compared with the BPA only-treated rats, and it may have a major contribution in protecting the testicular cells against BPA-induced reproductive toxicity because the number of apoptotic cells was low, while viable cells were higher in MLT þ BPAtreated rats than in BPA-treated rats. The small size and chemical nature of MLT give the extra advantage that it can easily cross biological membranes, thus, reaching all compartments of the cell including the cytosol, mitochondria, and nucleus to prevent damage of DNA (El-Sokkary et al., 2005) and promote signaling function (Luchetti et al., 2010). This study supports the association between MLT’s antioxidative and antiapoptotic function. In confirmation of this suggestion, MLT not only reduced oxidative stress but also upregulated Bcl-2 expression and attenuated the apoptotic effect of BPA, suggesting

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that the apoptosis-inhibiting effect of MEL involved the control of mirochondrial pathways. MLT also played a role in modulation of Bax, Bcl-2, and P53 expression levels to protect rat kidney (Pedreanez et al., 2004) peripheral blood lymphocytes (Mohseni et al., 2012) and murine embryonic stem cells against apoptosis (Yoo et al., 2011). Meanwhile, MLT was able to decrease superoxide anion production, mitochondrial damage, and caspase-dependent apoptosis by improved Bcl-2 levels and decreased cytochrome c release in the cytoplasm in human monocytic U937 cells irradiated with ultraviolet B light (Luchetti et al., 2009). Other reports indicated that Bcl-2 might contribute to the protection against oxidant-induced cell apoptosis or increase cellular redox capacity. It is reported that Bcl-2 exerted antiapoptotic and antinecrotic effects by its influence on cellular redox state and intracellular ROS (Kowaltowski and Fiskum, 2005). Bcl-2 expression also correlates with protection against the depletion of cellular GSH (Meredith et al., 1998). Furthermore, Bcl-2-overexpression increased the antioxidant capacity of neural cell lines through elevation of either catalase, glutathione peroxidase, glutathione reductase, or GSH and nicotinamide adenine dinucleotide phosphate-oxidase (Kowaltowski and Fiskum, 2005). Thus, MLT in the pharmacological dose protected germ cells from BPA-induced oxidative stress and apoptosis by regulating the redox state as well as modulating the antiapoptotic and redox-sensitive element Bcl-2. In conclusion, MLT protected the testis and epididymal sperm quality against BPA-induced oxidative damage and apoptosis. The protective mechanism of MLT involved integration between its ability to control cellular redox balance and its antiapoptotic action. It is anticipated that MLT supplementation might not only protect male fertility but also assist normal fertilization, and it prevents the transfer of defective paternal DNA to the progeny. Acknowledgments Facilities provided by Faculty of Science, Mansoura University, Egypt, are greatly acknowledged and appreciated.

Conflict of interest The authors declared no conflicts of interest.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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