Article
Protective effects of grape seed extract on cadmium-induced testicular damage, apoptosis, and endothelial nitric oxide synthases expression in rats
Toxicology and Industrial Health 1–9 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714566874 tih.sagepub.com
Mehmet Fatih So¨nmez and Simge Tascioglu Abstract This study aims to evaluate the protective effect of grape seed proanthocyanidin extract (GSPE) on cadmium (Cd)-induced testicular apoptosis, endothelial nitric oxide synthases (eNOS) expression, and toxicity in rats. A total of 24 male Wistar rats were divided into four groups, namely, control, Cd (2.5 mg/kg), Cd þ GSPE (100 mg/kg/day), and GSPE. Spermatogenesis and mean seminiferous tubule diameter were significantly decreased in the Cd groups. Furthermore, the GSPE-treated animals showed an improved histological appearance in the Cd group. The immunoreactivity of eNOS and the number of apoptotic cells were increased in Cd group. Our data indicate a significant reduction of terminal deoxynucleotide transferase-mediated 20 -deoxyuridine 50 -triphosphate nick end-labeling staining and a decrease in the expression of eNOS in the testes tissue of the Cd group treated with GSPE therapy. Therefore, our results suggest that GSPE acts as a potent protective agent against Cd-induced testicular toxicity in rats. Keywords Cadmium, grape seed proanthocyanidin extract, eNOS, apoptosis, testis
Introduction A great number of environmental toxicants have been shown to adversely affect spermatogenesis in rodents and humans, which can lead to low sperm count, abnormal sperm morphology, and poor semen quality (Aitken et al., 2004; Sharpe, 2010). Cadmium (Cd) is one of the major industrial and environmental toxicants. Growing evidence has clearly demonstrated that environmental exposure to Cd is associated with male infertility and poor semen quality in humans (Ji et al., 2011; Pant et al., 2003). Also Cd has been reported to cause apoptosis in testicular germ cells (Kim and Soh, 2009; Ozawa et al., 2002), interstitial edema, hemorrhage, and necrosis (Ji et al., 2012). According to several earlier studies, Cd-induced male reproductive damage was associated with oxidative stress in testes (Ates et al., 2004; Oteiza et al., 1999). Recent studies found that several antioxidants have been shown to protect against Cd-induced testicular oxidative stress and male reproductive damage (Acharya et al., 2008; Ji et al., 2012). Grape seed proanthocyanidin extract (GSPE) has been
demonstrated to exhibit a broad spectrum of pharmacological, therapeutic, and chemoprotective properties. GSPE is known to have greater antioxidant activity than several well-known antioxidants, including vitamin C, vitamin E, and gallic acid (Ariga, 2004). GSPE has been shown in animal models to prevent the development of many diseases, such as diabetic nephropathy, drug-induced renal toxicity, cancer metastasis, fungal infection, and ischemic cardiomyopathy (Bayatli et al., 2013; Guler et al., 2011; Li et al., 2008; Mantena et al., 2006; Ulusoy et al., 2012). However, its precise role in Cd-induced testicular damage and therapeutic potential has not been thoroughly investigated.
Department of Histology and Embryology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Corresponding author: Mehmet Fatih So¨nmez, Department of Histology and Embryology, Faculty of Medicine, Erciyes University, 38039 Kayseri, Turkey. Email: [email protected]
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Nitric oxide (NO) is a potent biologic mediator with different physiological and pathophysiological roles. NO is synthesized from L-arginine by a family of NO synthases (NOS) (Rath et al., 2014). There are three isoforms including the constitutively expressed neuronal NOS (nNOS/NOS-I) and endothelial NOS (eNOS/NOS-III) and a third inducible isoform NOS (iNOS/NOS-II) (Rath et al., 2014; Sonmez et al., 2009a, 2009b). NO exerts an inhibitory effect on testicular steroidogenesis (Adams et al., 1992). The eNOS is expressed in Leydig cells and Sertoli cells, spermatocytes, and spermatids in the testis (Zini et al., 1998). The increasing level of NOS expression causes excessive NO production (Taneli et al., 2005), and as a result, NO at high concentrations may lead to cell death (Lue et al., 2003; Sonmez et al., 2012). Therefore, testicular NOS concentration may define prognosis after Cd-induced injury. In this study, we investigated the effects of GSPE on Cd-induced testicular damage, eNOS expression, and germ cell apoptosis in rats. We demonstrate for the first time that GSPE has protective effects on Cd-induced germ cell apoptosis in rat testes.
Materials and methods Animals Sexually mature male, 8 weeks old, Wistar rats (245 + 10 g), obtained from the Hakan C¸etinsaya Experimental and Clinic Research Center, Erciyes University, Kayseri, Turkey, were used for the study. Animals were housed in plastic cages placed in a well-ventilated rat house and allowed ad libitum access to rat chow and water and subjected to natural photoperiod of 12-h light:12-h dark cycle. All the animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health, Bethesda, Maryland, United States. The ethic regulations have been followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments (Public Health Service (PHS), 1996). The rats were randomly assigned to four groups with six rats in each group. The groups include control, Cd, Cd þ GSPE, and GSPE. In the Cd group, 2.5 mg/kg Cd (intraperitoneal (i.p.)) was administered to rats (Santos et al., 2005); in the Cd þ GSPE group, 2.5 mg/kg (i.p) Cd with 100 mg/kg/day GSPE (oral gavage) were administered to rats; and in the GSPE group, 100 mg/kg/day
GSPE (oral gavage) was administered to rats. GSPE (General Nutrition Corporation, Pittsburgh, Pennsylvania, USA) was freshly prepared and administered daily and presented at the same time of the day (10:00 a.m.). On the 8th day of the study, Cd (20298, Sigma-Aldrich, Taufkirchen, Germany) was given to rats. On the 10th day (48 h later since Cd was administered), animals were killed by decapitation under i.p ketamine (75 mg/kg) þ xylazine (10 mg/ kg) anesthesia. After decapitation, testes tissues were quickly removed, fixed in formalin, and embedded in paraffin. Five micrometer thick sections were stained with hematoxylin–eosin and Masson’s trichrome and photographs were taken with a photomicroscope (Olympus BX-51, Japan).
Histopathological evaluation The testicular tissue was examined and evaluated in random order under blindfold conditions with standard light microscopy by a histologist. Three slides prepared from the upper, lower, and midportions of the testes were evaluated for each testis. Mean seminiferous tubule diameters (MSTDs) of each animal were measured in micrometers. More than 20 seminiferous tubular sections per testis were given a Johnsen’s score (mean testicular biopsy score (MTBS) from 1 to 10 as described previously (Johnsen, 1970). In this system of classification, all tubular sections in each section of the testicular biopsy are evaluated systematically and each is given a score from 1 to 10. Complete spermatogenesis with many spermatozoa present is evaluated as score 10.
TUNEL assay In situ detection of apoptosis was performed in the testes by terminal deoxynucleotide transferasemediated 20 -deoxyuridine 50 -triphosphate nick endlabeling (TUNEL) using In situ Cell Death Detection Kit, AP (Roche, Salt Lake City, Utah, USA) according to manufacturer’s instructions. Briefly, the paraffin-embedded sections of testes tissues were deparaffinized, rehydrated, and permeabilized with proteinase K (S3020, Dako, Glostrup, Denmark) that was added on each section, and the slides were incubated in a humidified atmosphere for 30 min at 37 C in the dark. The specimens were washed in phosphate-buffered saline (PBS) three times (5 min each time). Fifty microliters of TUNEL reaction mixture were added into each slide and incubated in a humidified atmosphere for 60 min at 37 C in the dark.
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The sections were then washed in PBS three times and incubated with Converter-AP (Roche, Salt Lake City, Utah, USA) humidified chamber for 30 min at 37 C. Then, sections were incubated with Fast Red (F4648, Sigma-Aldrich) for 4 min. Finally, the sections were rinsed in PBS and mounted with glycerol. In order to estimate the apoptotic index, TUNEL-positive cells in seminiferous tubules in 20 randomly chosen fields were counted. The apoptotic index was calculated as the percentage of TUNEL-positive cells.
Immunohistochemistry The expression of eNOS was detected immunohistochemically in the testes using a rabbit polyclonal antibody (sc.654, Santa Cruz Biotechnology, Santa Cruz, California, USA) and the streptavidin–biotin peroxidase technique as described previously (Ozan et al., 2007). The procedure was performed under identical conditions for all sections. Paraffin sections (5 mm) were deparaffinized in xylene. The sections were rehydrated, rinsed in deionized water, and antigen retrieval was carried out by microwave treatment in 0.01 M sodium citrate buffer (pH 6.0) at 95 C for 5 min, and then the slides were cooled rapidly to room temperature for 20 min. After sections were washed with PBS, endogenous peroxidase activity was inhibited with 3% hydrogen peroxide in methanol for 10 min. Five percent serum blocking was used to block the nonspecific staining. The histological sections were then incubated with the polyclonal antibody for eNOS (sc.654, Santa Cruz Biotechnology) at a dilution of 2.5 mg/mL in 5% serum blocking overnight at 4 C. After washing with PBS, sections were incubated with the biotinylated secondary antibodies. Then the immunoreaction was amplified with streptavidin–avidin–peroxidase complex, and the sections were visualized using 3,30 -diaminobenzidine tetrahydrochloride and lightly counterstained with hematoxylin. Negative controls, where incubation with the primary antisera was omitted, were completely unlabeled. Immunohistochemical staining was scored in a semiquantitative manner to determine the differences between the control group and the experimental groups. The intensity of the staining was recorded as weak (þ/), mild (þ), moderate (þþ), and strong (þþþ). This analysis was performed in at least 10 tubuli per testicular section, in five sections from each animal at 400.
Statistical analysis One-way analysis of variance and post hoc Tukey test were used to determine differences between groups. Results are presented as mean + SEM. Values were considered statistically significant if p < 0.05. The Statistical Package for Social Sciences (SPSS)/PC program (Version 15.0; SPSS Inc., Chicago, Illinois, USA) was used for the statistical analysis.
Results Histopathological findings Control (Figure 1(a)) and GSPE-administered (Figure 1(b)) groups showed the presence of normal testicular architecture and regular seminiferous tubular morphology with normal spermatogenesis and the presence of primary and secondary spermatocytes, spermatids, and spermatozoa. Congestion of vessels and hemorrhage, formation of the vacuoles in epithelial cells (Figure 1(c)), desquamation of epithelial cells in the lumen, disorder of seminiferous tubule germinal epithelium, multinucleated giant cells (Figure 1(d)), and necrosis of some seminiferous tubules (Figure 2(a)) were determined in the Cd group. Also mononuclear cell infiltration in some fields was observed (Figure 2(b)). Cd þ GSPE group showed lesser atrophic and degenerative changes to the tubular epithelium when compared with the Cd group. The seminiferous tubules in many of the sections showed near-normal architecture, with different layers of spermatogenic cells up to the spermatids. No vacuoles were observed in the germinal epithelium. GSPE clearly prevented Cd-induced histopathological damage in the testes (Figure 2(c)). The MSTD and Johnsen’s MTBS values for testes in each group are shown in Table 1. It was observed that the MSTD and MTBS of the Cd-induced testes were statistically significantly lower than the control rats. However, prevention effect was determined with the administration of GSPE in the Cd-induced group. The MSTD and Johnsen’s MTBS in the GSPE alone group were similar to that in the control group (Table 1).
Apoptotic findings Table 1 and Figure 2(d) illustrate apoptosis as demonstrated by TUNEL staining. The mean apoptotic index in the testis of control rats was found to be 0.20 + 0.05%. Cd-induced resulted in the increase of the number of TUNEL-positive cells and the apoptotic index was 1.78 + 0.18%. The increase in the
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Figure 1. Light microscopy of testicular tissue in different groups. (a) In control and (b) GSPE groups, normal testicular architecture was observed; (c) in Cd-induced group, congestion of vessels (thin arrow), hemorrhage (h), and formation of the vacuoles (thick arrow) in epithelial cells were exhibited; and (d) again in Cd-induced group, multinucleated giant cells (arrow) and the formation of the vacuoles (asterisk) in epithelial cells were observed. Testicular cross sections were stained with H&E and Masson’s trichrome. Cd: cadmium; GSPE: grape seed proanthocyanidin extract; H&E: hematoxylin–eosin.
apoptotic index was statistically significant in Cd group compared with the control group (p < 0.05). Cd þ GSPE group resulted in the decrease of the number of TUNEL-positive cells and the apoptotic index was 0.45 + 0.19%. The decrease in the apoptotic index was statistically significant in Cd þ GSPE group compared with the Cd group (p < 0.05). The apoptotic index in the testes of GSPE group was found to be 0.27 + 0.12%, and it was similar to the control.
in Leydig cells of Cd group (Figure 3(b)). GSPE treatment prevented testicular damage and eNOS immunoreactivity was close to the control ones (Figure 3(c)). The eNOS expression in the testes of GSPE group was similar to that in the control. Negative controls that were incubated with the primary antisera were completely unlabeled, as shown in Figure 3(d).
Discussion Immunohistochemical findings Table 2 summarized the expression of eNOS levels. Immunohistochemical studies demonstrated the presence of slight eNOS immunostaining in the germinal cells of the seminiferous tubules and moderate immunostaining of Leydig cells and vascular endothelial cells of the testes in the control group (Figure 3(a)). While eNOS immunoreactivity was significantly increased especially in germinal cells, it was decreased
This study reports the potential protective effects of GSPE against Cd-induced toxicity by studying the changes in seminiferous tubules and testicular interstitium, changes in MSTD and MTBS values, number of apoptotic germ cells, and eNOS expression. The latest studies have shown reduced male fertility, such as decreased sperm count and poor semen quality, in men exposed to Cd and/or other environmental toxicants (Benoff et al., 2000; Siu et al., 2009). In our
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Figure 2. Light microscopy of testicular tissue in different groups. (a) Necrosis of some seminiferous tubules (asterisk) were observed after Cd induction; (b) mononuclear cell infiltration (arrow) in some fields was noted after Cd induction; (c) GSPE pretreatment prevented testicular damage; and (d) after Cd induction, TUNEL-positive germ cells (arrow) were observed in the seminiferous epithelium. Cd: cadmium; GSPE: grape seed proanthocyanidin extract; TUNEL: terminal deoxynucleotide transferase-mediated 20 -deoxyuridine 50 -triphosphate nick end-labeling. Table 1. Apoptotic cells number, MSTD, and MTBS levels of control, Cd, Cd þ GSPE, and GSPE groups (n ¼ 6 for each group).a
MSTD (m) MTBS Apoptotic cells
Control
Cd
Cd þ GSPE
GSPE
319.66 + 27.76 9.36 + 1.79 0.20 + 0.05
225.73 + 55.52b 5.11 + 2.68b 1.78 + 0.18b
301.03 + 35.81c 8.31 + 1.74c 0.45 + 0.19c
336.45 + 42.80 9.16 + 1.59 0.27 + 0.12
Cd: cadmium; GSPE: grape seed proanthocyanidin extract; MSTD: mean seminiferous tubule diameter; MTBS: mean testicular biopsy score. a Values are expressed as mean + SD. b p < 0.05: compared with control group. c p < 0.05: compared with control and Cd-treated group.
study, we examined the effect of Cd on the testicular histology of the rats. Our results clearly showed that Cd severely damaged the seminiferous tubules and interstitium, including the congestion of vessels and hemorrhage, the formation of the vacuoles in epithelial cells, the desquamation of epithelial cells in the lumen, the disorder of seminiferous tubule germinal epithelium, multinucleated giant cells, and necrosis
of some seminiferous tubules. Also, MSTD and MTBS of the Cd group were statistically significantly lower compared with the control rats. The formation of multinucleated giant cells in Cd-intoxicated rats indicates the continuous degeneration of the spermatogenic epithelium and appears to represent a nonspecific reaction to injury. The giant cells are proposed to be a result of the inability of primary spermatocytes to
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Table 2. eNOS expression of control, Cd, Cd þ GSPE, and GSPE groups (n ¼ 6 for each group).a
eNOS (germ cells) eNOS (Leydig cells)
Cd þ GSPE GSPE
Control
Cd
þ þþ
þþþ þ
þ þþ
þ þþ
eNOS: endothelial nitric oxide synthases; Cd: cadmium; GSPE: grape seed proanthocyanidin extract. a Intensity of staining was recorded as weak (þ/), mild (þ), moderate (þþ), and strong (þþþ).
undergo meiotic divisions to generate haploid sperm cells, which undergo additional DNA replication and giving rise to multinucleated giant cells (Prabu et al., 2010). Our findings are consistent with other studies and further support their observations (Aktoz et al., 2010; Batra et al., 1998; Klaassen et al., 1999). At present, it is well established that testicular oxidative stress usually induced under different normal and/or pathophysiological conditions give rise to male infertility. Oxidative stress is a common factor in about half of the infertile men examined to date, demonstrating the importance of Cd as an inducer of oxidative stress (Siu et al., 2009). Therefore, it is reasonable to assume that antioxidant agents (enzymatic and nonenzymatic) may prevent or at least diminish the Cd toxicity to the testes. Indeed, several studies with substances having antioxidant activities, such as vitamin C, vitamin E, zinc, selenium, and melatonin, have also exhibited that the oxidative stress was associated with the Cd-induced testicular damage, as these substances reduced and/or prevented both the oxidative stress and the damage caused by Cd in the testes (Acharya et al., 2008; Amara et al., 2008; Hu et al., 2004; Kara et al., 2007; Niewenhuis and Fende, 1978) Proanthocyanidins are powerful naturally occurring polyphenolic antioxidants widely available in fruits, vegetables, seeds, nuts, flowers, and bark. Proanthocyanidins are known to possess antibacterial, antiviral, anti-inflammatory, antiallergic, and vasodilatory actions. They have also been shown to inhibit lipid peroxidation, platelet aggregation, capillary permeability, and fragility (Chen et al., 1996; Hanefeld and Herrmann, 1976). In this study, GSPE was employed as a novel antioxidant, and its protective effect was evaluated. In a recent study, GSPE were investigated on ischemia–reperfusion injury of the liver. Authors have identified that GSPE application has well-preservative effects on liver parenchyma (Sehirli et al., 2008). Su et al. (2011) in their study
investigated the effect of GSPE on testicular tissue that was already damaged with nickel sulfate. They identified that GSPE corrects the sperm motility and reduces the oxidative damage and blocks apoptosis (Su et al., 2011). Oxidative stress seems to be crucial in the etiology of Cd-induced male reproductive toxicity in humans and animals. Thus, antioxidant therapy is considered to be an important approach for the intervention of Cd toxicity. For this reason, in this study we have implemented GSPE for protective purposes. When Cd and GSPE were applied to the rats, the big portion of the seminiferous tubules of the testis was seen as normal. Seminiferous tubular diameters, tubule, and biopsy scores were determined to be close to the controls with the implementation of the Cd þ GSPE group. Our results are in agreement with earlier studies indicated protective effect of GSPE. Apoptosis is a highly regulated process that is used to eliminate unwanted or damaged cells in multicellular organisms. Several studies showed that Cd administration into adult rodents resulted in germ cell apoptosis in testes (Aktas et al., 2012; Ozawa et al., 2002; Zhou et al., 1999). In this study, we investigated the effects of GSPE on Cd-induced germ cell apoptosis in testes. As anticipated, a single dose of Cd clearly elevated the percentage of tubules with apoptotic germ cells. In addition, the number of apoptotic germ cells per tubule was significantly increased in testes of rats injected with Cd. Pretreatment with GSPE significantly alleviated Cd-induced germ cell apoptosis in testes. These results are in agreement with those from a recent study, in which GSPE protected against Cdinduced germ cell apoptosis in rat testes (Su et al., 2011). Three isoforms of NOS, namely nNOS, iNOS, and eNOS, are found in the testis and are known to be associated with infertility, spermatogenesis, and sperm maturation. The increased expression of eNOS in ischemia and reperfusion injury of testis has been reported previously (Aktoz et al., 2010). However, there is no study on the expression of eNOS in Cd-induced testes. Kolluru et al. (2006) found that micromolar concentrations of Cd led to endothelial dysfunction by blocking eNOS activity. Soyupek et al. (2012) demonstrated that Cd exposure causes increased renal eNOS, iNOS, and nNOS isoforms. Similarly, we found a significant elevation in eNOS expression of germinal cells and a reduction in eNOS expression of Leydig cells in testes after Cd exposure. Ji et al. (2011) demonstrated that maternal Cd
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Figure 3. Representative photomicrographs of testicular immunostaining for eNOS in different groups. (a) In normal controls, weak eNOS immunostaining in germinal cells in the seminiferous tubules and moderate eNOS immunostaining of Leydig cells (arrow) were noted; (b) in Cd-induced group, eNOS expression was increased in the germ cells (arrow) of the tubules and was slightly decreased in the Leydig cells; (c) in Cd þ GSPE group, weak eNOS immunostaining in germinal cells in the seminiferous tubules and moderate eNOS immunostaining of Leydig cells (arrow) were exhibited; and (d) in negative control group no detectable signals were detected. The immunoreaction was amplified with immunoperoxidase and the sections were stained with hematoxylin counterstain. eNOS: endothelial nitric oxide synthases; Cd: cadmium; GSPE: grape seed proanthocyanidin extract.
exposure during the late pregnancy period inhibited testicular testosterone production in male offspring. In addition, they also showed reduced number of Leydig cells in male offspring after maternal Cd exposure. In another study, Aktas et al. (2012) showed the loss of Leydig cells after Cd treatment. In our study, the reduction of eNOS expression in the interstitial field after Cd application may be associated with the reduction of Leydig cells in this area. We also investigated the effects of GSPE on Cd-induced eNOS expression in testes. We found that the pretreatment of GSPE decreased eNOS immunoreactivity in Cd-induced testes of the rats. We have demonstrated the protective effect of GSPE on Cd-induced testicular injury for the first time. The administration of GSPE, a novel antioxidant, improved the histopathological parameters,
decreased immunoexpression of Cd-induced testicular eNOS, and decreased germ cell apoptosis in Cdtreated testes. Thus, antioxidants may be useful as pharmacological agents to protect against Cdinduced testicular injury. Acknowledgments The authors wish to thank Dr Eser Kilic¸ and Dr C ¸ ag˘rı Sakalar for technical support.
Conflict of interest The authors declared no conflicts of interest.
Funding This work was supported by a research grant from the Erciyes University Scientific Research Projects Unit (EUBAP, TSY-09-775).
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Protective effects of grape seed extract on cadmium-induced testicular damage, apoptosis, and endothelial nitric oxide synthases expression in rats
Toxicology and Industrial Health 1–9 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714566874 tih.sagepub.com
Mehmet Fatih So¨nmez and Simge Tascioglu Abstract This study aims to evaluate the protective effect of grape seed proanthocyanidin extract (GSPE) on cadmium (Cd)-induced testicular apoptosis, endothelial nitric oxide synthases (eNOS) expression, and toxicity in rats. A total of 24 male Wistar rats were divided into four groups, namely, control, Cd (2.5 mg/kg), Cd þ GSPE (100 mg/kg/day), and GSPE. Spermatogenesis and mean seminiferous tubule diameter were significantly decreased in the Cd groups. Furthermore, the GSPE-treated animals showed an improved histological appearance in the Cd group. The immunoreactivity of eNOS and the number of apoptotic cells were increased in Cd group. Our data indicate a significant reduction of terminal deoxynucleotide transferase-mediated 20 -deoxyuridine 50 -triphosphate nick end-labeling staining and a decrease in the expression of eNOS in the testes tissue of the Cd group treated with GSPE therapy. Therefore, our results suggest that GSPE acts as a potent protective agent against Cd-induced testicular toxicity in rats. Keywords Cadmium, grape seed proanthocyanidin extract, eNOS, apoptosis, testis
Introduction A great number of environmental toxicants have been shown to adversely affect spermatogenesis in rodents and humans, which can lead to low sperm count, abnormal sperm morphology, and poor semen quality (Aitken et al., 2004; Sharpe, 2010). Cadmium (Cd) is one of the major industrial and environmental toxicants. Growing evidence has clearly demonstrated that environmental exposure to Cd is associated with male infertility and poor semen quality in humans (Ji et al., 2011; Pant et al., 2003). Also Cd has been reported to cause apoptosis in testicular germ cells (Kim and Soh, 2009; Ozawa et al., 2002), interstitial edema, hemorrhage, and necrosis (Ji et al., 2012). According to several earlier studies, Cd-induced male reproductive damage was associated with oxidative stress in testes (Ates et al., 2004; Oteiza et al., 1999). Recent studies found that several antioxidants have been shown to protect against Cd-induced testicular oxidative stress and male reproductive damage (Acharya et al., 2008; Ji et al., 2012). Grape seed proanthocyanidin extract (GSPE) has been
demonstrated to exhibit a broad spectrum of pharmacological, therapeutic, and chemoprotective properties. GSPE is known to have greater antioxidant activity than several well-known antioxidants, including vitamin C, vitamin E, and gallic acid (Ariga, 2004). GSPE has been shown in animal models to prevent the development of many diseases, such as diabetic nephropathy, drug-induced renal toxicity, cancer metastasis, fungal infection, and ischemic cardiomyopathy (Bayatli et al., 2013; Guler et al., 2011; Li et al., 2008; Mantena et al., 2006; Ulusoy et al., 2012). However, its precise role in Cd-induced testicular damage and therapeutic potential has not been thoroughly investigated.
Department of Histology and Embryology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Corresponding author: Mehmet Fatih So¨nmez, Department of Histology and Embryology, Faculty of Medicine, Erciyes University, 38039 Kayseri, Turkey. Email: [email protected]
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Nitric oxide (NO) is a potent biologic mediator with different physiological and pathophysiological roles. NO is synthesized from L-arginine by a family of NO synthases (NOS) (Rath et al., 2014). There are three isoforms including the constitutively expressed neuronal NOS (nNOS/NOS-I) and endothelial NOS (eNOS/NOS-III) and a third inducible isoform NOS (iNOS/NOS-II) (Rath et al., 2014; Sonmez et al., 2009a, 2009b). NO exerts an inhibitory effect on testicular steroidogenesis (Adams et al., 1992). The eNOS is expressed in Leydig cells and Sertoli cells, spermatocytes, and spermatids in the testis (Zini et al., 1998). The increasing level of NOS expression causes excessive NO production (Taneli et al., 2005), and as a result, NO at high concentrations may lead to cell death (Lue et al., 2003; Sonmez et al., 2012). Therefore, testicular NOS concentration may define prognosis after Cd-induced injury. In this study, we investigated the effects of GSPE on Cd-induced testicular damage, eNOS expression, and germ cell apoptosis in rats. We demonstrate for the first time that GSPE has protective effects on Cd-induced germ cell apoptosis in rat testes.
Materials and methods Animals Sexually mature male, 8 weeks old, Wistar rats (245 + 10 g), obtained from the Hakan C¸etinsaya Experimental and Clinic Research Center, Erciyes University, Kayseri, Turkey, were used for the study. Animals were housed in plastic cages placed in a well-ventilated rat house and allowed ad libitum access to rat chow and water and subjected to natural photoperiod of 12-h light:12-h dark cycle. All the animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health, Bethesda, Maryland, United States. The ethic regulations have been followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments (Public Health Service (PHS), 1996). The rats were randomly assigned to four groups with six rats in each group. The groups include control, Cd, Cd þ GSPE, and GSPE. In the Cd group, 2.5 mg/kg Cd (intraperitoneal (i.p.)) was administered to rats (Santos et al., 2005); in the Cd þ GSPE group, 2.5 mg/kg (i.p) Cd with 100 mg/kg/day GSPE (oral gavage) were administered to rats; and in the GSPE group, 100 mg/kg/day
GSPE (oral gavage) was administered to rats. GSPE (General Nutrition Corporation, Pittsburgh, Pennsylvania, USA) was freshly prepared and administered daily and presented at the same time of the day (10:00 a.m.). On the 8th day of the study, Cd (20298, Sigma-Aldrich, Taufkirchen, Germany) was given to rats. On the 10th day (48 h later since Cd was administered), animals were killed by decapitation under i.p ketamine (75 mg/kg) þ xylazine (10 mg/ kg) anesthesia. After decapitation, testes tissues were quickly removed, fixed in formalin, and embedded in paraffin. Five micrometer thick sections were stained with hematoxylin–eosin and Masson’s trichrome and photographs were taken with a photomicroscope (Olympus BX-51, Japan).
Histopathological evaluation The testicular tissue was examined and evaluated in random order under blindfold conditions with standard light microscopy by a histologist. Three slides prepared from the upper, lower, and midportions of the testes were evaluated for each testis. Mean seminiferous tubule diameters (MSTDs) of each animal were measured in micrometers. More than 20 seminiferous tubular sections per testis were given a Johnsen’s score (mean testicular biopsy score (MTBS) from 1 to 10 as described previously (Johnsen, 1970). In this system of classification, all tubular sections in each section of the testicular biopsy are evaluated systematically and each is given a score from 1 to 10. Complete spermatogenesis with many spermatozoa present is evaluated as score 10.
TUNEL assay In situ detection of apoptosis was performed in the testes by terminal deoxynucleotide transferasemediated 20 -deoxyuridine 50 -triphosphate nick endlabeling (TUNEL) using In situ Cell Death Detection Kit, AP (Roche, Salt Lake City, Utah, USA) according to manufacturer’s instructions. Briefly, the paraffin-embedded sections of testes tissues were deparaffinized, rehydrated, and permeabilized with proteinase K (S3020, Dako, Glostrup, Denmark) that was added on each section, and the slides were incubated in a humidified atmosphere for 30 min at 37 C in the dark. The specimens were washed in phosphate-buffered saline (PBS) three times (5 min each time). Fifty microliters of TUNEL reaction mixture were added into each slide and incubated in a humidified atmosphere for 60 min at 37 C in the dark.
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The sections were then washed in PBS three times and incubated with Converter-AP (Roche, Salt Lake City, Utah, USA) humidified chamber for 30 min at 37 C. Then, sections were incubated with Fast Red (F4648, Sigma-Aldrich) for 4 min. Finally, the sections were rinsed in PBS and mounted with glycerol. In order to estimate the apoptotic index, TUNEL-positive cells in seminiferous tubules in 20 randomly chosen fields were counted. The apoptotic index was calculated as the percentage of TUNEL-positive cells.
Immunohistochemistry The expression of eNOS was detected immunohistochemically in the testes using a rabbit polyclonal antibody (sc.654, Santa Cruz Biotechnology, Santa Cruz, California, USA) and the streptavidin–biotin peroxidase technique as described previously (Ozan et al., 2007). The procedure was performed under identical conditions for all sections. Paraffin sections (5 mm) were deparaffinized in xylene. The sections were rehydrated, rinsed in deionized water, and antigen retrieval was carried out by microwave treatment in 0.01 M sodium citrate buffer (pH 6.0) at 95 C for 5 min, and then the slides were cooled rapidly to room temperature for 20 min. After sections were washed with PBS, endogenous peroxidase activity was inhibited with 3% hydrogen peroxide in methanol for 10 min. Five percent serum blocking was used to block the nonspecific staining. The histological sections were then incubated with the polyclonal antibody for eNOS (sc.654, Santa Cruz Biotechnology) at a dilution of 2.5 mg/mL in 5% serum blocking overnight at 4 C. After washing with PBS, sections were incubated with the biotinylated secondary antibodies. Then the immunoreaction was amplified with streptavidin–avidin–peroxidase complex, and the sections were visualized using 3,30 -diaminobenzidine tetrahydrochloride and lightly counterstained with hematoxylin. Negative controls, where incubation with the primary antisera was omitted, were completely unlabeled. Immunohistochemical staining was scored in a semiquantitative manner to determine the differences between the control group and the experimental groups. The intensity of the staining was recorded as weak (þ/), mild (þ), moderate (þþ), and strong (þþþ). This analysis was performed in at least 10 tubuli per testicular section, in five sections from each animal at 400.
Statistical analysis One-way analysis of variance and post hoc Tukey test were used to determine differences between groups. Results are presented as mean + SEM. Values were considered statistically significant if p < 0.05. The Statistical Package for Social Sciences (SPSS)/PC program (Version 15.0; SPSS Inc., Chicago, Illinois, USA) was used for the statistical analysis.
Results Histopathological findings Control (Figure 1(a)) and GSPE-administered (Figure 1(b)) groups showed the presence of normal testicular architecture and regular seminiferous tubular morphology with normal spermatogenesis and the presence of primary and secondary spermatocytes, spermatids, and spermatozoa. Congestion of vessels and hemorrhage, formation of the vacuoles in epithelial cells (Figure 1(c)), desquamation of epithelial cells in the lumen, disorder of seminiferous tubule germinal epithelium, multinucleated giant cells (Figure 1(d)), and necrosis of some seminiferous tubules (Figure 2(a)) were determined in the Cd group. Also mononuclear cell infiltration in some fields was observed (Figure 2(b)). Cd þ GSPE group showed lesser atrophic and degenerative changes to the tubular epithelium when compared with the Cd group. The seminiferous tubules in many of the sections showed near-normal architecture, with different layers of spermatogenic cells up to the spermatids. No vacuoles were observed in the germinal epithelium. GSPE clearly prevented Cd-induced histopathological damage in the testes (Figure 2(c)). The MSTD and Johnsen’s MTBS values for testes in each group are shown in Table 1. It was observed that the MSTD and MTBS of the Cd-induced testes were statistically significantly lower than the control rats. However, prevention effect was determined with the administration of GSPE in the Cd-induced group. The MSTD and Johnsen’s MTBS in the GSPE alone group were similar to that in the control group (Table 1).
Apoptotic findings Table 1 and Figure 2(d) illustrate apoptosis as demonstrated by TUNEL staining. The mean apoptotic index in the testis of control rats was found to be 0.20 + 0.05%. Cd-induced resulted in the increase of the number of TUNEL-positive cells and the apoptotic index was 1.78 + 0.18%. The increase in the
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Figure 1. Light microscopy of testicular tissue in different groups. (a) In control and (b) GSPE groups, normal testicular architecture was observed; (c) in Cd-induced group, congestion of vessels (thin arrow), hemorrhage (h), and formation of the vacuoles (thick arrow) in epithelial cells were exhibited; and (d) again in Cd-induced group, multinucleated giant cells (arrow) and the formation of the vacuoles (asterisk) in epithelial cells were observed. Testicular cross sections were stained with H&E and Masson’s trichrome. Cd: cadmium; GSPE: grape seed proanthocyanidin extract; H&E: hematoxylin–eosin.
apoptotic index was statistically significant in Cd group compared with the control group (p < 0.05). Cd þ GSPE group resulted in the decrease of the number of TUNEL-positive cells and the apoptotic index was 0.45 + 0.19%. The decrease in the apoptotic index was statistically significant in Cd þ GSPE group compared with the Cd group (p < 0.05). The apoptotic index in the testes of GSPE group was found to be 0.27 + 0.12%, and it was similar to the control.
in Leydig cells of Cd group (Figure 3(b)). GSPE treatment prevented testicular damage and eNOS immunoreactivity was close to the control ones (Figure 3(c)). The eNOS expression in the testes of GSPE group was similar to that in the control. Negative controls that were incubated with the primary antisera were completely unlabeled, as shown in Figure 3(d).
Discussion Immunohistochemical findings Table 2 summarized the expression of eNOS levels. Immunohistochemical studies demonstrated the presence of slight eNOS immunostaining in the germinal cells of the seminiferous tubules and moderate immunostaining of Leydig cells and vascular endothelial cells of the testes in the control group (Figure 3(a)). While eNOS immunoreactivity was significantly increased especially in germinal cells, it was decreased
This study reports the potential protective effects of GSPE against Cd-induced toxicity by studying the changes in seminiferous tubules and testicular interstitium, changes in MSTD and MTBS values, number of apoptotic germ cells, and eNOS expression. The latest studies have shown reduced male fertility, such as decreased sperm count and poor semen quality, in men exposed to Cd and/or other environmental toxicants (Benoff et al., 2000; Siu et al., 2009). In our
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Figure 2. Light microscopy of testicular tissue in different groups. (a) Necrosis of some seminiferous tubules (asterisk) were observed after Cd induction; (b) mononuclear cell infiltration (arrow) in some fields was noted after Cd induction; (c) GSPE pretreatment prevented testicular damage; and (d) after Cd induction, TUNEL-positive germ cells (arrow) were observed in the seminiferous epithelium. Cd: cadmium; GSPE: grape seed proanthocyanidin extract; TUNEL: terminal deoxynucleotide transferase-mediated 20 -deoxyuridine 50 -triphosphate nick end-labeling. Table 1. Apoptotic cells number, MSTD, and MTBS levels of control, Cd, Cd þ GSPE, and GSPE groups (n ¼ 6 for each group).a
MSTD (m) MTBS Apoptotic cells
Control
Cd
Cd þ GSPE
GSPE
319.66 + 27.76 9.36 + 1.79 0.20 + 0.05
225.73 + 55.52b 5.11 + 2.68b 1.78 + 0.18b
301.03 + 35.81c 8.31 + 1.74c 0.45 + 0.19c
336.45 + 42.80 9.16 + 1.59 0.27 + 0.12
Cd: cadmium; GSPE: grape seed proanthocyanidin extract; MSTD: mean seminiferous tubule diameter; MTBS: mean testicular biopsy score. a Values are expressed as mean + SD. b p < 0.05: compared with control group. c p < 0.05: compared with control and Cd-treated group.
study, we examined the effect of Cd on the testicular histology of the rats. Our results clearly showed that Cd severely damaged the seminiferous tubules and interstitium, including the congestion of vessels and hemorrhage, the formation of the vacuoles in epithelial cells, the desquamation of epithelial cells in the lumen, the disorder of seminiferous tubule germinal epithelium, multinucleated giant cells, and necrosis
of some seminiferous tubules. Also, MSTD and MTBS of the Cd group were statistically significantly lower compared with the control rats. The formation of multinucleated giant cells in Cd-intoxicated rats indicates the continuous degeneration of the spermatogenic epithelium and appears to represent a nonspecific reaction to injury. The giant cells are proposed to be a result of the inability of primary spermatocytes to
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Table 2. eNOS expression of control, Cd, Cd þ GSPE, and GSPE groups (n ¼ 6 for each group).a
eNOS (germ cells) eNOS (Leydig cells)
Cd þ GSPE GSPE
Control
Cd
þ þþ
þþþ þ
þ þþ
þ þþ
eNOS: endothelial nitric oxide synthases; Cd: cadmium; GSPE: grape seed proanthocyanidin extract. a Intensity of staining was recorded as weak (þ/), mild (þ), moderate (þþ), and strong (þþþ).
undergo meiotic divisions to generate haploid sperm cells, which undergo additional DNA replication and giving rise to multinucleated giant cells (Prabu et al., 2010). Our findings are consistent with other studies and further support their observations (Aktoz et al., 2010; Batra et al., 1998; Klaassen et al., 1999). At present, it is well established that testicular oxidative stress usually induced under different normal and/or pathophysiological conditions give rise to male infertility. Oxidative stress is a common factor in about half of the infertile men examined to date, demonstrating the importance of Cd as an inducer of oxidative stress (Siu et al., 2009). Therefore, it is reasonable to assume that antioxidant agents (enzymatic and nonenzymatic) may prevent or at least diminish the Cd toxicity to the testes. Indeed, several studies with substances having antioxidant activities, such as vitamin C, vitamin E, zinc, selenium, and melatonin, have also exhibited that the oxidative stress was associated with the Cd-induced testicular damage, as these substances reduced and/or prevented both the oxidative stress and the damage caused by Cd in the testes (Acharya et al., 2008; Amara et al., 2008; Hu et al., 2004; Kara et al., 2007; Niewenhuis and Fende, 1978) Proanthocyanidins are powerful naturally occurring polyphenolic antioxidants widely available in fruits, vegetables, seeds, nuts, flowers, and bark. Proanthocyanidins are known to possess antibacterial, antiviral, anti-inflammatory, antiallergic, and vasodilatory actions. They have also been shown to inhibit lipid peroxidation, platelet aggregation, capillary permeability, and fragility (Chen et al., 1996; Hanefeld and Herrmann, 1976). In this study, GSPE was employed as a novel antioxidant, and its protective effect was evaluated. In a recent study, GSPE were investigated on ischemia–reperfusion injury of the liver. Authors have identified that GSPE application has well-preservative effects on liver parenchyma (Sehirli et al., 2008). Su et al. (2011) in their study
investigated the effect of GSPE on testicular tissue that was already damaged with nickel sulfate. They identified that GSPE corrects the sperm motility and reduces the oxidative damage and blocks apoptosis (Su et al., 2011). Oxidative stress seems to be crucial in the etiology of Cd-induced male reproductive toxicity in humans and animals. Thus, antioxidant therapy is considered to be an important approach for the intervention of Cd toxicity. For this reason, in this study we have implemented GSPE for protective purposes. When Cd and GSPE were applied to the rats, the big portion of the seminiferous tubules of the testis was seen as normal. Seminiferous tubular diameters, tubule, and biopsy scores were determined to be close to the controls with the implementation of the Cd þ GSPE group. Our results are in agreement with earlier studies indicated protective effect of GSPE. Apoptosis is a highly regulated process that is used to eliminate unwanted or damaged cells in multicellular organisms. Several studies showed that Cd administration into adult rodents resulted in germ cell apoptosis in testes (Aktas et al., 2012; Ozawa et al., 2002; Zhou et al., 1999). In this study, we investigated the effects of GSPE on Cd-induced germ cell apoptosis in testes. As anticipated, a single dose of Cd clearly elevated the percentage of tubules with apoptotic germ cells. In addition, the number of apoptotic germ cells per tubule was significantly increased in testes of rats injected with Cd. Pretreatment with GSPE significantly alleviated Cd-induced germ cell apoptosis in testes. These results are in agreement with those from a recent study, in which GSPE protected against Cdinduced germ cell apoptosis in rat testes (Su et al., 2011). Three isoforms of NOS, namely nNOS, iNOS, and eNOS, are found in the testis and are known to be associated with infertility, spermatogenesis, and sperm maturation. The increased expression of eNOS in ischemia and reperfusion injury of testis has been reported previously (Aktoz et al., 2010). However, there is no study on the expression of eNOS in Cd-induced testes. Kolluru et al. (2006) found that micromolar concentrations of Cd led to endothelial dysfunction by blocking eNOS activity. Soyupek et al. (2012) demonstrated that Cd exposure causes increased renal eNOS, iNOS, and nNOS isoforms. Similarly, we found a significant elevation in eNOS expression of germinal cells and a reduction in eNOS expression of Leydig cells in testes after Cd exposure. Ji et al. (2011) demonstrated that maternal Cd
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Figure 3. Representative photomicrographs of testicular immunostaining for eNOS in different groups. (a) In normal controls, weak eNOS immunostaining in germinal cells in the seminiferous tubules and moderate eNOS immunostaining of Leydig cells (arrow) were noted; (b) in Cd-induced group, eNOS expression was increased in the germ cells (arrow) of the tubules and was slightly decreased in the Leydig cells; (c) in Cd þ GSPE group, weak eNOS immunostaining in germinal cells in the seminiferous tubules and moderate eNOS immunostaining of Leydig cells (arrow) were exhibited; and (d) in negative control group no detectable signals were detected. The immunoreaction was amplified with immunoperoxidase and the sections were stained with hematoxylin counterstain. eNOS: endothelial nitric oxide synthases; Cd: cadmium; GSPE: grape seed proanthocyanidin extract.
exposure during the late pregnancy period inhibited testicular testosterone production in male offspring. In addition, they also showed reduced number of Leydig cells in male offspring after maternal Cd exposure. In another study, Aktas et al. (2012) showed the loss of Leydig cells after Cd treatment. In our study, the reduction of eNOS expression in the interstitial field after Cd application may be associated with the reduction of Leydig cells in this area. We also investigated the effects of GSPE on Cd-induced eNOS expression in testes. We found that the pretreatment of GSPE decreased eNOS immunoreactivity in Cd-induced testes of the rats. We have demonstrated the protective effect of GSPE on Cd-induced testicular injury for the first time. The administration of GSPE, a novel antioxidant, improved the histopathological parameters,
decreased immunoexpression of Cd-induced testicular eNOS, and decreased germ cell apoptosis in Cdtreated testes. Thus, antioxidants may be useful as pharmacological agents to protect against Cdinduced testicular injury. Acknowledgments The authors wish to thank Dr Eser Kilic¸ and Dr C ¸ ag˘rı Sakalar for technical support.
Conflict of interest The authors declared no conflicts of interest.
Funding This work was supported by a research grant from the Erciyes University Scientific Research Projects Unit (EUBAP, TSY-09-775).
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