Quinestrol induces spermatogenic apoptosis in vivo via increasing pro-apoptotic proteins in adult male mice

Tissue and Cell 46 (2014) 318–325

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Quinestrol induces spermatogenic apoptosis in vivo via increasing pro-apoptotic proteins in adult male mice Jian Li a,b , Funing Chen c , Charles Li d , Yaoxing Chen a,∗ a

Laboratory of Veterinary Anatomy, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Laboratory of Veterinary Anatomy, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471000, China Yanjing Medical College, Capital Medical University, Beijing 101300, China d Department of Environmental Toxicology, University of California, Davis, CA 95616, USA b c

a r t i c l e

i n f o

Article history: Received 6 March 2014 Received in revised form 7 May 2014 Accepted 25 May 2014 Available online 27 June 2014 Keywords: Quinestrol Spermatogenic cell Apoptosis Oxidative stress Adult male mice

a b s t r a c t The effects of quinestrol on spermatogenesis were investigated in adult male mice by daily intragastric administration of quinestrol with various doses of 5, 10, 50 and 100 mg/kg body weight for 10 days. The sperm counts declined while the number of abnormal spermatozoa went up in mice treated with quinestrol. The testicular weight and seminiferous tubular area gradually declined with increasing dosages of quinestrol to 50 and 100 mg/kg. Rarefied germ cells showed irregular distributions in the seminiferous tubules of mice treated with 50 and 100 mg/kg quinestrol. Apoptosis was highly pronounced in spermatogonia, spermatocytes, spermatids and Leydig cells. Antioxidant enzyme activities – superoxide dismutase and glutathione peroxidase – as well as total antioxidant capacity significantly reduced, while malondialdehyde contents increased. The number of germ cells expressing caspase-3, p53, Bax and FasL significantly increased whereas cells expressing Bcl-2 significantly decreased in groups treated with 50 and 100 mg/kg quinestrol compared with the control. The concentration of nitrogen monoxidum also increased significantly under these dosages. The results suggest that quinestrol stimulates oxidative stress to induce apoptosis in spermatogenic cells through the mitochondrial and death receptor pathways in adult male mice. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Xenoestrogens cause male reproductive tract abnormalities, including testicular hypoplasia, cryptorchidism, hypospadias, tumours of the rete testis and interstitial cells, erectile dysfunction, sperm granulomas and decreased total sperm counts and fertility (McLachlan, 2001; Oliva et al., 2002). Quinestrol, a synthetic oestrogen homolog used as a fertility control, is the major component of long-term oral contraceptives for women (Singh et al., 1971; Dipasquale et al., 1974). After ingestion, it can be deposited in adipose tissue as proto type and then released slowly into the circulation. Quinestrol could inhibit the hypothalamic release of gonadotropin releasing hormone, causing disturbance of the hypothalamus–pituitary–ovary axis and depressing follicle development and ovulation (Nudemberg et al., 1973; Zhao et al., 2007). Previous studies also showed that quinestrol has been

∗ Corresponding author at: Laboratory of Veterinary Anatomy, College of Veterinary Medicine, China Agricultural University, Haidian, Beijing 100193, China. Tel.: +86 10 62733778; fax: +86 10 62733199. E-mail address: [email protected] (Y. Chen). http://dx.doi.org/10.1016/j.tice.2014.05.012 0040-8166/© 2014 Elsevier Ltd. All rights reserved.

commercially used not only as a contraceptive for humans, but also as a rodenticide in wild rodents (Shen et al., 2012). Up to the present time, quinestrol has been reported to alter the structure of the reproductive organs and exerts anti-fertility effects on males (Singh et al., 1971; Zhao et al., 2007; Li et al., 2014). However, the mechanism by which quinestrol induces these changes in the male mice reproductive tract following adult exposure has rarely been studied. Apoptosis is a normal feature of spermatogenic lineages. Excessive or ectopic apoptosis jeopardises the male reproductive health. The excessive or ectopic apoptosis of testicular germ cells might be associated with pathological conditions such as radiation (Samuni et al., 2004) and chemical exposure to environmental estrogens or anti-androgenic compounds (Ma et al., 2008). Apoptosis are regulated by specific pro- and anti-apoptotic proteins through the extrinsic and the intrinsic pathways and features death receptor, endoplasmic reticulum, mitochondria-dependent apoptotic and mitogen-activated protein kinase (MAPK) pathways (Hikim et al., 2003). However, very little is known about the underlying mechanisms by which quinestrol, an environmental oestrogen induce spermatogenic cell apoptosis in adult male mice. Thus, it is essential and urgent to determine whether quinestrol changes the

J. Li et al. / Tissue and Cell 46 (2014) 318–325

expression of apoptosis-related proteins in spermatogenic cell. The aim of the present study is to fill this gap by investigating the reproductive toxicity and the expression of caspase-3, p53, Bcl-2, Bax and FasL.

2. Materials and methods 2.1. Animal treatment Twenty-five eight-week-old adult male imprinting control region mice weighing 30 g were obtained from Vitalriver Laboratory Animal Technology Co. Ltd. (Beijing, China) for this study. Mice were housed in polypropylene cages with cellulose fibre chip bedding at an ambient temperature of 25 ◦ C and humidity of 30–40% and kept on a 14-h light:10-h dark cycle. All animals were given access ad libitum to feed and water in glass bottles with rubber stoppers. After 1 week of acclimation, the mice were randomly divided into five treatment groups (n = 5). The treatment groups were intragastrically administered 0.05 mL of an olive oil vehicle to deliver daily doses of 5, 10, 50 or 100 mg/kg body weight (BW). The control group was only treated with vehicle. Dosing continued for 10 days, and individual BW was recorded daily. Animal handling and treatment protocols were performed in compliance with Chinese national guidelines.

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2.5. Immunohistochemical staining for caspase-3, p53, FasL, Bcl-2 and Bax Sections were immunostained with primary antibodies for caspase-3, p53, FasL, Bcl-2 and Bax rabbit polyclonal antibodies (Sigma) at 4 ◦ C for overnight and then incubated with antirabbit biotin-labelled secondary antibodies for 2 h followed by streptavidin–biotin–peroxidase for 90 min each at room temperature. Immunoreactivity was visualised by incubating the sections in 0.01 mol PBS containing 0.05% 3,3 -diaminobenzidine tetrahydrochloride (Sigma) and 0.003% hydrogen peroxide for 10 min followed by counterstaining with haematoxylin. Control slides without primary antibody were examined in all cases. Positive cells exhibited a brown stain in the nucleus for caspase-3, p53, Bcl-2 and Bax and in the cytoplasm for FasL.

2.6. Measurements of antioxidant activity and lipid peroxidation NO content, total antioxidant capacity (T-AOC), malondialdehyde (MDA), and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were assayed by commercial kits (Jiancheng, Nanjing, China) according to the method used previously (Ma et al., 2008). Results were expressed as ␮mol/L for NO, units (U) per mg protein for T-AOC, SOD and GSH-Px and nmol/mg protein for MDA.

2.2. Tissue harvesting and processing 2.7. Morphometric measurements Mice were euthanised under deep Nembutal anaesthesia (20 mg/kg BW) on the 10th day after the 10-day treatment. Both right and left testes were immediately removed and their weights and size were recorded. The left testis was quickly immersed in 4% paraformaldehyde and divided into serial cross-sections with 5–6 ␮m thickness for histological examination and immunohistochemical staining. The right testis was prepared for biochemical assays of nitrogen monoxidum (NO) and antioxidant enzymes activities. The caudal epididymides were removed for sperm analysis.

2.3. Sperm parameter analysis The caudal epididymides from individual mouse were minced and suspended in 2 mL of 37 ◦ C 0.9% sodium chloride. The mixture was homogenously stirred by vortex and overlaid with mineral oil. Preparation of the sperm suspension was performed according to the method as described by Amann and Lambiase Jr. (1969). The viability of spermatozoa was observed under a microscope by eosine–nigrosine staining (Dott and Foster, 1972). Spermatozoal morphology and acrosomal integrity were further assessed by sperm suspension smearing and Giemsa staining (Watson, 1975). Morphologically abnormal sperms were recorded as described previously (Wyrobek and Bruce, 1975). The number of spermatozoa was counted using a haemocytometer (Seed et al., 1996). The sperm counts were expressed as million/mL of suspension.

2.4. Methyl green-pyronin staining After deparaffination and rehydration, the sections were immersed in methyl green-pyronin solution for 10–20 min at room temperature, then in acetone and ethanol (1:1, v/v) and then in xylene and acetone (1:1, v/v) for 10–40 s. The cytoplasm of apoptotic cells showed cardinal red staining under an optical microscope.

BW and testis were measured, and the relative testis weights [testis weight (mg)/BW (g)] were calculated (n = 5). The lengths of the long and short axes of the testis also were measured. Seminiferous tubular (ST) areas, including epithelium and lumen, were measured by randomly selecting 100 round STs from ten cross-sections of each animal (n = 5). The percentage of cell subpopulations, including spermatogonia, spermatocytes and spermatids, was determined by counting 1000 spermatogenic cells from the ST cross-sections with H&E staining of each animal. The numbers of cells positive for caspase-3, p53, FasL, Bcl-2 and Bax were counted for 1000 spermatogenic cells and Leydig cells from ST cross-sections (n = 5) according to the method used previously (Yin et al., 1998).

2.8. Statistical analysis Data were analysed by one-way ANOVA using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). Results were evaluated as mean ± SD. P < 0.05 and P < 0.01 were considered statistically significant.

3. Results 3.1. Effect of quinestrol on semen quality Table 1 shows that the semen quality responded to quinestrol in a dose-dependent relationship. The sperm counts significantly declined by 86–99% in all animals exposed to the chemical at all dose levels (P < 0.01) compared with the control group. The change in sperm counts indicated that the percentage of live sperm decreased and the number of abnormal spermatozoa increased significantly in all treatment groups (P < 0.01). A significant reduction in the percentage of acrosomal integrity of spermatozoa was observed in all mice treated with quinestrol.

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Table 1 Effects of quinestrol on semen quality of mice (means ± SD, n = 5 per group). Quinestrol (mg/kg BW)

Sperm counts (106 /mL)

100 50 10 5 Control

0.31 0.77 2.48 4.58 32.50

**

± ± ± ± ±

Percent of live sperm (%)

0.14** 0.47** 0.87** 3.04** 4.14

67.00 73.60 72.20 74.98 90.34

± ± ± ± ±

Abnormal morphology (%)

3.92** 8.79** 3.40** 8.20** 1.97

23.42 22.78 22.24 20.78 12.60

± ± ± ± ±

Integrity of acrosome (%)

3.17** 5.76** 4.24** 2.07** 2.20

76.68 79.44 80.24 81.06 91.36

± ± ± ± ±

5.53** 3.49** 3.66** 3.16** 3.69

P < 0.01 as compared with control.

Table 2 Effects of quinestrol on weight and size of testes, seminiferous tubular area and percentage of germ cells in adult mice. Quinestrol (mg/kg BW)

BW (g)

Testis weight (mg) Absolute weight (mg)

100 50 10 5 Control * **

32.53 34.20 31.39 32.81 32.96

± ± ± ± ±

3.46 2.21 1.77 1.62 1.49

133.58 186.94 203.48 225.46 245.84

± ± ± ± ±

Long axis (mm)

Short axis (mm)

Relative weight (mg/g b.w.)

66.38** 27.22 58.12 24.97 31.50

4.11 5.47 6.51 6.89 7.45

± ± ± ± ±

2.13** 0.75* 1.90 0.88 0.84

6.75 7.57 7.82 8.37 8.24

± ± ± ± ±

1.13** 0.81 0.91 0.68 0.84

4.45 5.07 5.24 5.20 5.32

± ± ± ± ±

0.42** 0.59 0.63 0.40 0.74

Seminiferous tubular area (␮m2 ) 15.45 18.05 22.63 22.21 22.82

± ± ± ± ±

3.18** 3.57* 2.88 2.42 2.18

Percentage of germ cells (%) Spermatogonia 32.14 29.35 25.75 24.59 23.38

± ± ± ± ±

3.64** 4.92* 3.96 2.03 2.09

Spermatocytes 28.52 25.54 22.49 24.51 23.48

± ± ± ± ±

3.99* 2.55 4.37 1.21 2.15

Spermatids 39.33 45.11 51.76 50.90 53.14

± ± ± ± ±

6.17** 7.39* 8.18 2.53 3.12

P < 0.05 as compared with control. P < 0.01 as compared with control.

3.2. Morphological changes in testis and STs Dose-dependent morphological changes in testis were observed without BW alteration in all mice after quinestrol administration. Both absolute and relative testicular weights decreased by 46% and 45% (P < 0.01), respectively, after treatment with 100 mg/kg quinestrol (Fig. 1A–D and Table 2). Similarly, the relative testicular weight of mice declined by 27% (P < 0.05) after treatment with 50 mg/kg quinestrol. Long and short axial dimensions were reduced by 16–18%, in agreement with the drop in testicular weight after treatment with 100 mg/kg quinestrol. These effects, however, were insignificant at lower dose levels. Quinestrol exposure also altered the size of STs. Most STs were atrophic and the tubular area was approximately 21% and 32% lower than that of the control at doses of 50 and 100 mg/kg, respectively (Table 2). Size of STs did not change after treatment at lower does, 5 and 10 mg/kg quinestrol. Consistent with the reduction in size of STs, quinestrol administration significantly changed the histology of the seminiferous epithelium. Numerous spermatogonia, spermatocytes, spermatids and Leydig cells swelled or shrank after treatment with 50 and 100 mg/kg quinestrol (Fig. 1G and H). Furthermore, the total number of spermatogenic cells declined and the relative distribution of spermatogonia, spermatocytes and spermatids obviously changed. The ratios of spermatogonia and spermatocytes in mice from the 100 mg/kg treatment group were about 1.4- and 1.2-fold greater than those of the control group. By contrast, the ratio of spermatids in the treated groups was lower than that in the control group (Table 2). Compared with the controls, no significant differences were observed in the histology of seminiferous epithelium after treatment with 5 and 10 mg/kg quinestrol. 3.3. Apoptosis of spermatogenic cells Apoptotic cells were observed by caspase-3 staining (positive cells exhibit a brown nuclear staining) or methyl green-pyronin staining (cytoplasm of apoptotic cells shows cardinal red staining) according to the methods used by Rodríguez-Hernández et al. (2006) and Al-Hazzaa and Bowen (1998), respectively. Both caspase-3 and methyl green-pyronin staining results showed similar trends (Fig. 1I–P). The change in numbers of apoptotic spermatogenic cells in mice was dose-dependent. Compared with the control group, the numbers of apoptotic cells in the 100 mg/kg

treatment group was the highest (∼8-fold), followed by the 50 mg/kg treatment group (∼6-fold) and then the 5 and 10 mg/kg treatment groups (2∼2-fold) (Fig. 3A). Apoptotic cells were found in the spermatogonia, spermatocytes, spermatids and Leydig cells. Degeneration of spermatocytes (27–29% of positive cells) and spermatids (30–31% of positive cells) mainly contributed to the increase in number of apoptosis-positive cells in the 50 and 100 mg/kg treatment groups. By contrast, most apoptotic cells in the 5 and 10 mg/kg groups were observed in spermatids (22–29% of positive cells) and Leydig cells (31–33% of positive cells). 3.4. Expression of apoptosis-related proteins in spermatogenic cells The numbers of p53-positive cells markedly increased by 2-, 4and 6-fold from 10, 50 and 100 mg/kg treatment groups than that of the control (Figs. 2A and 3B). Those p53-positive cells resulted mainly from degeneration of spermatocytes (26–27% of positive cells) and spermatids (32–37% of positive cells). P53-positive cells from the 5 mg/kg treatment group only were observed in Leydig cells. A similar effect was observed for the pro-apoptotic proteins Bax (Figs. 2C and 3D) and FasL (Figs. 2D and 3E). Bax-positive cells significantly increased by approximately 2-, 4- and 6-fold for the 10, 50 and 100 mg/kg treatment groups than control group. Apoptosis-positive cells mainly were resulted from degeneration of spermatids and Leydig cells in the 5 and 10 mg/kg treatment groups and degeneration of spermatogonia, spermatocytes and spermatids in the 50 and 100 mg/kg treatment groups. A similar trend was found for FasL expression, which increased significantly in the spermatogonia, spermatocytes, spermatids and Leydig cells of the 10 mg/kg to 100 mg/kg treatment groups. In contrast to p53 and Bax, the anti-apoptotic protein Bcl-2 significantly reduced by 38% and 42% in the 50 and 100 mg/kg treatment groups, respectively (Figs. 2B and 3C). 3.5. NO contents in testis NO contents in the experiments increased to 0.75 and 1.00 ␮mol/L in the 50 and 100 mg/kg treatment groups, respectively, compared with the control (0.36 ␮mol/L). No significant change was observed for the lower doses of quinestrol compared to control (Fig. 3F).

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Fig. 1. Morphological changes in the testes and STs in adult mice treated with quinestrol. A to D show the gross changes in the testes. E to H show the gross changes in the STs. I to L show the depletion of spermatogenic cells in the lumens, as indicated by methyl green-pyronin staining. M to N show the negative control slides without primary antibody. O to P show apoptotic spermatogenic cells in the lumens, as indicated by caspase-3 staining. (A, E, I) control; (B, F, J) 10 mg quinestrol; (C, G, K) 50 mg quinestrol; (D, H, L) 100 mg quinestrol. (M, O) control; (N, P) 100 mg quinestrol. STs: seminiferous tubule; sg: spermatogonium; sc: spermatocyte; sp: spermatid; lc: Leydig cells.

3.6. Changes in antioxidant enzymes activity The antioxidant enzymes activity and lipid peroxidase concentration in the testes of mice were altered by quinestrol treatment. SOD activity in the testes significantly inactivated in all treatment groups (Fig. 4A). Significantly reduced GSH-Px and T-AOC activities were also observed in the high-dose treatment

groups compared with the control group (Fig. 4B and C). MDA levels increased with decreasing antioxidant capacity (Fig. 4D). MDA contents in the testes of mice from the 50 and 100 mg/kg treatment groups increased to 1.84 and 2.52 nmol/mg protein while that in the testes of control mice was 1.22 nmol/mg protein, though these changes were not significant at lower dose levels.

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Fig. 2. Apoptosis-related protein changes in spermatogenic cells of the testes of adult mice after quinestrol exposure. A and E show p53 expression, B and F show Bcl-2 expression, C and G show Bax expression and D and H show FasL expression. (A, B, C, D) 100 mg quinestrol; (E, F, G, H) control. p53: tumour suppressor gene; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2 associated X protein; FasL: factor associated suicide ligand; sg: spermatogonium; sc: spermatocyte; sp: spermatid; lc: Leydig cells.

Fig. 3. Effects of quinestrol on the expression of (A) caspase-3, (B) p53, (C) Bcl-2, (D) Bax and (E) FasL in the testes of adult mice. Numbers of caspase-3, p53, Bcl-2, Bax and FasL positive-cells were determined by counting 1000 spermatogenic and Leydig cells. F shows the content of NO in the testes of adult mice after quinestrol treatment. Values indicate mean ± S.D. (n = 5 per group). *P < 0.05; **P < 0.01 compared with the control. p53: tumour suppressor gene; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2 associated X protein; FasL: factor associated suicide ligand; NO: nitrogen monoxidum.

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Fig. 4. Effects of quinestrol on (A) SOD, (B) GSH-Px, (C) T-AOC and (D) MDA levels in the testes of adult mice. Values indicate mean ± S.D. (n = 5 per group). *P < 0.05; **P < 0.01 compared with the control. SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; T-AOC: total antioxidant capacity; MDA: malondialdehyde.

4. Discussion 4.1. Quinestrol-induced abnormal spermatogenesis in testis of adult mice The present study shows that epididymal sperm counts, sperm viability and acrosomal integrity of spermatozoa decreased while the ratio of teratospermia increased in a dose-dependent manner in adult mice exposed to 5–100 mg/kg quinestrol for 10 days. These results suggest that quinestrol may adversely affect semen quality. However, semen quality could be also affected by some procedures involving spermatogenesis in testis, maturity in epididymis and secretion of seminal vesicle and prostate. As a synthetic oestrogen, quinestrol inhibits the spermatogenesis in rat (Singh et al., 1971) and Brandt’s vole (Zhao et al., 2007). Our results further suggest that quinestrol disrupts the structure and organisation of testis in mice. Atrophy of the testes significantly reduced ST areas and germ cell numbers in mice in the 50 and 100 mg/kg treatment groups. Numerous spermatogenic cells have positive responses to caspase3 and methyl green-pyronin staining. Higher doses of quinestrol could sufficiently induce apoptosis of spermatogenic cells in adult mice. This result indicates that the quinestrol-induced apoptosis of spermatogenic cells is important for the alteration of semen quality in adult mice. Apoptotic cells mostly are resulted from degeneration of spermatogonia, spermatocytes, spermatids and Leydig cells that are the primary source of intratesticular and circulating androgens (Sun et al., 2009), which are essential for normal spermatogenesis and male fertility (Welsh et al., 2009). Spermatogenic cell apoptosis also occurs in response to androgen deficiency (Tapanainen et al., 1993). Xenoestrogen exposure might reduce plasma testosterone levels (Rivas et al., 2003). Thus, Leydig cell apoptosis may promote spermatogenic cell apoptosis by inducing androgen deficiency. Consequently, quinestrol could induce abnormal spermatogenesis both directly and indirectly.

4.2. Quinestrol-induced expression of apoptosis-related proteins in germ cells Apoptosis depends on the regulation of many specific proand anti-apoptotic proteins and features mitochondria-dependent apoptotic, death receptor, endoplasmic reticulum and mitogenactivated protein kinase (MAPK) pathways (Hikim et al., 2003). Different pathological stresses may initiate different pathway(s). Members of the Bcl-2 family play a major role in the mitochondriadependent apoptotic pathway. Bcl-2 functions as an anti-apoptotic protein while Bax functions as a pro-apoptotic protein (Oltvai et al., 1993; Chittenden et al., 1995). p53, a sequence specific transcription factor, functions as a transcription factor and induces cell cycle arrest or apoptosis (Basu and Haldar, 1998). FasL, a member of the Fas system, triggers cell death by activating and interacting with Fas receptor (Nagata, 1997). In this study, we found that quinestrol treatment increases the rate of apoptotic cell. Moreover, the accumulation of p53 and Bax proteins and activation of the p53 and Bax-dependent apoptotic pathways were both observed in the 50 and 100 mg/kg treatment groups instead of the 5 and 10 mg/kg treatment groups. FasL also was involved in the promotion of quinestrol-induced spermatogenic cell apoptosis through the death receptor pathway in the 10, 50 and 100 mg/kg treatment groups. Changes in the Bcl-2 expression indicated a reverse tendency that is similar to those of pro-apoptotic proteins. The expression of p53, Bax, FasL and Bcl-2 was found in spermatogonia, spermatocytes, spermatids and Leydig cells. Only spermatids and (or) Leydig cells, which expressed p53 and Bax, increased in the 5 and 10 mg/kg treatment groups. By contrast, spermatogonia, spermatocytes, spermatids and Leydig cells all increased markedly in the 50 and 100 mg/kg treatment groups. FasL expression in spermatogonia, spermatocytes, spermatids and Leydig cells increased in the 10, 50 and 100 mg/kg treatment groups but not in the 5 mg/kg group. These results are consistent with the changes in semen quality after

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quinestrol treatment at higher doses. Overall, the mitochondrial and death receptor pathways are the two key pathways in the quinestrol-induced spermatogenic cell apoptosis. 4.3. Role of oxidative stress on quinestrol-induced germ cell apoptosis Reactive oxygen species (ROS) is a potential signal for apoptosis (Green and Reed, 1998). NO, a ubiquitous free radical moiety, acts as a pro-oxidant at high concentrations (Gulati et al., 2007). Moderate accumulation of ROS and overproduction of NO are involved in the classical pathways of apoptosis (Lewen´ et al., 2000; Lu et al., 2003). High degree of lipid peroxidation may induce cell death by altering the cellular membrane structure and then blocking cellular metabolism (Ennamany et al., 1995). Recent studies have implicated NO in the inhibition of testicular testosterone production and antioxidant enzyme activities (Kostic et al., 2000). NO also might induce spermatozoal dysfunction and infertility (Rosselli et al., 1998). Therefore, oxidative stress is important in the initiation of spermatogenic cell apoptosis. However, the effects of ROS may be tempered by antioxidant protective systems (i.e., thioredoxin, SOD and GSH-Px) in normal cells. In the present study, the testicular concentration of NO increased remarkably and the activities of SOD and GSH-Px decreased significantly in the experiments after exposure to 10–100 mg/kg quinestrol for 10 days, which suggests that quinestrol could cause oxidative stress and ROS-mediated damage in the testes at relatively higher levels and result in germ cell apoptosis. The increased NO concentrations are consistent with the reduced activities of SOD and GSH-Px. Severe lipid peroxidation may be generated when ROS is produced excessively in the testicular compartment (Aitken and Baker, 2002). Consistent with the reductions in the activities of SOD and GSH-Px, the results show a significantly increase in MDA concentrations and decrease in T-AOC contents in the 50 and 100 mg/kg treatment groups. The results indicate that exposure to quinestrol at higher doses could generate more ROS in testis. Similarly, Mishra and Shaha (2005) found that oxidative stress was involved in oestrogen-induced spermatogenic cell apoptosis. These results further suggest that relatively higher doses of quinestrol increase NO and ROS contents. These findings indicated that oxidative stress was involved in quinestrol-induced apoptosis in spermatogenic cells. Increased O2 − concentration and its downstream consequences such as hydrogen peroxide, peroxynitrite, hydroxyl radicals could lead to oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction (Geng et al., 2014). In agreement with the earlier report, quinestrol changed the expressions of caspase-3, p53, FasL, Bcl-2 and Bax. These results suggest that quinestrol induced oxidative stress firstly, then initiated oxidative stress and meanwhile promoted apoptosis in spermatogenic cells through the mitochondrial and death receptor pathways. These findings could contribute to understanding the mechanism of quinestrol on male infertility. 5. Conclusion The results in the present study suggest that quinestrol stimulated oxidative stress and induced apoptosis in testicular spermatogenic cells through the mitochondrial and death receptor pathways in adult male mice. Further investigations will be helpful to understand and provide insights concerning the mechanisms by which quinestrol induce male reproductive toxicity, apoptosis and abnormal spermatogenesis. Acknowledgement This work was supported by National High-tech Research and Development Projects (863) of China (No. 2013AA10230603).

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