Nitric Oxide 46 (2015) 165–171
Contents lists available at ScienceDirect
Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Hydrogen sulfide protects testicular germ cells against heat-induced injury Guang Li a,1, Zhi-Zhong Xie a,1,2, Jason M.W. Chua a, P.C. Wong b, Jinsong Bian a,* a b
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore
A R T I C L E
I N F O
Article history: Available online 24 October 2014 Keywords: Heat stress Hydrogen sulfide Testicular germ cell Apoptosis Infertility Antioxidant
A B S T R A C T
Objective: The present study was designed to investigate whether H2S can protect testicular germ cells against heat exposure induced injury and the underlying mechanisms. Results: It was found that all three H2S generating enzymes, cystathionine β-synthase (CBS), cystathionine γ-lysase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST), were expressed in mouse testicular tissue. Three episodes of heat exposure (42 °C, 30 min/day, 3 days) significantly decreased endogenous H2S production and down-regulated the expression of CBS and CSE in testes. In primary cultured testicular germ cells, exogenous application of NaHS (an H2S donor) attenuated heat stress (42 °C, 30 min) induced cell death and apoptosis. This was mediated by the inhibitory effects of H2S on cytochrome C release and the ratio of the Bax/Bcl-2. NaHS also improved mitochondrial function by decreasing oxygen consumption and increasing ATP production. NaHS treatment also stimulated SOD activity and reduced ROS production. Conclusions: Our results revealed both physiological and pharmacological roles of H2S in testicular germ cells. Exogenous application of H2S may protect germ cells by preservation of mitochondrial function and stimulation of anti-oxidant activity. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Almost 15% of couples trying to conceive are affected by infertility worldwide. Male infertility accounts for approximately half of these cases and is increasing faster than female [1]. Of the many factors that affect male infertility, heat stress of the scrotum is commonly accepted as one major contributor affecting spermatogenesis [2]. Heat stress can be caused by saunas, excessive exercise and heat exposure working environment. In addition, cryptorchidism has also been reported to induce heat stress and affect up to 3% of boys [3,4]. Accumulated evidence suggests that heat stress cause apoptosis of testicular germ cells [5,6] via altering the interplay of Bcl-2 and Bax protein [7] and generation of reactive oxygen species (ROS) [8].
Abbreviations: H2S, hydrogen sulfide; CBS, cystathionine β-synthase; CSE, cystathionine γ-lysase; 3MST, 3-mercaptopyruvate sulfurtransferase; SOD, superoxide dismutase; ROS, reactive oxygen species; PBS, phosphate buffered saline; H2DCFDA, 2′,7′ –dichlorodihydrofluoresceindiacetate. * Corresponding author. Departments of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical Drive, MD 11, CRC Building, #5-30A, Singapore 117597. Fax: +65 6873 7690. E-mail address: [email protected] (J. Bian). 1 These authors contributed equally to this work. 2 Present address: Institute of Pharmacy & Pharmacology, University of South China, Hengyang City, Hunan Province 421001, China. http://dx.doi.org/10.1016/j.niox.2014.10.005 1089-8603/© 2014 Elsevier Inc. All rights reserved.
Hydrogen sulfide (H2S) is now believed to be the third human endogenous gaseous transmitter. We have demonstrated in our previous studies that H2S produces significant anti-oxidant effect in various systems including brain, heart, kidney and bone [9–14]. Endogenous H2S are mainly generated by cystathionine β-synthase (CBS), cystathionine γ-lysase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). CBS and 3MST are highly expressed in the brain and nervous system, whereas CSE is reported to be predominant in the cardiovascular system [15]. It is also reported that both CBS and CSE are expressed in rat testes [16], but little is known about 3MST expression, and its role in the reproductive system is still unknown. In this study, we investigated the expression of intrinsic H2S system after short term exposure to heat and observed whether exogenous application of H2S may protect germ cells against heat –induced injury. 2. Materials and methods 2.1. Animal treatment All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore. After 4-week acclimatization to the home cage, 8-week-old male C57 BL/6J mice (22 ± 3 g) were randomly divided into four groups:
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control, heat stress, NaHS alone, and NaHS treatment groups. NaHS (5.6 mg/kg) or the same volume of 0.9% NaCl (control) was administered to mice via intraperitoneal injection. After 30 min, the mice were then anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and their scrota were immersed in a thermostatically controlled water bath at 22 °C (control) or 42 °C for 30 min. After drying the hair, they were sent back to their cages. This process was carried out once or repeated for 3 days. Testicular cells were collected 6 h after the last heat stress. 2.2. Primary testicular cell culture The isolation and culture of testicular cells were performed as described before with minor modification [17]. Briefly, testes of mice were removed and cut into pieces. Seminiferous tubules were incubated in phosphate buffered saline (PBS) containing 0.25% collagenase (type I) for 15 min at 32.5 °C followed by further 15 min digestion in PBS containing 0.25% trypsin with gentle shaking. The cell suspension was filtered, centrifuged, resuspended in F12DMEM medium plus 10% (v/v) fetal bovine serum and cultured at 32.5 °C in 5% CO2 for further use. 2.3. Cell viability assay Cell viability was evaluated with the MTT method as described previously with modification [13]. In brief, primary cultured cells were pre-treated with NaHS for 30 min. After washing out NaHS, cells were continued to be cultured with or without heat treatment (42 °C) for 30 min. MTT (0.5 mg/ml) was then added and incubated at 32.5 °C for another 4 h. The cultured medium containing MTT was removed and dimethyl sulfoxide (100 μl) was then added into each well. The absorbance at 570 nm was measured with a spectrophotometric plate reader (Safire2, Tecan Group Ltd.). 2.4. H2S measurement The H2S level was measured as described previously with minor modification [11]. Briefly, 10% testicular homogenate was prepared in 50 mM ice-cold potassium phosphate buffer (pH 6.8) using Dounce tissue grinders. The homogenate was centrifuged (1000 × g, 3 min, 4 °C) and 800 μl supernatants were collected. Then 8 μl NaOH (1 N), 80 μl of N,N-dimethyl-p-phenylenediamine sulfate (20 mM in 7.2 M HCl), and 80 μl of FeCl3 (30 mM in 1.2 M HCl) were added sequentially. The mixtures were incubated at room temperature for 20 min and the absorbance was detected at 668 nm with a 96well microplate reader (TECAN infinite 200, Tecan Group Ltd). The H2S concentration was assessed with a standard curve of NaHS.
2.7. ROS production The use of the 2′,7′-dichlorodihydrofluoresceindiacetate (H2DCFDA) assay was carried out to determine ROS production level. Cultured testicular germ cells were first seeded in black 96-well plates and cultured in their respective conditions. The medium were aspirated and substituted with phenol-red-free DMEM together with 10 μl MH2DCF-DA for 30 min before any treatment. The ROS levels were determined by a fluorescence reader (Safire2, Tecan Group Ltd.) with the emission wavelength at 529 nM and excitation wavelength at 504 nM. 2.8. Determination of superoxide dismutase (SOD) activity After treatment, primary cultured cells were homogenized and the supernatant SOD activities were measured using a SOD assay kit (Cat No.706002, Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer’s instructions. Cells were normalized according to their protein level using the BCA protein assay (Pierce). Each sample was assayed in duplicate, and the assay was repeated a minimum of three times. 2.9. Intracellular ATP assay Intracellular ATP content was performed according to the manufacturer’s instructions of the ATP assay kit (BioThema, Handen, Sweden) after heat treatment of primary cultured cells. Each sample was assayed in duplicate, and the assay was repeated a minimum of three times. 2.10. Oxygen consumption analysis Oxygen consumption rate was measured with MitoXpress probe (Luxcel, Cork, Ireland). In brief, germ cells were cultured in 96-well plate. After heat treatment, MitoXpress-Xtra probe stock solution and substrate solution were added sequentially. One hundred microliters of mineral oil was added to overlay each well. The measurement was carried out immediately with a Safire microplate reader (TECAN, excitation at 380 nm and emission at 650 nm) and the slope of linear regressed read out represents the oxygen consumption rate. 2.11. Caspase-8 activity assay Caspase-8 activity of primary cultured cells was determined using the Apo Target Kit (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions after heat treatment. Each sample was assayed in duplicate, and the assay was repeated a minimum of three times.
2.5. TUNEL staining 2.12. Western blotting analysis Six hours after heat treatment, the mice were killed and testes were immersion fixed in Bouin solution overnight, embedded in paraffin and sectioned at 5 μm. Germ cell apoptosis was verified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique using a TUNEL Apoptosis Assay Kit (Biovision). The rate of germ cell apoptosis or apoptotic index (AI) was expressed as the number of apoptotic germ cells per 100 Sertoli cells [18,19]. 2.6. Detection of apoptotic cells by Hoechst 33342 staining To visualize the morphology changes in nucleus, primary cultured testicular germ cells were seeded on coverslips. After treatment, the cells were fixed with 4% buffered paraformaldehyde and stained with 2.5 μg/ml Hoechst DNA dye. The cells with uniformly stained nuclei were scored as healthy, viable cells, whereas those stained with condensed or fragmented nuclei were scored as apoptotic cells.
Six hours after heat treatment, the testicular germ cells were isolated, washed with chilled PBS solution twice and harvested for protein extraction. RIPA buffer containing protease-inhibitor cocktail (Thermo Scientific) was used for total protein extraction. For cytosolic protein extraction, the lysis buffer contains 250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 10 mM KCl, 1.5 mM Na-EGTA, 1.5 mM Na-EDTA, 1 mM MgCl2, 1 mM dithiothreitol, and a cocktail of protease inhibitors (Sigma). Protein concentrations were determined by Lowry’s method. Equal amount of protein samples were separated by 8–12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Whatman, Dassel, Germany). After blocking in 10% milk in TBST buffer for 1 h, the membrane was incubated with primary antibodies at 4 °C overnight and washed three times in TBST buffer, then followed by incubation with the secondary peroxidaseconjugated antibodies for 1 h at room temperature and subjected
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to immunodetection analyses using the ECL Kit. The density of the band was finally quantified using Image J software.
2.13. Statistical analysis All data are presented as means ± SEM (standard error of the mean) with the number of experiments in each group as mentioned in each of the figure legends. Statistical analysis was performed using oneway ANOVA followed by a post hoc (Bonferroni) test for multiple group comparison or Student t test between two groups. p < 0.05 was considered as statistically significant.
3. Results 3.1. Heat stress inhibited endogenous H2S production We first determined the expression of different H2S generating enzymes (CBS, CSE and 3MST) in the testicular tissue. As shown in Fig. 1A–C, all the above three enzymes were expressed in testicular germ cells. Interestingly, heat stress (42 °C, 30 min/day, 3 days) significantly decreased the expression levels of CBS (Fig. 1A) and CSE (Fig. 1B), but not that of 3-MST (Fig. 1C). We further examined the effect of heat stress on endogenous H2S production. As shown in Fig. 1D, heat stress significantly decreased testicular H2S level by ~34% compared with the value in the control group (Fig. 1D). These data suggest that heat stress may down-regulate CBS and CSE expression and inhibit H2S production.
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3.2. Exogenous application of H2S alleviates heat-induced apoptosis in testicular germ cells We further examined the effect of exogenous application of H2S on mouse testicular germ cell viability. As shown in Fig. 2A, treatment with NaHS (an H2S donor) at the concentration range of 1–200 μM for 30 min had no significant effect on germ cell viability. However, when NaHS concentration increased to 1 mM, it induced cell injury obviously. For this reason, NaHS at the concentration range of 1–50 μM was used in the following experiments. We next examined whether exogenous application of H2S can protect testicular germ cells against heat stress-induced apoptosis in vitro. As shown in Fig. 2B, pretreatment with NaHS at concentrations of 1 (a), 10 (b) and 50 μM (c) concentration-dependently protected testicular germ cells against heat-induced cell injury. Apoptotic death is recognized to be responsible for the heatinduced loss of germ cells and spermatogenesis damage [20]. We further examined the effect of NaHS on heat-induced programmed cell death in vivo with a modified TUNEL technique. As shown in Fig. 2C, the heat exposure significantly activated germ cell TUNEL positive apoptosis. However, pretreatment with NaHS (5.6 mg/kg) markedly decreased the number of apoptotic cells per 100 Sertoli cells from 73.7 ± 7.8 in heat treated group to 41.2 ± 7.7 in NaHS treatment group (p < 0.01). Testicular germ cells apoptosis was further visualized using a Hoechst 33342 staining method. In primary cultured testicular germ cells, heat exposure induced characteristic condensed and fragmented nuclei during apoptosis. This effect was significantly
Fig. 1. Effect of heat stress on endogenous H2S system in testis. (A–C) Representative Western blots and densitometric analysis showing that three episodes of heat stress (42 °C, 30 min/day, 3 days) inhibited expression levels of intrinsic CBS (A) and CSE (B), but not that of 3MST (C). (D) The same treatment also decreased testicular H2S generation. The values were presented as mean ± SEM from four to six independent experiments, ***p < 0.001 vs control group.
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Fig. 2. Effects of NaHS on heat-induced testicular germ cells apoptosis in vivo and in vitro. (A) Concentration-dependent effect of NaHS (1–1000 μM) on testicular germ cells. NaHS only at 1000 μM produced significant cell injury. n = 8. (B) Effect of NaHS on heat-induced cell injury. Pretreatment with NaHS (1, 10, 50 μM) for 30 min alleviated heat-induced cell death in a dose-dependent manner. (C) Representative images and quantification analysis showing that pretreatment with NaHS at 5.6 mg/kg inhibited heat-induced germ cell apoptosis. n = 8. (D) Representative images and quantification analysis showing that pretreatment with NaHS at 10 μM inhibited heat-induced germ cells apoptosis in vitro. Values are normalized by the value in the control group and presented as mean ± SEM from three to five independent experiments, §p > 0.05 vs. control, ***p < 0.001 vs. control, ##p < 0.01, ###p < 0.001 vs. heat treatment group.
attenuated by pretreatment with NaHS (Fig. 2D). Taken together, H2S may protect germ cells via its anti-apoptotic effects. 3.3. Effects of H2S on heat stress-stimulated apoptotic pathways We next examined the signaling mechanism by which H2S inhibited germ apoptosis. As shown in Fig. 3A, heat stress led to release of cytochrome C into the cytosol in cultured testicular germ cells. Pretreatment with NaHS (10 μM) statistically attenuated this effect. In addition, NaHS pretreatment decreased the ratio of protein
expression of Bax/Bcl-2 (Fig. 3B), but had no effect on the activity of caspases 8 (Fig. 3C). These results indicate that inactivation of intrinsic pathway, rather than extrinsic pathway, mediates the anti-apoptotic effect of H2S. 3.4. Effects of H2S on mitochondrial function and ROS generation in heat-treated germ cells Mitochondrial function plays a central role in apoptosis. In cultured testicular germ cells, mitochondria function was examined
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Fig. 3. Effects of NaHS on apoptotic pathways activated by heat stress. (A) Pretreatment with NaHS (10 μM) inhibited heat exposure-induced cytochrome c release into the cytosol. (B) Representative images and densitometric analysis showing that short term exposure to heat (42 °C, 30 min) increased the ratio of protein expression of Bax/ Bcl-2, which was largely reversed by pretreatment with 10 μM NaHS for 30 min before heat exposure. (C) Heat exposure with or without NaHS pretreatment had no effect on the activity of caspase 8. Values are normalized by the value in the control group and presented as mean ± SEM from four to six independent experiments, **p < 0.01, ***p < 0.001 vs control, #p < 0.05 vs heat treatment group.
by measuring oxygen consumption and ATP level. As shown in Fig. 4A and B, heat exposure resulted in higher oxygen consumption and lower ATP level. Pretreatment with NaHS decreased the rate of oxygen consumption (158.8% ± 9.9% in heat-treated group vs. 119.3% ± 6.5% in NaHS pre-treated group, p < 0.01, Fig. 4A) and reversed the ATP depletion from 43.5% ± 3.1% in heat-treated group to 78.8% ± 8.4% in NaHS pre-treated group (p < 0.001, Fig. 4B). To further study the mechanisms for the effect of H2S on heatinduced apoptosis, two parameters, ROS production and SOD activity were detected in the cultured cells. As shown in Fig. 4C and D, heat exposure significantly increased intracellular ROS production (Fig. 4C) and suppressed SOD activity (Fig. 4D). H2S pretreatment attenuated these effects, suggesting that H2S may protect germ cells by stimulation of the activity of anti-oxidant. 4. Discussion In normal condition, human testicular temperature is maintained 3 °C lower than the core body temperature. This is necessary as germ cells and Sertoli cells are highly sensitive to elevated
temperature. Exposure of the testis to abnormal elevated temperature, either from hot environment or naturally occurring cryptorchidism, causes extensive degeneration of the germ cells by heat stress. In this study, we examined the physiological and pharmacological functions of H2S, an endogenous biological gas, in the testicular germ cells. We first determined the protein expression of different H2S generating enzymes. CBS and CSE were reported to be the two major enzymes to generate H2S in rat testes [16,21]. However, little is known about the distribution of 3MST in testicular germ cells. In this study, we found that all three enzymes (CBS, CSE and 3MST) are expressed in testicular germ cells. Interestingly, only the expressions of CBS and CSE were significantly decreased after exposure to heat at 42 °C for 30 min. No significant change was found in the expression of 3-MST upon heat challenge. We further examined endogenous H2S level. Similarly, endogenous H2S was largely decreased upon three episodes of heat treatment. These data suggest that endogenous H2S production may be impaired during heat stress. To confirm the effect of H2S on heat stress-caused male subfertility, NaHS, an H2S donor, was used. We found that pretreatment with NaHS
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Fig. 4. Effects of NaHS on heat-induced mitochondrial function damage and ROS generation. (A) Short term exposure to heat (42 °C, 30 min) led to a faster rate of oxygen consumption, which was blocked by NaHS pretreatment (10 μM, 30 min before heat exposure). (B) NaHS alleviated the ATP depletion caused by the heat exposure. (C) NaHS pretreatment inhibited heat exposure-induced ROS production in testicular germ cells. (D) NaHS pretreatment reversed heat exposure-induced SOD deficiency in testicular germ cells. Values are normalized by the value in the control group and presented as mean ± SEM. n = 6–8, ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs heat treatment group.
(10 μM, 30 min) significantly protected heat stress-induced cell death. Accumulating evidence showed that excessive germ cell apoptosis/ abrupt spermatogenesis is the major pathology upon local testicular heat treatment [6,22,23]. Consistent with previous findings, we also found that exposure to heat at 42 °C for 30 min significantly increased the number of apoptotic cells by increasing the ratio of Bax/ Bcl-2 and release of cytochrome C. However, heat stress had no effect on the activity of caspases 8. These results imply that heat triggers apoptosis via an intrinsic pathway in testicular germ cells. Compared with somatic cells, sperm has limited cellular cytoplasm and is especially susceptible to oxidative stress. The ROS attack the lipids in the sperm plasma membrane, oxidizes the polyunsaturated fatty acids of the mitochondria sheath, and had widely been accepted as a significant contributory factor to sperm apoptosis [24,25]. The mitochondrial respiratory chain is a major source of ROS. Uncoupled respiration and ATP synthesis potentially halt the electron flux and the leakage will be transferred to oxygen or other electron acceptors to generate ROS [26]. In this study, testicular germ cells undergoing heat-triggered apoptosis exhibits a faster oxygen consumption rate and a lower intracellular ATP content, consistent with a higher intracellular ROS level, which indicated
an increased oxidative stress associated with the mitochondria dysfunction. Application of NaHS, however, decreased oxygen consumption, reversed heat-induced ATP depletion, and led to less production of intracellular ROS. Furthermore, NaHS treatment also ameliorated the activity of SOD, one of the most important antioxidative enzymes on scavenging of free radicals. Results obtained in these studies suggest that the H2S protected testicular germ cells against heat-induced apoptosis via an anti-oxidant mechanism (Fig. 5).
5. Conclusions Our results showed the important roles of intrinsic H2S system on heat-induced testicular germ cells injury. We also demonstrated for the first time that application of H2S ameliorates heatinduced testicular cell apoptosis likely via its anti-oxidant and anti-apoptotic actions. Our results provided evidence for the potential therapeutic value of H2S on male infertility. However, this effect can only be achieved when H2S concentration is at a low concentration range. One should bear in mind that H2S at higher
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Fig. 5. Schematic illustration showing the mechanisms underlying the protective effect of H2S. H2S protects germ cells against heat injury via anti-apoptotic and antioxidant effects through activation of SOD activity and its subsequent ROS scavenging effect. OCR: oxygen consumption rate.
concentration may also produce toxic effects as we observed in the present study. Acknowledgment This work was supported by NUHS B2B research grant (NUHSRO/ 2011/012/STB/B2B-08) References [1] I.D. Sharlip, J.P. Jarow, A.M. Belker, L.I. Lipshultz, M. Sigman, A.J. Thomas, et al., Best practice policies for male infertility, Fertil. Steril. 77 (2002) 873–882. [2] B. Kim, K. Park, K. Rhee, Heat stress response of male germ cells, Cell. Mol. Life Sci. 70 (2013) 2623–2636. [3] M.P. De Miguel, J.M. Marino, P. Gonzalez-Peramato, M. Nistal, J. Regadera, Epididymal growth and differentiation are altered in human cryptorchidism, J. Androl. 22 (2001) 212–225. [4] Y. Yin, K.L. Hawkins, W.C. DeWolf, A. Morgentaler, Heat stress causes testicular germ cell apoptosis in adult mice, J. Androl. 18 (1997) 159–165. [5] I.T. Koksal, M. Usta, I. Orhan, S. Abbasoglu, A. Kadioglu, Potential role of reactive oxygen species on testicular pathology associated with infertility, Asian J. Androl. 5 (2003) 95–99.
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Contents lists available at ScienceDirect
Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Hydrogen sulfide protects testicular germ cells against heat-induced injury Guang Li a,1, Zhi-Zhong Xie a,1,2, Jason M.W. Chua a, P.C. Wong b, Jinsong Bian a,* a b
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore
A R T I C L E
I N F O
Article history: Available online 24 October 2014 Keywords: Heat stress Hydrogen sulfide Testicular germ cell Apoptosis Infertility Antioxidant
A B S T R A C T
Objective: The present study was designed to investigate whether H2S can protect testicular germ cells against heat exposure induced injury and the underlying mechanisms. Results: It was found that all three H2S generating enzymes, cystathionine β-synthase (CBS), cystathionine γ-lysase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST), were expressed in mouse testicular tissue. Three episodes of heat exposure (42 °C, 30 min/day, 3 days) significantly decreased endogenous H2S production and down-regulated the expression of CBS and CSE in testes. In primary cultured testicular germ cells, exogenous application of NaHS (an H2S donor) attenuated heat stress (42 °C, 30 min) induced cell death and apoptosis. This was mediated by the inhibitory effects of H2S on cytochrome C release and the ratio of the Bax/Bcl-2. NaHS also improved mitochondrial function by decreasing oxygen consumption and increasing ATP production. NaHS treatment also stimulated SOD activity and reduced ROS production. Conclusions: Our results revealed both physiological and pharmacological roles of H2S in testicular germ cells. Exogenous application of H2S may protect germ cells by preservation of mitochondrial function and stimulation of anti-oxidant activity. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Almost 15% of couples trying to conceive are affected by infertility worldwide. Male infertility accounts for approximately half of these cases and is increasing faster than female [1]. Of the many factors that affect male infertility, heat stress of the scrotum is commonly accepted as one major contributor affecting spermatogenesis [2]. Heat stress can be caused by saunas, excessive exercise and heat exposure working environment. In addition, cryptorchidism has also been reported to induce heat stress and affect up to 3% of boys [3,4]. Accumulated evidence suggests that heat stress cause apoptosis of testicular germ cells [5,6] via altering the interplay of Bcl-2 and Bax protein [7] and generation of reactive oxygen species (ROS) [8].
Abbreviations: H2S, hydrogen sulfide; CBS, cystathionine β-synthase; CSE, cystathionine γ-lysase; 3MST, 3-mercaptopyruvate sulfurtransferase; SOD, superoxide dismutase; ROS, reactive oxygen species; PBS, phosphate buffered saline; H2DCFDA, 2′,7′ –dichlorodihydrofluoresceindiacetate. * Corresponding author. Departments of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical Drive, MD 11, CRC Building, #5-30A, Singapore 117597. Fax: +65 6873 7690. E-mail address: [email protected] (J. Bian). 1 These authors contributed equally to this work. 2 Present address: Institute of Pharmacy & Pharmacology, University of South China, Hengyang City, Hunan Province 421001, China. http://dx.doi.org/10.1016/j.niox.2014.10.005 1089-8603/© 2014 Elsevier Inc. All rights reserved.
Hydrogen sulfide (H2S) is now believed to be the third human endogenous gaseous transmitter. We have demonstrated in our previous studies that H2S produces significant anti-oxidant effect in various systems including brain, heart, kidney and bone [9–14]. Endogenous H2S are mainly generated by cystathionine β-synthase (CBS), cystathionine γ-lysase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). CBS and 3MST are highly expressed in the brain and nervous system, whereas CSE is reported to be predominant in the cardiovascular system [15]. It is also reported that both CBS and CSE are expressed in rat testes [16], but little is known about 3MST expression, and its role in the reproductive system is still unknown. In this study, we investigated the expression of intrinsic H2S system after short term exposure to heat and observed whether exogenous application of H2S may protect germ cells against heat –induced injury. 2. Materials and methods 2.1. Animal treatment All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore. After 4-week acclimatization to the home cage, 8-week-old male C57 BL/6J mice (22 ± 3 g) were randomly divided into four groups:
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control, heat stress, NaHS alone, and NaHS treatment groups. NaHS (5.6 mg/kg) or the same volume of 0.9% NaCl (control) was administered to mice via intraperitoneal injection. After 30 min, the mice were then anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and their scrota were immersed in a thermostatically controlled water bath at 22 °C (control) or 42 °C for 30 min. After drying the hair, they were sent back to their cages. This process was carried out once or repeated for 3 days. Testicular cells were collected 6 h after the last heat stress. 2.2. Primary testicular cell culture The isolation and culture of testicular cells were performed as described before with minor modification [17]. Briefly, testes of mice were removed and cut into pieces. Seminiferous tubules were incubated in phosphate buffered saline (PBS) containing 0.25% collagenase (type I) for 15 min at 32.5 °C followed by further 15 min digestion in PBS containing 0.25% trypsin with gentle shaking. The cell suspension was filtered, centrifuged, resuspended in F12DMEM medium plus 10% (v/v) fetal bovine serum and cultured at 32.5 °C in 5% CO2 for further use. 2.3. Cell viability assay Cell viability was evaluated with the MTT method as described previously with modification [13]. In brief, primary cultured cells were pre-treated with NaHS for 30 min. After washing out NaHS, cells were continued to be cultured with or without heat treatment (42 °C) for 30 min. MTT (0.5 mg/ml) was then added and incubated at 32.5 °C for another 4 h. The cultured medium containing MTT was removed and dimethyl sulfoxide (100 μl) was then added into each well. The absorbance at 570 nm was measured with a spectrophotometric plate reader (Safire2, Tecan Group Ltd.). 2.4. H2S measurement The H2S level was measured as described previously with minor modification [11]. Briefly, 10% testicular homogenate was prepared in 50 mM ice-cold potassium phosphate buffer (pH 6.8) using Dounce tissue grinders. The homogenate was centrifuged (1000 × g, 3 min, 4 °C) and 800 μl supernatants were collected. Then 8 μl NaOH (1 N), 80 μl of N,N-dimethyl-p-phenylenediamine sulfate (20 mM in 7.2 M HCl), and 80 μl of FeCl3 (30 mM in 1.2 M HCl) were added sequentially. The mixtures were incubated at room temperature for 20 min and the absorbance was detected at 668 nm with a 96well microplate reader (TECAN infinite 200, Tecan Group Ltd). The H2S concentration was assessed with a standard curve of NaHS.
2.7. ROS production The use of the 2′,7′-dichlorodihydrofluoresceindiacetate (H2DCFDA) assay was carried out to determine ROS production level. Cultured testicular germ cells were first seeded in black 96-well plates and cultured in their respective conditions. The medium were aspirated and substituted with phenol-red-free DMEM together with 10 μl MH2DCF-DA for 30 min before any treatment. The ROS levels were determined by a fluorescence reader (Safire2, Tecan Group Ltd.) with the emission wavelength at 529 nM and excitation wavelength at 504 nM. 2.8. Determination of superoxide dismutase (SOD) activity After treatment, primary cultured cells were homogenized and the supernatant SOD activities were measured using a SOD assay kit (Cat No.706002, Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer’s instructions. Cells were normalized according to their protein level using the BCA protein assay (Pierce). Each sample was assayed in duplicate, and the assay was repeated a minimum of three times. 2.9. Intracellular ATP assay Intracellular ATP content was performed according to the manufacturer’s instructions of the ATP assay kit (BioThema, Handen, Sweden) after heat treatment of primary cultured cells. Each sample was assayed in duplicate, and the assay was repeated a minimum of three times. 2.10. Oxygen consumption analysis Oxygen consumption rate was measured with MitoXpress probe (Luxcel, Cork, Ireland). In brief, germ cells were cultured in 96-well plate. After heat treatment, MitoXpress-Xtra probe stock solution and substrate solution were added sequentially. One hundred microliters of mineral oil was added to overlay each well. The measurement was carried out immediately with a Safire microplate reader (TECAN, excitation at 380 nm and emission at 650 nm) and the slope of linear regressed read out represents the oxygen consumption rate. 2.11. Caspase-8 activity assay Caspase-8 activity of primary cultured cells was determined using the Apo Target Kit (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions after heat treatment. Each sample was assayed in duplicate, and the assay was repeated a minimum of three times.
2.5. TUNEL staining 2.12. Western blotting analysis Six hours after heat treatment, the mice were killed and testes were immersion fixed in Bouin solution overnight, embedded in paraffin and sectioned at 5 μm. Germ cell apoptosis was verified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique using a TUNEL Apoptosis Assay Kit (Biovision). The rate of germ cell apoptosis or apoptotic index (AI) was expressed as the number of apoptotic germ cells per 100 Sertoli cells [18,19]. 2.6. Detection of apoptotic cells by Hoechst 33342 staining To visualize the morphology changes in nucleus, primary cultured testicular germ cells were seeded on coverslips. After treatment, the cells were fixed with 4% buffered paraformaldehyde and stained with 2.5 μg/ml Hoechst DNA dye. The cells with uniformly stained nuclei were scored as healthy, viable cells, whereas those stained with condensed or fragmented nuclei were scored as apoptotic cells.
Six hours after heat treatment, the testicular germ cells were isolated, washed with chilled PBS solution twice and harvested for protein extraction. RIPA buffer containing protease-inhibitor cocktail (Thermo Scientific) was used for total protein extraction. For cytosolic protein extraction, the lysis buffer contains 250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 10 mM KCl, 1.5 mM Na-EGTA, 1.5 mM Na-EDTA, 1 mM MgCl2, 1 mM dithiothreitol, and a cocktail of protease inhibitors (Sigma). Protein concentrations were determined by Lowry’s method. Equal amount of protein samples were separated by 8–12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Whatman, Dassel, Germany). After blocking in 10% milk in TBST buffer for 1 h, the membrane was incubated with primary antibodies at 4 °C overnight and washed three times in TBST buffer, then followed by incubation with the secondary peroxidaseconjugated antibodies for 1 h at room temperature and subjected
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to immunodetection analyses using the ECL Kit. The density of the band was finally quantified using Image J software.
2.13. Statistical analysis All data are presented as means ± SEM (standard error of the mean) with the number of experiments in each group as mentioned in each of the figure legends. Statistical analysis was performed using oneway ANOVA followed by a post hoc (Bonferroni) test for multiple group comparison or Student t test between two groups. p < 0.05 was considered as statistically significant.
3. Results 3.1. Heat stress inhibited endogenous H2S production We first determined the expression of different H2S generating enzymes (CBS, CSE and 3MST) in the testicular tissue. As shown in Fig. 1A–C, all the above three enzymes were expressed in testicular germ cells. Interestingly, heat stress (42 °C, 30 min/day, 3 days) significantly decreased the expression levels of CBS (Fig. 1A) and CSE (Fig. 1B), but not that of 3-MST (Fig. 1C). We further examined the effect of heat stress on endogenous H2S production. As shown in Fig. 1D, heat stress significantly decreased testicular H2S level by ~34% compared with the value in the control group (Fig. 1D). These data suggest that heat stress may down-regulate CBS and CSE expression and inhibit H2S production.
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3.2. Exogenous application of H2S alleviates heat-induced apoptosis in testicular germ cells We further examined the effect of exogenous application of H2S on mouse testicular germ cell viability. As shown in Fig. 2A, treatment with NaHS (an H2S donor) at the concentration range of 1–200 μM for 30 min had no significant effect on germ cell viability. However, when NaHS concentration increased to 1 mM, it induced cell injury obviously. For this reason, NaHS at the concentration range of 1–50 μM was used in the following experiments. We next examined whether exogenous application of H2S can protect testicular germ cells against heat stress-induced apoptosis in vitro. As shown in Fig. 2B, pretreatment with NaHS at concentrations of 1 (a), 10 (b) and 50 μM (c) concentration-dependently protected testicular germ cells against heat-induced cell injury. Apoptotic death is recognized to be responsible for the heatinduced loss of germ cells and spermatogenesis damage [20]. We further examined the effect of NaHS on heat-induced programmed cell death in vivo with a modified TUNEL technique. As shown in Fig. 2C, the heat exposure significantly activated germ cell TUNEL positive apoptosis. However, pretreatment with NaHS (5.6 mg/kg) markedly decreased the number of apoptotic cells per 100 Sertoli cells from 73.7 ± 7.8 in heat treated group to 41.2 ± 7.7 in NaHS treatment group (p < 0.01). Testicular germ cells apoptosis was further visualized using a Hoechst 33342 staining method. In primary cultured testicular germ cells, heat exposure induced characteristic condensed and fragmented nuclei during apoptosis. This effect was significantly
Fig. 1. Effect of heat stress on endogenous H2S system in testis. (A–C) Representative Western blots and densitometric analysis showing that three episodes of heat stress (42 °C, 30 min/day, 3 days) inhibited expression levels of intrinsic CBS (A) and CSE (B), but not that of 3MST (C). (D) The same treatment also decreased testicular H2S generation. The values were presented as mean ± SEM from four to six independent experiments, ***p < 0.001 vs control group.
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Fig. 2. Effects of NaHS on heat-induced testicular germ cells apoptosis in vivo and in vitro. (A) Concentration-dependent effect of NaHS (1–1000 μM) on testicular germ cells. NaHS only at 1000 μM produced significant cell injury. n = 8. (B) Effect of NaHS on heat-induced cell injury. Pretreatment with NaHS (1, 10, 50 μM) for 30 min alleviated heat-induced cell death in a dose-dependent manner. (C) Representative images and quantification analysis showing that pretreatment with NaHS at 5.6 mg/kg inhibited heat-induced germ cell apoptosis. n = 8. (D) Representative images and quantification analysis showing that pretreatment with NaHS at 10 μM inhibited heat-induced germ cells apoptosis in vitro. Values are normalized by the value in the control group and presented as mean ± SEM from three to five independent experiments, §p > 0.05 vs. control, ***p < 0.001 vs. control, ##p < 0.01, ###p < 0.001 vs. heat treatment group.
attenuated by pretreatment with NaHS (Fig. 2D). Taken together, H2S may protect germ cells via its anti-apoptotic effects. 3.3. Effects of H2S on heat stress-stimulated apoptotic pathways We next examined the signaling mechanism by which H2S inhibited germ apoptosis. As shown in Fig. 3A, heat stress led to release of cytochrome C into the cytosol in cultured testicular germ cells. Pretreatment with NaHS (10 μM) statistically attenuated this effect. In addition, NaHS pretreatment decreased the ratio of protein
expression of Bax/Bcl-2 (Fig. 3B), but had no effect on the activity of caspases 8 (Fig. 3C). These results indicate that inactivation of intrinsic pathway, rather than extrinsic pathway, mediates the anti-apoptotic effect of H2S. 3.4. Effects of H2S on mitochondrial function and ROS generation in heat-treated germ cells Mitochondrial function plays a central role in apoptosis. In cultured testicular germ cells, mitochondria function was examined
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Fig. 3. Effects of NaHS on apoptotic pathways activated by heat stress. (A) Pretreatment with NaHS (10 μM) inhibited heat exposure-induced cytochrome c release into the cytosol. (B) Representative images and densitometric analysis showing that short term exposure to heat (42 °C, 30 min) increased the ratio of protein expression of Bax/ Bcl-2, which was largely reversed by pretreatment with 10 μM NaHS for 30 min before heat exposure. (C) Heat exposure with or without NaHS pretreatment had no effect on the activity of caspase 8. Values are normalized by the value in the control group and presented as mean ± SEM from four to six independent experiments, **p < 0.01, ***p < 0.001 vs control, #p < 0.05 vs heat treatment group.
by measuring oxygen consumption and ATP level. As shown in Fig. 4A and B, heat exposure resulted in higher oxygen consumption and lower ATP level. Pretreatment with NaHS decreased the rate of oxygen consumption (158.8% ± 9.9% in heat-treated group vs. 119.3% ± 6.5% in NaHS pre-treated group, p < 0.01, Fig. 4A) and reversed the ATP depletion from 43.5% ± 3.1% in heat-treated group to 78.8% ± 8.4% in NaHS pre-treated group (p < 0.001, Fig. 4B). To further study the mechanisms for the effect of H2S on heatinduced apoptosis, two parameters, ROS production and SOD activity were detected in the cultured cells. As shown in Fig. 4C and D, heat exposure significantly increased intracellular ROS production (Fig. 4C) and suppressed SOD activity (Fig. 4D). H2S pretreatment attenuated these effects, suggesting that H2S may protect germ cells by stimulation of the activity of anti-oxidant. 4. Discussion In normal condition, human testicular temperature is maintained 3 °C lower than the core body temperature. This is necessary as germ cells and Sertoli cells are highly sensitive to elevated
temperature. Exposure of the testis to abnormal elevated temperature, either from hot environment or naturally occurring cryptorchidism, causes extensive degeneration of the germ cells by heat stress. In this study, we examined the physiological and pharmacological functions of H2S, an endogenous biological gas, in the testicular germ cells. We first determined the protein expression of different H2S generating enzymes. CBS and CSE were reported to be the two major enzymes to generate H2S in rat testes [16,21]. However, little is known about the distribution of 3MST in testicular germ cells. In this study, we found that all three enzymes (CBS, CSE and 3MST) are expressed in testicular germ cells. Interestingly, only the expressions of CBS and CSE were significantly decreased after exposure to heat at 42 °C for 30 min. No significant change was found in the expression of 3-MST upon heat challenge. We further examined endogenous H2S level. Similarly, endogenous H2S was largely decreased upon three episodes of heat treatment. These data suggest that endogenous H2S production may be impaired during heat stress. To confirm the effect of H2S on heat stress-caused male subfertility, NaHS, an H2S donor, was used. We found that pretreatment with NaHS
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Fig. 4. Effects of NaHS on heat-induced mitochondrial function damage and ROS generation. (A) Short term exposure to heat (42 °C, 30 min) led to a faster rate of oxygen consumption, which was blocked by NaHS pretreatment (10 μM, 30 min before heat exposure). (B) NaHS alleviated the ATP depletion caused by the heat exposure. (C) NaHS pretreatment inhibited heat exposure-induced ROS production in testicular germ cells. (D) NaHS pretreatment reversed heat exposure-induced SOD deficiency in testicular germ cells. Values are normalized by the value in the control group and presented as mean ± SEM. n = 6–8, ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs heat treatment group.
(10 μM, 30 min) significantly protected heat stress-induced cell death. Accumulating evidence showed that excessive germ cell apoptosis/ abrupt spermatogenesis is the major pathology upon local testicular heat treatment [6,22,23]. Consistent with previous findings, we also found that exposure to heat at 42 °C for 30 min significantly increased the number of apoptotic cells by increasing the ratio of Bax/ Bcl-2 and release of cytochrome C. However, heat stress had no effect on the activity of caspases 8. These results imply that heat triggers apoptosis via an intrinsic pathway in testicular germ cells. Compared with somatic cells, sperm has limited cellular cytoplasm and is especially susceptible to oxidative stress. The ROS attack the lipids in the sperm plasma membrane, oxidizes the polyunsaturated fatty acids of the mitochondria sheath, and had widely been accepted as a significant contributory factor to sperm apoptosis [24,25]. The mitochondrial respiratory chain is a major source of ROS. Uncoupled respiration and ATP synthesis potentially halt the electron flux and the leakage will be transferred to oxygen or other electron acceptors to generate ROS [26]. In this study, testicular germ cells undergoing heat-triggered apoptosis exhibits a faster oxygen consumption rate and a lower intracellular ATP content, consistent with a higher intracellular ROS level, which indicated
an increased oxidative stress associated with the mitochondria dysfunction. Application of NaHS, however, decreased oxygen consumption, reversed heat-induced ATP depletion, and led to less production of intracellular ROS. Furthermore, NaHS treatment also ameliorated the activity of SOD, one of the most important antioxidative enzymes on scavenging of free radicals. Results obtained in these studies suggest that the H2S protected testicular germ cells against heat-induced apoptosis via an anti-oxidant mechanism (Fig. 5).
5. Conclusions Our results showed the important roles of intrinsic H2S system on heat-induced testicular germ cells injury. We also demonstrated for the first time that application of H2S ameliorates heatinduced testicular cell apoptosis likely via its anti-oxidant and anti-apoptotic actions. Our results provided evidence for the potential therapeutic value of H2S on male infertility. However, this effect can only be achieved when H2S concentration is at a low concentration range. One should bear in mind that H2S at higher
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Fig. 5. Schematic illustration showing the mechanisms underlying the protective effect of H2S. H2S protects germ cells against heat injury via anti-apoptotic and antioxidant effects through activation of SOD activity and its subsequent ROS scavenging effect. OCR: oxygen consumption rate.
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