ANDROLOGY
ISSN: 2047-2919
ORIGINAL ARTICLE
Correspondence: Myung-Geol Pang, Department of Animal Science and Technology, Chung-Ang University, Anseong, 456-756 Gyeonggi-do, Korea. E-mail: [email protected]
Sodium nitroprusside suppresses male fertility in vitro 1
Keywords: acrosome reaction, fertilization, embryo development, ROS, sodium nitroprusside
M. S. Rahman, 1W.-S. Kwon, 1J.-S. Lee, 1J. Kim, 1S.-J. Yoon, 1Y.-J. Park, 1 Y.-A. You, 2S. Hwang and 1M.-G. Pang 1
Department of Animal Science and Technology, Chung-Ang University, Anseong, and 2Animal Biotechnology Division, National Institute of Animal Science, RDA, Suwon, Gyeonggi-do, Korea
Received: 2-Jul-2014 Revised: 30-Jul-2014 Accepted: 11-Aug-2014 doi: 10.1111/j.2047-2927.2014.00273.x
SUMMARY Sodium nitroprusside is a nitric oxide donor involved in the regulation of the motility, hyperactivation, capacitation, and acrosome reaction (AR) of spermatozoa. However, the molecular mechanism underlying this regulation has not yet been elucidated. Therefore, this study was designed to evaluate the molecular basis for the effects of sodium nitroprusside on different processes in spermatozoa and its consequences on subsequent oocyte fertilization and embryo development. In this in vitro study, mouse spermatozoa were incubated with various concentrations of sodium nitroprusside (1, 10, and 100 lM) for 90 min. Our results showed that sodium nitroprusside inhibited sperm motility and motion kinematics in a dose-dependent manner by significantly enhancing intracellular iron and reactive oxygen species (ROS), and decreasing Ca2+, and adenosine triphosphate levels in spermatozoa. Moreover, short-term exposure of spermatozoa to sodium nitroprusside increased the tyrosine phosphorylation of sperm proteins involved in PKA-dependent regulation of intracellular calcium levels, which induced a robust AR. Finally, sodium nitroprusside significantly decreased the rates of fertilization and blastocyst formation during embryo development. Based on these results, we propose that sodium nitroprusside increases ROS production and precocious AR may alter overall sperm physiology, leading to poor fertilization and compromised embryonic development.
INTRODUCTION Approximately only one in 25 000 spermatozoa reaches the fallopian tube on their way to fertilize an oocyte. In fact, mammalian spermatozoa are unable to fertilize an oocyte before achieving functional maturation, which includes motility, hyperactivation, capacitation, and the acrosome reaction (AR) (Rahman et al., 2014). Sperm motility and hyperactivation are considered important factors in successful pregnancies as only hyperactivated motile spermatozoa are able to fertilize oocytes (Suarez, 2008; Rahman et al., 2013). Therefore, measuring the fraction of spermatozoa in a population that is motile is one of the most extensively used measures of semen quality (Park et al., 2012), and inadequate sperm motility is a common cause of subfertility or infertility (Suarez, 2008). Under normal circumstances, both in vivo and in vitro, spermatozoa require a period of preparation, termed capacitation, to gain the capacity to fertilize oocytes (Rodriguez et al., 2005). Capacitation is followed by an exocytotic event known as the AR that allows spermatozoa to penetrate the zona pellucida (ZP) and fuse with the oocyte © 2014 American Society of Andrology and European Academy of Andrology
plasma membrane (Yanagimachi, 1994; Fraser, 1998; Buzadzic et al., 2014). During these processes, complex coordination of multiple factors, including changes in the fluidity of the plasma membrane, intracellular ion concentrations (e.g. K+, Na+, Ca2+), energy metabolism, protein phosphorylation, bicarbonate, cyclic adenosine monophosphate (cAMP), protein kinase activity, adenosine triphosphate (ATP), pH, etc., is required (Wennemuth et al., 2003; Rodriguez et al., 2005; Visconti, 2009). It has been demonstrated that sodium nitroprusside, a nitric oxide (NO) donor, regulates a variety of cell signals in spermatozoa, including capacitation and the AR (Buzadzic et al., 2014), and may potentially regulate male fertility (Rosselli et al., 1995; Nobunaga et al., 1996; Rodriguez et al., 2005; Buzadzic et al., 2014); however, the underlying molecular mechanism remains largely unknown. A literature review showed that sodium nitroprusside has a biphasic role in the regulation of sperm function; low, controlled NO levels produced by sodium nitroprusside have a positive effect on sperm function, whereas higher concentrations have Andrology, 1–11
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an opposite effect (Revelli et al., 2001; Miraglia et al., 2011; Buzadzic et al., 2014). Consistent with this finding, higher NO levels have been reported in the semen of asthenozoospermic infertile men than those of the normozoospermic fertile counterpart (Perera et al., 1996; Balercia et al., 2004; Bolanos et al., 2008). Mitochondria play a central role in energy metabolism and regulate the sperm motility, AR, and spermatozoa–oocyte fusion (Gallon et al., 2006; Agarwal et al., 2008; Otasevic et al., 2013). It has been demonstrated that while higher levels of NO (lM) inhibit mitochondrial respiration in spermatozoa (Rosselli et al., 1995; Weinberg et al., 1995), lower concentrations (nM) have been strongly associated with energy production (Hellstrom et al., 1994). In addition, other research groups showed that higher levels of NO involve in sperm plasma membrane lipid peroxidation (Baker et al., 1996), axoneme, DNA, RNA, and proteins damages (Agarwal & Prabakaran, 2005). Therefore, NO must be maintained at certain levels to ensure normal sperm function. An intensive literature search showed that only two studies have attempted to evaluate the effect of NO on embryo development followed by in vivo fertilization (Joo et al., 1999) and human spermatozoa–zona binding ability (Wu et al., 2004). Therefore, although the effect of sodium nitroprusside on spermatozoa/ male fertility has been researched for last few decades (Nobunaga et al., 1996; Zhang & Zheng, 1996 Revelli et al., 2001; Miraglia et al., 2011), several fundamental questions remain unanswered. For example, how does sodium nitroprusside effect different key processes in spermatozoa, including motility, hyperactivation, capacitation, and the AR? Moreover, what is the underlying molecular mechanism through which sodium nitroprusside controls these processes and consequent fertilization and embryo development? To answer these questions, we first investigated the effect of sodium nitroprusside on mouse spermatozoa. Secondly, to clarify the role of sodium nitroprusside in specific sperm processes, we measured the effect of sodium nitroprusside on various parameters related to sperm motility, capacitation, and the AR, including intracellular iron, reactive oxygen species (ROS), calcium ([Ca2+]i), ATP, lactose dehydrogenase (LDH; as a measure of cytotoxicity), tyrosine phosphorylation, and protein kinase A (PKA) activity levels. Finally, to elucidate the potential regulatory role of sodium nitroprusside in fertility and embryo development, the effect of sodium nitroprusside on fertilization and early embryogenesis was assayed using an in vitro fertilization (IVF) system.
MATERIALS AND METHODS Ethical statement All animal procedures were performed in accordance with the guidelines for the ethical treatment of animals, and were approved by the Institutional Animal Care and Use Committee of Chung-Ang University, Seoul, Republic of Korea. Media and chemicals Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Modified Tyrode’s medium was used as a basic medium (BM), and was prepared fresh before each experiment (Quinn et al., 1982). The BM contained 97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2H2O, 0.36 mM NaH2PO4H2O, 5.56 mM D-glucose, 25 mM NaHCO3, 1.78 mM 2
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ANDROLOGY CaCl2H2O, 24.9 mM Na-lactate, 0.47 mM Na-pyruvate, and 50 lg/mL gentamycin. The medium was pre-incubated for 1 day prior to the experiment, and bovine serum albumin (BSA; 4 mg/mL) was added to induce capacitation of the spermatozoa. The sodium nitroprusside stock solution was diluted with distilled water and stored in an aluminum foil-wrapped plastic container at 4 °C in the dark. Experiments using sodium nitroprusside were performed in a safety cabinet under a red light. The stock solution was diluted with BM to reach the desired final molar concentrations of 1, 10, and 100 lM. Preparation and treatment of mouse spermatozoa Experimental mice were housed in a temperature (22 2 °C), ventilation, and light-controlled (12-h light/dark) room and were provided ad libitum access to laboratory feed (Cargill Agripurina, Seongnom, Korea) and water. A mouse sperm suspension was prepared using 8- to 12-week-old male ICR mice (Nara Biotech, Seoul, Korea). Spermatozoa were collected from the mice using a standard procedure (Tayama et al., 2006). In brief, both cauda epididymides from each mouse were separated, and the associated fat was removed. The sample was placed on a piece of filter paper to remove the excess liquid. The cauda epididymides were placed in cell culture dishes with BM containing 0.4% BSA, and then punctured using a sterile needle to facilitate the release of spermatozoa. The released spermatozoa were incubated for approximately 10 min with 5% CO2 in air at 37 °C to facilitate dispersal (Kwon et al., 2013a,b; Shukla et al., 2013). Finally, the sperm suspension was incubated for 90 min under the same conditions to induce capacitation in BM supplemented with 1, 10, and 100 lM sodium nitroprusside in separate falcon tubes. Computer-assisted sperm analysis (CASA) Sperm motility and kinematic parameters were evaluated using CASA (SAIS plus version 10.1; Medical Supply, Seoul, Korea) (Kwon et al., 2013a; Shukla et al., 2013). In brief, 10 lL of a sample was placed in a Makler chamber (Makler, Haifa, Israel) on a heated (37 °C) stage (Kawano et al., 2014). The 109 phase contrast objective was used by the SAIS software to relay and analyze the spermatozoa. Five fields of each sample were randomly selected to evaluate the movement of at least 250 sperm cells. The particular settings of the program were previously determined (frames acquired, 20; frame rate, 30 Hz; minimum contrast, 7; minimum size, 5; low/high size gates, 0.4–1.5; low/ high intensity gates, 0.4–1.5; non-motile head size, 16; nonmotile brightness, 14). For each of the six independent experiments, three male mice per replicate were considered for the final results. Measurement of intracellular iron concentration The intracellular iron concentration was measured using the QuantiChrom Iron Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions (Raulfs et al., 2008). Briefly, spermatozoa were harvested and washed, and then 50 lL was placed into each well of a clear, flat-bottomed, 96-well plate. A working solution was prepared and equilibrated to room temperature (RT) for 15 min prior to the assay. Working solution (200 lL) was added to each well and incubated for 40 min at RT. Finally, the optical density (OD) at 590 nm was measured. The data reported are the ratio of the OD of the sodium nitroprusside-treated sample to that of the untreated © 2014 American Society of Andrology and European Academy of Andrology
SODIUM NITROPRUSSIDE DECREASES MALE FERTILITY
ANDROLOGY
control. For each of the six independent experiments, three male mice per replicate were considered for the final results.
control. For each of the four independent experiments, three male mice per replicate were considered for the final results.
Measurement of cellular reactive oxygen species Cellular ROS was measured using the oxidation-sensitive fluorescent dye 20 ,70 -dichlorofluorescin diacetate (DCFDA) (Abcam, Cambridge, UK) according to the manufacturer’s directions and a previously described method (Elkharaz et al., 2013). The DCFDA mix, 19 buffer, and 19 supplemental buffer were prepared according to the manufacturer’s directions. After 90-min incubation, the samples were washed once by centrifugation at 200 g for 10 min, and then resuspended in 1 mL of DCFDA mix and incubated at 37 °C for 30 min. After incubation, the samples were washed again in 19 buffer solution by centrifugation at 200 g for 10 min, and then resuspended in 19 supplemental buffer. Finally, the cell suspension (500 lL) was seeded in a dark 96-well plate. The sperm suspension was subjected to excitation at 485 nm and emitted fluorescence was measured at 535 nm. Fluorescence was detected with a microplate fluorometer (Gemini Em; Molecular Devices, Sunnyvale, CA, USA) and calculated with SoftMax Pro 5 (Molecular Devices). The fluorescence of all treatments and the control were calculated as a ratio (485/535) and the fluorescence is reported as the ratio of the fluorescence of the sodium nitroprusside-treated samples to that of the control. For each of the six independent experiments, three male mice per replicate were considered for the final results.
Detection of lactate dehydrogenase (LDH) To determine cytotoxicity, we used the CytoTox 96â Nonradioactive Cytotoxicity assay kit (Promega, Fitchburg, WI, USA), which is based on the calorimetric detection of released LDH. Target cells were harvested, washed, counted, and diluted 1 : 5000, and then placed in a 96-well plate at 50 lL/well. To ensure contact between effectors and target cells, the plate was centrifuged at 250 g for 4 min. The sample was then incubated for 4 h in incubator at 37 °C in 5% CO2. Aliquots (50 lL) were placed in a 96-well plate, and LDH activity was measured as the absorbance at 490 nm using a luminometer (Gemini Em) and calculated using SoftMax Pro 5 software. Activity is reported as the ratio of the fluorescence of sodium nitroprusside-treated samples to that of the control. For each of the six independent experiments, three male mice per replicate were considered for the final results.
Detection of intracellular ATP generation ATP in cells was quantitatively detected using an ATP Bioluminescence Assay Kit (CLS II; Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol (Shukla et al., 2013). Briefly, the cells were diluted to 105–108 cells/lL, and 25 lL of the diluted cells was placed in a 96-well plate. An equal volume of cell lysis reagent was added to each well and incubated at RT for 5 min. Finally, ATP dilutions in a 50-lL volume were added to 50 lL of luciferase reagent in a 96well plate, and luminescence was detected using a Microplate Multimode Reader (GloMaxâ-Multi Microplate Multimode Reader; Promega, Madison, WI, USA). For each of the four independent experiments, three male mice per replicate were considered for the final results. Detection of intracellular calcium ion concentration ([Ca2+]i) [Ca2+]i was measured according to a previously described method (Herrick et al., 2005; Kwon et al., 2013a; Shukla et al., 2013). Briefly, after incubating the spermatozoa in capacitation medium containing sodium nitroprusside for 60 min, 5 lM Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) was added. Dye was added to the spermatozoa for the last 30 min of the capacitation induction to passively load the probe. The sample was centrifuged at 200 g for 10 min, and then resuspended in DPBS. Two excitation wavelengths (340 and 380 nm) were used and emitted fluorescence was detected at 510 nm (Pinto et al., 2009). [Ca2+]i was calculated as the ratio of the fluorescence at 340 to that at 380 nm (F340/F380). A microplate fluorometer (Gemini Em) was utilized to detect the fluorescence, and SoftMax Pro 5 software (Molecular Devices) was used for the calculations. All the treatments and the control were calculated as the ratio of F340/F380, and the data are reported as the ratio of sodium nitroprusside-treated fluorescence to that of the © 2014 American Society of Andrology and European Academy of Andrology
Combined Hoechst 33258/chlortetracycline fluorescence assessment of capacitation status (H33258/CTC) The capacitation status of the spermatozoa was determined by a dual-staining method (Kwon et al., 2013a; Shukla et al., 2013). Briefly, 135 lL of treated spermatozoa was added to 15 lL of H33258 solution (10 lg H33258/mL DPBS) and incubated for 2 min at RT. Excess dye was removed by layering the mixture over 250 lL of 2% (w/v) polyvinylpyrrolidone in Dulbecco’s phosphate-buffered saline (DPBS). After centrifugation at 100 g for 2.5 min, the excess supernatant was removed, and the pellet was resuspended in 100 lL of DPBS, and then 100 lL of a freshly prepared chlortetracycline fluorescence (CTC) solution (750 mM CTC in 5 lL buffer: 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4) was added. Samples were observed under a Microphot-FXA microscope (Nikon, Tokyo, Japan) with epifluorescence illumination using ultraviolet BP 340–380/LP 425 and BP 450– 490/LP 515 excitation/emission filters for H33258 and CTC, respectively. This analysis revealed four separate patterns of capacitation status: dead (D pattern, blue fluorescence distributed uniformly over the entire sperm head), live non-capacitated (F pattern, bright green fluorescence distributed uniformly over the entire sperm head), live capacitated (B pattern, bright green fluorescence over the acrosomal region and a dark post-acrosomal region), or live acrosome-reacted (AR pattern, no fluorescence over the head, or green fluorescence only in the postacrosomal region) according to the criteria of Maxwell and Johnson (Maxwell & Johnson, 1999). At least 400 spermatozoa were evaluated per slide, under each condition. For each of the six independent experiments, three male mice per replicate were considered for the final results. Western blot analysis of phospho-PKA substrates and tyrosine phosphorylation of sperm proteins Western blot analysis of PKA activity and tyrosine phosphorylation in mouse spermatozoa was carried out according to a previously described method (Kwon et al., 2013b). Briefly, after treatment, each sample was washed twice with DPBS and centrifuged at 10 000 g for 10 min. Sperm pellets were resuspended in Laemmli sample buffer (63 mM Tris, 10% glycerol, 10% sodium dodecyl sulfate, and 5% bromophenol blue) containing Andrology, 1–11
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5% 2-mercaptoethanol and incubated for 10 min at RT (final concentration 500 9 106 cells/mL). Finally, the supernatants were separated by centrifugation at 10 000 g for 10 min. Samples were then subjected to SDS-PAGE using a 12% mini-gel system (Amersham, Piscataway, NJ, USA), and then the separated proteins were transferred to a polyvinylidene fluoride membrane (Amersham). The membrane was blocked by incubating it in a blocking agent (3%; Amersham) for 1 h at RT. To detect phospho-PKA substrates, the membrane was incubated with an antiphospho-PKA substrate rabbit monoclonal antibody [1 : 10 000 (diluted in blocking solution); Cell Signaling Technology, Danvers, MA, USA] overnight at 4 °C. The membrane was then incubated with an horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG [1 : 5000 (diluted in blocking solution); Abcam] for 1 h at RT. Tyrosine phosphorylation was detected using an HRP-conjugated mouse monoclonal anti-phosphotyrosine antibody [PY20, 1 : 2500 (diluted in blocking solution); Abcam] overnight at 4 °C. a-Tubulin was used as an internal control [detected with a monoclonal anti a-tubulin mouse antibody, 1 : 10 000 (diluted in blocking solution); Abcam] for 2 h at RT. Finally, the membranes were washed three times with PBST. Protein–antibody complexes on the membrane were visualized using an enhanced chemiluminescence technique. The bands were scanned with a GS-800-calibrated imaging densitometer (Bio-Rad, Hercules, CA, USA) and analyzed using Quantity One software (Bio-Rad). Finally, the ratios of phospho-PKA substrate/a-tubulin and phosphotyrosine/a-tubulin were calculated. For each of the four independent experiments, three male mice per replicate were considered for western blot analysis of phospho-PKA substrates and tyrosine phosphorylation. In vitro fertilization Eight- to twelve-week-old B6D2F1/CrljOri hybrid female mice were purchased from Nara Biotech and superovulated by an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin (hCG) 48 h later (Kwon et al., 2013a,b; Shukla et al., 2013). Cumulus–oocyte complexes (COCs) were collected from oviducts into DPBS after 15 h of hCG treatment. COCs were placed in 50 lL of BM supplemented with 10% FBS under mineral oil, and then incubated at 37 °C with 5% CO2 in air for 90 min before insemination. After capacitation, treated spermatozoa were washed with BM supplemented with 0.4% BSA, and 1 9 106/mL spermatozoa were
gently inseminated into the incubated COCs and further incubated for 6 h at 37 °C under the same conditions. After fertilization, normal zygotes were collected, and then incubated in 50 lL of BM supplemented with 0.4% BSA. Eighteen hours after insemination, the fertilization rate was assessed by determining the number of two-cell embryos. All two-cell embryos were separated and cultured in 50 lL of BM supplemented with 0.4% BSA for 4 days at 37 °C in 5% CO2. All embryos that developed to the blastocyst stage were counted. The final results are representative of three independent experiments. Statistical analysis Data were analyzed using one-way ANOVA in SPSS (Version 12.0; SPSS Inc., Chicago, IL, USA), and Tukey’s test was used to locate differences. All numerical data are presented as mean SEM. Tests were interpreted at a confidence level of 95% (p < 0.05).
RESULTS Effect of sodium nitroprusside on sperm motility and motion kinematics To explore the effect of sodium nitroprusside on the motility and motion kinematics of spermatozoa, CASA was performed following a 90-min incubation. The motility and motion kinematic parameters of spermatozoa treated with sodium nitroprusside (1, 10, and 100 lM) and the untreated control are shown in Table 1. The percentage of motile spermatozoa decreased significantly in the presence of higher sodium nitroprusside concentrations. A variety of motion parameters, including hyperactivated motility (%), curvilinear velocity, velocity straight line, velocity average path, mean angular displacement, dance, dance mean, and mean lateral head displacement, were decreased by sodium nitroprusside in a dose-dependent manner, and particularly noticeable effects were observed at the highest concentration (100 lM) of sodium nitroprusside (Table 1). The effect of sodium nitroprusside on the intracellular iron and ROS level in spermatozoa To evaluate the effect of sodium nitroprusside on intracellular iron and ROS levels in spermatozoa, both were quantitatively detected as described in the Materials and methods.
Table 1 Effect of sodium nitroprusside on sperm motility and motion kinematics Parameter
Control
1 MOT (%) HYP (%) VCL (lm/sec) VSL (lm/sec) VAP (lm/sec) MAD (degree) DNC DNM (lm) ALH
65.18 9.68 127.87 55.11 61.97 14.91 704.90 12.53 5.36
p-value
Sodium nitroprusside (lM)
3.00a 1.56a 8.0a 4.52a 4.08a 1.50a 77.50a 0.55a 0.32a
52.99 5.95 106.74 41.80 48.26 10.97 459.73 11.37 4.36
10
2.08a 2.04a,b 8.18a,b 4.16a,b 4.36a,b 1.65a,b 64.17a,b 0.57a 0.30a,b
39.04 3.18 76.33 26.68 33.87 5.78 258.19 9.62 3.25
100
2.31a,b 1.05a,b 6.88b,c 2.68b,c 3.01b,c 1.0b,c 42.19b,c 0.86a 0.29b,c
17.27 0c 40.79 15.17 19.19 2.50 87.66 4.60 1.68
5.28c 11.40c 4.32c 5.43c 0.86c 24.71c 1.30b 0.47c
0.001 0.006 0.001 0.001 0.001 0.008 0.002 0.01 0.007
Spermatozoa were incubated at 37 °C with 5% CO2 in air for 90 min in BM supplemented with sodium nitroprusside (1, 10, and 100 lM). Sperm motility (%) and kinematic parameters, including % hyperactivated motility (HYP), curvilinear velocity (VCL), velocity straight line (VSL), velocity average path (VAP), mean angular displacement (MAD), dance (DNC), dance mean (DNM), and mean lateral head displacement (ALH), were measured using the CASA system. Data are the mean of six replicates SEM. Different superscripts letters within the same row indicate significant differences (p < 0.05) by one-way ANOVA.
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SODIUM NITROPRUSSIDE DECREASES MALE FERTILITY
Spermatozoa intracellular iron levels were significantly (p = 0.001) increased by treatment with higher concentrations (10 and 100 lM) of sodium nitroprusside (Fig. 1A). In different cells, extracellular iron catalyzes the production of ROS through the Fenton reaction (Borkowska et al., 2011); therefore, we evaluated the effect of sodium nitroprusside on ROS production in spermatozoa using fluorescent dye assays. Significant increases in ROS production were observed in spermatozoa treated with all concentrations of sodium nitroprusside compared with the control (p = 0.001) (Fig. 1B). Effect of sodium nitroprusside on ATP and [Ca2+]i levels in spermatozoa It has been demonstrated that the fertilizing capability of spermatozoa depends on the appropriate and time-dependent occurrence of several fundamental events in spermatozoa, with [Ca2+]i and ATP extensively involved in almost every step (Tateno et al., 2013; Rahman et al., 2014; Krapf et al., 2014). Therefore, we evaluated the effect of sodium nitroprusside treatment on ATP and [Ca2+]i levels in spermatozoa. A significant decrease in ATP production was noted in spermatozoa treated with the highest concentration of sodium nitroprusside compared with the control (p = 0.001; Fig. 2A). Sodium nitroprusside treatment also decreased [Ca2+]i levels in a dose-dependent manner (p = 0.001; Fig. 2B). Effect of sodium nitroprusside on LDH levels in spermatozoa A literature search demonstrated that sodium nitroprusside as a NO donor has been reported to play an important role in cellular signal transduction as well as a cytotoxic effector molecule (Buzadzic et al., 2014). Therefore, next, we evaluated the effect of sodium nitroprusside treatment on LDH production as a measure of cytotoxicity. Significantly increased LDH levels were observed in spermatozoa treated with the highest concentration of sodium nitroprusside (p = 0.002) (Fig. 3).
Involvement of PKA-dependent phosphorylation of tyrosine proteins in the sodium nitroprusside-induced AR in spermatozoa To measure the effect of sodium nitroprusside on sperm capacitation, we performed combined Hoechst 33258/chlortetracycline fluorescence staining. Increased numbers of spontaneous acrosome-reacted (AR pattern, Fig. 4A) spermatozoa were noted after treatment with all tested concentrations of sodium nitroprusside (p = 0.001) (Fig. 4E). In contrast, the numbers of capacitated (B pattern, Fig. 4B) spermatozoa in the 1 and 100 lM sodium nitroprusside-treated groups did not differ from that of the control group; however, the 10 lM sodium nitroprusside-treated group did differ from the control group (p = 0.048) (Fig. 4F). Sodium nitroprusside treatment resulted in a dose-dependent decrease in numbers of non-capacitated (F pattern, Fig. 4C) spermatozoa (p = 0.001) (Fig. 4G). In addition, to evaluate the regulatory mechanism underlying the sodium nitroprusside-induced AR, we measured spermatozoa PKA activity and phosphotyrosine levels (Kwon et al., 2013a) by western blotting (Fig. 5A and B). We observed dose-dependent increases in the levels of five different PKA substrate species (~16, ~21, ~22, ~55, and ~65 kDa) compared with their levels in the control (p < 0.05), with particularly noticeable increases in the ~21 and ~65 kDa species (Fig. 5A, B). Similarly, we observed significant increases in the ~20 and ~56 kDa tyrosine-phosphorylated species in sodium nitroprusside-treated spermatozoa (p < 0.05) (Fig. 5C, D). Effect of sodium nitroprusside on fertilization and embryonic development Next, we used an IVF system to evaluate the effect of sodium nitroprusside on fertilization and embryonic development. The rate of fertilization was significantly decreased in spermatozoa treated with higher concentrations of sodium nitroprusside (10 and 100 lM; p = 0.001) (Fig. 6A). Similarly, the rate of blastocyst formation was significantly decreased by the treatment of spermatozoa with sodium nitroprusside in a dose-dependent manner (p = 0.001) (Fig. 6B).
Figure 1 Effect of sodium nitroprusside on intracellular iron ([Fe2+]i) and reactive oxygen species (ROS) levels in spermatozoa. (A) Differences in [Fe2+]i levels in spermatozoa after incubation with different concentrations of sodium nitroprusside. The [Fe2+]i level of the control was set to one, and the levels in the sodium nitroprusside-treated samples are expressed relative to the control. Data represent the mean of six replicates SEM. Data are the means of six replicates SEM. Values with different lower case letters are significantly different between the control and treatment samples by one-way ANOVA (p = 0.001). (B) Differences in ROS levels in control and sodium nitroprusside-treated samples. Data are the means of six replicates SEM. Values with different superscripts are significantly different between the control and treatment samples by one-way ANOVA (p = 0.001).
(A)
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(B)
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Figure 2 Effect of sodium nitroprusside on ATP and [Ca2+]i levels in spermatozoa. (A) Differences in ATP levels between control and sodium nitroprussidetreated samples. Data are the means of four replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001). (B) Differences in [Ca2+]i in control and sodium nitroprusside-treated samples. Data are the means of four replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001).
(A)
DISCUSSION A variety of biological and pharmacological agents can regulate sperm function (Thundathil et al., 2002). The presence of NO in diverse cells of the male reproductive organs (Rosselli, 1997) suggests a major role in the regulation of male fertility/ reproduction. Among NO donors, sodium nitroprusside is regarded as the most effective for the study of the action of NO (Joo et al., 1999). It has been demonstrated that sodium nitroprusside-induced low and controlled concentrations of NO play an important role in sperm physiology (Herrero et al., 2003; Rodriguez et al., 2005; Zalazar et al., 2012). However, opposite effects have been reported following exposure to higher concentrations of sodium nitroprusside (Rosselli et al., 1995; Nobunaga et al., 1996). Our findings revealed that the higher concentrations of sodium nitroprusside increase ROS production and premature AR may alter overall sperm function, leading to poor fertilization and compromised early embryonic development.
Figure 3 Effect of sodium nitroprusside on lactate dehydrogenase (LDH) levels in spermatozoa. Differences in LDH levels in control and sodium nitroprusside-treated samples. Data are the mean of six replicates SEM. Values with different superscripts are significantly different between control and treatment groups by one-way ANOVA (p = 0.002).
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In this study, sodium nitroprusside dose dependently decreased the motility and hyperactivation of spermatozoa, which were significantly decreased at the highest concentration of sodium nitroprusside tested (100 lM) (Table 1). Similar findings were reported in several previous studies (Rosselli et al., 1995; Weinberg et al., 1995; Nobunaga et al., 1996; Joo et al., 1999). Interestingly, in another study on bovine spermatozoa, no decrease in motility or hyperactivation was noted after incubation with 1–200 lM sodium nitroprusside (Rodriguez et al., 2005). The differences in these results may be because of the different culture conditions or different species used. In contrast, low doses of sodium nitroprusside have been reported to increase sperm motility in hamster (Yeoman et al., 1998), ram (Hassanpour et al., 2007), mice (Sliwa & Stochmal, 2000), and human (Zhang & Zheng, 1996). Consistent results were also reported for the effect of sodium nitroprusside on sperm kinematic parameters assessed in this study (Table 1). Clinically, higher levels of NO have been reported in infertile men with decreased sperm motility, whereas inhibition of NO synthase improved sperm motility (Nobunaga et al., 1996; Perera et al., 2002; Wu et al., 2004). According to our data, the sodium nitroprusside-mediated decreases in sperm motility and motion kinematics were associated with increased intracellular iron/ROS and decreased Ca2+/ ATP levels (Figs 1 & 2). The mammalian spermatozoa must be progressively motile for a certain period of time to fertilize an oocyte (Mukai & Okuno, 2004). It has been proposed that the energy required to regulate sperm motility is provided by ATP, which is predominantly produced via mitochondrial respiration nchez et al., 2003; Miki et al., 2004; Mukai & Okuno, (Dıez-Sa 2004). Therefore, if mitochondrial respiration is unable to synthesize ATP, it could decrease the sperm motility. Sodium nitroprusside produces NO and iron (Kim et al., 2006). Excess iron has a harmful effect as a catalyst for the production of ROS which is responsible for oxidative stress (Cozzi et al., 2000). Spermatozoa, which have a relatively low antioxidant capacity, are accordingly more susceptible to oxidative stress (Sawyer et al., 2003). In clinical cases, iron overload in b-thalassemia patients predisposes spermatozoa to oxidative injury, DNA damage, and motility loss (Perera et al., 2002). Loss of motility in © 2014 American Society of Andrology and European Academy of Andrology
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SODIUM NITROPRUSSIDE DECREASES MALE FERTILITY
Figure 4 Effect of sodium nitroprusside on the capacitation status of spermatozoa. (A) AR pattern (live acrosome-reacted spermatozoa, no fluorescence over the head, or green fluorescence only in the post-acrosomal region). (B) B pattern (live capacitated spermatozoa, bright green fluorescence over the acrosomal region, and a dark post-acrosomal region). (C) F pattern (live non-capacitated spermatozoa, bright green fluorescence distributed uniformly over the entire sperm head). (D) D pattern spermatozoa (dead spermatozoa, blue fluorescence distributed over the head). (E) Acrosome-reacted (AR pattern) spermatozoa in the control and sodium nitroprusside-treated samples. Data are the mean of six replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001). (F) Differences in the number of capacitated (B pattern) spermatozoa in response to sodium nitroprusside treatment. Data are the mean of six replicates SEM. Values with different superscripts (a,b) were significantly different by one-way ANOVA (p = 0.048). (G) Differences in the number of non-capacitated (F pattern) spermatozoa in response to sodium nitroprusside. Data are the mean of six replicates SEM. Values with different superscripts are significantly different (between the control and treatment groups) by one-way ANOVA (p = 0.001).
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human spermatozoa exposed to extracellular iron and ROS was directly correlated with lipid peroxidation (Gomez et al., 1998; Wennemuth et al., 2003; Buzadzic et al., 2014). Indeed, the mechanism by which lipid peroxidation leads to sperm motility loss involves change in the membrane fluidity, alteration of the plasma membrane integrity, and failure to maintain optimal flagella movement. Recent publications have suggested that the ROS can damage sperm axoneme, impair mitochondrial function, DNA, RNA, and proteins, subsequently decreases the sperm motility (de Lamirande & Gagnon, 1992; Agarwal & Prabakaran, 2005). Exposure of ROS causes ATP depletion and inhibition of the motility of intact human spermatozoa (de Lamirande & Gagnon, 1992). In addition, it has been reported that incubation of spermatozoa with an extracellular Ca2+ source induces motility and hyperactivation in mammalian spermatozoa (Xia et al., 2007; Marquez & Suarez, 2008). That said, measuring [Ca2+]i levels with the fluorescent Ca2+ indicator indo-1 suggested that spermatozoa motility and hyperactivation are regulated by Ca2+ influx. Therefore, the decreased sperm motility observed in this study might also be the direct result of reduced [Ca2+]i in spermatozoa. Based on the results of several published studies and our own, it is tempting to hypothesize that higher concentrations of sodium nitroprusside decrease sperm motility © 2014 American Society of Andrology and European Academy of Andrology
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parameters through multiple signaling cascades that are regulated via not only increased intracellular iron and ROS but also decreased Ca2+ and ATP production. Successful fertilization requires that spermatozoa complete capacitation and the AR (Rahman et al., 2013, 2014). Capacitation adjusts the ability of a spermatozoon to gain hyperactive motility to interact with the oocyte ZP, undergo the AR, and finally initiate oocyte plasma membrane fusion (Yanagimachi, 1994). Both changes are associated with alterations in sperm membrane fluidity and intracellular levels of, ions, cAMP, tyrosine phosphorylation, and PKA activity (Gomez et al., 1998; Baldi et al., 2000). We showed that the addition of sodium nitroprusside to spermatozoa leads to a significant dose-dependent induction of the AR (Fig. 4). Although similar findings have been reported in several studies on human (Joo et al., 1999; Revelli et al., 2001), bovine (Rodriguez et al., 2005), and mouse (Herrero et al., 1997; Zalazar et al., 2012) spermatozoa, the underlining molecular basis has not been clearly elucidated. We demonstrated herein that decrease in ATP/[Ca2+]i and induced corresponding increase in PKA/tyrosine phosphorylation activity accompany the AR in spermatozoa (Figs 2 & 5). Rogers & Morton (1973) reported that ATP levels in capacitated/AR hamster spermatozoa are ~20–35% of Andrology, 1–11
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Figure 5 Effect of sodium nitroprusside on PKA activity and tyrosine phosphorylation in spermatozoa. (A) Density of PKA substrates (open bar, ~65 kDa, p = 0.022; striped bar, ~55 kDa, p = 0.001; dotted bar, ~22 kDa, p = 0.001; gray bar, ~21 kDa, p = 0.006; and black bar, ~16 kDa, p = 0.001). Data are the mean of four replicates SEM. Values with different indicators (A,B,a,b,c,d,a,b,I,II,m,n,o) are significantly different between the control and treatment groups by one-way ANOVA. (B) Phospho-PKA substrates were probed with an anti-phospho-PKA antibody. Lane 1, Control; lane 2, 1 lM sodium nitroprusside; lane 3, 10 lM sodium nitroprusside; lane 4, 100 lM sodium nitroprusside. (C) Density of tyrosine-phosphorylated proteins (open bar, ~20 kDa, p = 0.001; black bar, ~56 kDa, p = 0.001) in control and sodium nitroprusside-treated samples. Data are the mean of four replicates SEM. Values with different upper and lower case letters (A,B,C,a,b) are significantly different between control and treatment groups by one-way ANOVA. (D) Tyrosinephosphorylated proteins were probed with PY 20. Lane 1, Control; lane 2, 1 lM sodium nitroprusside; lane 3, 10 lM sodium nitroprusside; lane 4, 100 lM sodium nitroprusside.
Figure 6 Effect of sodium nitroprusside on fertilization and embryonic development. (A) Cleavage rate in control and sodium nitroprusside-treated samples. Data represent the mean of three replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001). (B) Blastocyst formation rate in control and sodium nitroprusside-treated samples. Data represent the mean of three replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001).
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those in non-capacitated spermatozoa. ATP depletion during capacitation has been attributed to the loss of ATP needed to maintain sperm motility and activation of cAMP (Rogers & Morton, 1973). In contrast, for capacitation, extracellular Ca2+ influx into spermatozoa through the calcium-specific CatSper channel increases [Ca2+]i (Kwon et al., 2013a,b; Rahman et al., 2014). However, AR spermatozoa discharge Ca2+ from the cell interior, resulting in decreased [Ca2+]i (Kwon et al., 2013a,b). In addition, tyrosine phosphorylation also has an important role in the regulation of various physiological processes in spermatozoa, including the AR (Kwon et al., 2013a; Shukla 8
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et al., 2013). In fact, PKA activity directly affects the tyrosine phosphorylation status of sperm proteins during capacitation and the AR (Kwon et al., 2013a). The capacitation- and the AR-associated increases in the levels of phosphorylated Ser and Thr residues provide a linkage between the early increases in PKA activity and the increases in tyrosine phosphorylation (Jha & Shivaji, 2002). Based on our data and data from other published reports, we hypothesize that sodium nitroprusside modulates cellular tyrosine phosphorylation by controlling ATP and [Ca2+]i levels via a PKA-dependent pathway, culminating in the AR in spermatozoa. © 2014 American Society of Andrology and European Academy of Andrology
SODIUM NITROPRUSSIDE DECREASES MALE FERTILITY
Another novel finding is the significant inhibitory effect of sodium nitroprusside on fertilization and early embryonic development (Fig. 6), which might be mediated by a robust AR in these spermatozoa. The AR is important for fertilization (Kawano et al., 2014); however, studies have demonstrated that a premature AR may result in altered mitochondrial function and it can lead to a reduction in motility and failure of chromatin decondensation which have a tragedy consequences for sperm viability and fertility (Chaveiro et al., 2006; Wongtawan et al., 2006). Under normal circumstances, a component of the ZP of the unfertilized egg induces the AR in spermatozoa following binding. However, spermatozoa in which the AR has taken place prematurely prior to the spermatozoa reaching the oocyte are unable to penetrate the ZP resulting in a loss in fertilizing capacity (Florman & Ducibella, 2006). Similar conclusions regarding the effect of premature AR on spermatozoa-fertilizing ability have been drawn in two previous studies (Joo et al., 1999; Wu et al., 2004). In contrast, it has recently been documented that fertilizing mouse spermatozoa begins the AR before contacting the ZP during IVF (Inoue et al., 2011; Jin et al., 2011). Therefore, the increased ROS (Fig. 1) and LDH (Fig. 3) production because of sodium nitroprusside exposure might lead directly to low fertility. Increased ROS is associated with lipid peroxidation and mitochondrial damage (Chaveiro et al., 2006; Wongtawan et al., 2006), which ultimately decrease fertilization. Alternatively, the germ cell-specific disruption of LDH leads to male infertility through a rapid decline in cellular ATP levels, progressive motility, and hyperactivated motility (Odet et al., 2011). Collectively, our findings support the notion that compromised fertilization and embryonic development following exposure of spermatozoa to high concentrations of sodium nitroprusside are related to increased ROS, LDH, and a premature AR together with decreased sperm motility and hyperactivation. Moreover, we suggest that sodium nitroprusside induces increases in cellular tyrosine phosphorylation through the control of ATP and [Ca2+]i via a PKA-dependent pathway, ultimately resulting in a robust AR in spermatozoa. In summary, a high dose of sodium nitroprusside has a profound effect on critical sperm functions and fertilization, and has considerable implications for male fertility.
FUNDING This work was supported by the “Cooperative Research Program for Agricultural Science and Technology Development” (project no. PJ008415) of the Rural Development Administration, Korea. Jin Kim was supported through the “Chung-Ang University Excellent Student Scholarship”, Chung-Ang University, Korea.
AUTHOR CONTRIBUTIONS M.S.R., W.S.K., J.S.L., J.K., S.J.Y., Y.J.P., Y.A.Y., and S.H. performed experiments, analyzed data, and drafted the manuscript. M.G.P. supervised the design of study and the data analysis, and revised the manuscript. All authors critically reviewed the manuscript for intellectual content and gave final approval for the version to be published.
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ISSN: 2047-2919
ORIGINAL ARTICLE
Correspondence: Myung-Geol Pang, Department of Animal Science and Technology, Chung-Ang University, Anseong, 456-756 Gyeonggi-do, Korea. E-mail: [email protected]
Sodium nitroprusside suppresses male fertility in vitro 1
Keywords: acrosome reaction, fertilization, embryo development, ROS, sodium nitroprusside
M. S. Rahman, 1W.-S. Kwon, 1J.-S. Lee, 1J. Kim, 1S.-J. Yoon, 1Y.-J. Park, 1 Y.-A. You, 2S. Hwang and 1M.-G. Pang 1
Department of Animal Science and Technology, Chung-Ang University, Anseong, and 2Animal Biotechnology Division, National Institute of Animal Science, RDA, Suwon, Gyeonggi-do, Korea
Received: 2-Jul-2014 Revised: 30-Jul-2014 Accepted: 11-Aug-2014 doi: 10.1111/j.2047-2927.2014.00273.x
SUMMARY Sodium nitroprusside is a nitric oxide donor involved in the regulation of the motility, hyperactivation, capacitation, and acrosome reaction (AR) of spermatozoa. However, the molecular mechanism underlying this regulation has not yet been elucidated. Therefore, this study was designed to evaluate the molecular basis for the effects of sodium nitroprusside on different processes in spermatozoa and its consequences on subsequent oocyte fertilization and embryo development. In this in vitro study, mouse spermatozoa were incubated with various concentrations of sodium nitroprusside (1, 10, and 100 lM) for 90 min. Our results showed that sodium nitroprusside inhibited sperm motility and motion kinematics in a dose-dependent manner by significantly enhancing intracellular iron and reactive oxygen species (ROS), and decreasing Ca2+, and adenosine triphosphate levels in spermatozoa. Moreover, short-term exposure of spermatozoa to sodium nitroprusside increased the tyrosine phosphorylation of sperm proteins involved in PKA-dependent regulation of intracellular calcium levels, which induced a robust AR. Finally, sodium nitroprusside significantly decreased the rates of fertilization and blastocyst formation during embryo development. Based on these results, we propose that sodium nitroprusside increases ROS production and precocious AR may alter overall sperm physiology, leading to poor fertilization and compromised embryonic development.
INTRODUCTION Approximately only one in 25 000 spermatozoa reaches the fallopian tube on their way to fertilize an oocyte. In fact, mammalian spermatozoa are unable to fertilize an oocyte before achieving functional maturation, which includes motility, hyperactivation, capacitation, and the acrosome reaction (AR) (Rahman et al., 2014). Sperm motility and hyperactivation are considered important factors in successful pregnancies as only hyperactivated motile spermatozoa are able to fertilize oocytes (Suarez, 2008; Rahman et al., 2013). Therefore, measuring the fraction of spermatozoa in a population that is motile is one of the most extensively used measures of semen quality (Park et al., 2012), and inadequate sperm motility is a common cause of subfertility or infertility (Suarez, 2008). Under normal circumstances, both in vivo and in vitro, spermatozoa require a period of preparation, termed capacitation, to gain the capacity to fertilize oocytes (Rodriguez et al., 2005). Capacitation is followed by an exocytotic event known as the AR that allows spermatozoa to penetrate the zona pellucida (ZP) and fuse with the oocyte © 2014 American Society of Andrology and European Academy of Andrology
plasma membrane (Yanagimachi, 1994; Fraser, 1998; Buzadzic et al., 2014). During these processes, complex coordination of multiple factors, including changes in the fluidity of the plasma membrane, intracellular ion concentrations (e.g. K+, Na+, Ca2+), energy metabolism, protein phosphorylation, bicarbonate, cyclic adenosine monophosphate (cAMP), protein kinase activity, adenosine triphosphate (ATP), pH, etc., is required (Wennemuth et al., 2003; Rodriguez et al., 2005; Visconti, 2009). It has been demonstrated that sodium nitroprusside, a nitric oxide (NO) donor, regulates a variety of cell signals in spermatozoa, including capacitation and the AR (Buzadzic et al., 2014), and may potentially regulate male fertility (Rosselli et al., 1995; Nobunaga et al., 1996; Rodriguez et al., 2005; Buzadzic et al., 2014); however, the underlying molecular mechanism remains largely unknown. A literature review showed that sodium nitroprusside has a biphasic role in the regulation of sperm function; low, controlled NO levels produced by sodium nitroprusside have a positive effect on sperm function, whereas higher concentrations have Andrology, 1–11
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an opposite effect (Revelli et al., 2001; Miraglia et al., 2011; Buzadzic et al., 2014). Consistent with this finding, higher NO levels have been reported in the semen of asthenozoospermic infertile men than those of the normozoospermic fertile counterpart (Perera et al., 1996; Balercia et al., 2004; Bolanos et al., 2008). Mitochondria play a central role in energy metabolism and regulate the sperm motility, AR, and spermatozoa–oocyte fusion (Gallon et al., 2006; Agarwal et al., 2008; Otasevic et al., 2013). It has been demonstrated that while higher levels of NO (lM) inhibit mitochondrial respiration in spermatozoa (Rosselli et al., 1995; Weinberg et al., 1995), lower concentrations (nM) have been strongly associated with energy production (Hellstrom et al., 1994). In addition, other research groups showed that higher levels of NO involve in sperm plasma membrane lipid peroxidation (Baker et al., 1996), axoneme, DNA, RNA, and proteins damages (Agarwal & Prabakaran, 2005). Therefore, NO must be maintained at certain levels to ensure normal sperm function. An intensive literature search showed that only two studies have attempted to evaluate the effect of NO on embryo development followed by in vivo fertilization (Joo et al., 1999) and human spermatozoa–zona binding ability (Wu et al., 2004). Therefore, although the effect of sodium nitroprusside on spermatozoa/ male fertility has been researched for last few decades (Nobunaga et al., 1996; Zhang & Zheng, 1996 Revelli et al., 2001; Miraglia et al., 2011), several fundamental questions remain unanswered. For example, how does sodium nitroprusside effect different key processes in spermatozoa, including motility, hyperactivation, capacitation, and the AR? Moreover, what is the underlying molecular mechanism through which sodium nitroprusside controls these processes and consequent fertilization and embryo development? To answer these questions, we first investigated the effect of sodium nitroprusside on mouse spermatozoa. Secondly, to clarify the role of sodium nitroprusside in specific sperm processes, we measured the effect of sodium nitroprusside on various parameters related to sperm motility, capacitation, and the AR, including intracellular iron, reactive oxygen species (ROS), calcium ([Ca2+]i), ATP, lactose dehydrogenase (LDH; as a measure of cytotoxicity), tyrosine phosphorylation, and protein kinase A (PKA) activity levels. Finally, to elucidate the potential regulatory role of sodium nitroprusside in fertility and embryo development, the effect of sodium nitroprusside on fertilization and early embryogenesis was assayed using an in vitro fertilization (IVF) system.
MATERIALS AND METHODS Ethical statement All animal procedures were performed in accordance with the guidelines for the ethical treatment of animals, and were approved by the Institutional Animal Care and Use Committee of Chung-Ang University, Seoul, Republic of Korea. Media and chemicals Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Modified Tyrode’s medium was used as a basic medium (BM), and was prepared fresh before each experiment (Quinn et al., 1982). The BM contained 97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2H2O, 0.36 mM NaH2PO4H2O, 5.56 mM D-glucose, 25 mM NaHCO3, 1.78 mM 2
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ANDROLOGY CaCl2H2O, 24.9 mM Na-lactate, 0.47 mM Na-pyruvate, and 50 lg/mL gentamycin. The medium was pre-incubated for 1 day prior to the experiment, and bovine serum albumin (BSA; 4 mg/mL) was added to induce capacitation of the spermatozoa. The sodium nitroprusside stock solution was diluted with distilled water and stored in an aluminum foil-wrapped plastic container at 4 °C in the dark. Experiments using sodium nitroprusside were performed in a safety cabinet under a red light. The stock solution was diluted with BM to reach the desired final molar concentrations of 1, 10, and 100 lM. Preparation and treatment of mouse spermatozoa Experimental mice were housed in a temperature (22 2 °C), ventilation, and light-controlled (12-h light/dark) room and were provided ad libitum access to laboratory feed (Cargill Agripurina, Seongnom, Korea) and water. A mouse sperm suspension was prepared using 8- to 12-week-old male ICR mice (Nara Biotech, Seoul, Korea). Spermatozoa were collected from the mice using a standard procedure (Tayama et al., 2006). In brief, both cauda epididymides from each mouse were separated, and the associated fat was removed. The sample was placed on a piece of filter paper to remove the excess liquid. The cauda epididymides were placed in cell culture dishes with BM containing 0.4% BSA, and then punctured using a sterile needle to facilitate the release of spermatozoa. The released spermatozoa were incubated for approximately 10 min with 5% CO2 in air at 37 °C to facilitate dispersal (Kwon et al., 2013a,b; Shukla et al., 2013). Finally, the sperm suspension was incubated for 90 min under the same conditions to induce capacitation in BM supplemented with 1, 10, and 100 lM sodium nitroprusside in separate falcon tubes. Computer-assisted sperm analysis (CASA) Sperm motility and kinematic parameters were evaluated using CASA (SAIS plus version 10.1; Medical Supply, Seoul, Korea) (Kwon et al., 2013a; Shukla et al., 2013). In brief, 10 lL of a sample was placed in a Makler chamber (Makler, Haifa, Israel) on a heated (37 °C) stage (Kawano et al., 2014). The 109 phase contrast objective was used by the SAIS software to relay and analyze the spermatozoa. Five fields of each sample were randomly selected to evaluate the movement of at least 250 sperm cells. The particular settings of the program were previously determined (frames acquired, 20; frame rate, 30 Hz; minimum contrast, 7; minimum size, 5; low/high size gates, 0.4–1.5; low/ high intensity gates, 0.4–1.5; non-motile head size, 16; nonmotile brightness, 14). For each of the six independent experiments, three male mice per replicate were considered for the final results. Measurement of intracellular iron concentration The intracellular iron concentration was measured using the QuantiChrom Iron Assay Kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions (Raulfs et al., 2008). Briefly, spermatozoa were harvested and washed, and then 50 lL was placed into each well of a clear, flat-bottomed, 96-well plate. A working solution was prepared and equilibrated to room temperature (RT) for 15 min prior to the assay. Working solution (200 lL) was added to each well and incubated for 40 min at RT. Finally, the optical density (OD) at 590 nm was measured. The data reported are the ratio of the OD of the sodium nitroprusside-treated sample to that of the untreated © 2014 American Society of Andrology and European Academy of Andrology
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control. For each of the six independent experiments, three male mice per replicate were considered for the final results.
control. For each of the four independent experiments, three male mice per replicate were considered for the final results.
Measurement of cellular reactive oxygen species Cellular ROS was measured using the oxidation-sensitive fluorescent dye 20 ,70 -dichlorofluorescin diacetate (DCFDA) (Abcam, Cambridge, UK) according to the manufacturer’s directions and a previously described method (Elkharaz et al., 2013). The DCFDA mix, 19 buffer, and 19 supplemental buffer were prepared according to the manufacturer’s directions. After 90-min incubation, the samples were washed once by centrifugation at 200 g for 10 min, and then resuspended in 1 mL of DCFDA mix and incubated at 37 °C for 30 min. After incubation, the samples were washed again in 19 buffer solution by centrifugation at 200 g for 10 min, and then resuspended in 19 supplemental buffer. Finally, the cell suspension (500 lL) was seeded in a dark 96-well plate. The sperm suspension was subjected to excitation at 485 nm and emitted fluorescence was measured at 535 nm. Fluorescence was detected with a microplate fluorometer (Gemini Em; Molecular Devices, Sunnyvale, CA, USA) and calculated with SoftMax Pro 5 (Molecular Devices). The fluorescence of all treatments and the control were calculated as a ratio (485/535) and the fluorescence is reported as the ratio of the fluorescence of the sodium nitroprusside-treated samples to that of the control. For each of the six independent experiments, three male mice per replicate were considered for the final results.
Detection of lactate dehydrogenase (LDH) To determine cytotoxicity, we used the CytoTox 96â Nonradioactive Cytotoxicity assay kit (Promega, Fitchburg, WI, USA), which is based on the calorimetric detection of released LDH. Target cells were harvested, washed, counted, and diluted 1 : 5000, and then placed in a 96-well plate at 50 lL/well. To ensure contact between effectors and target cells, the plate was centrifuged at 250 g for 4 min. The sample was then incubated for 4 h in incubator at 37 °C in 5% CO2. Aliquots (50 lL) were placed in a 96-well plate, and LDH activity was measured as the absorbance at 490 nm using a luminometer (Gemini Em) and calculated using SoftMax Pro 5 software. Activity is reported as the ratio of the fluorescence of sodium nitroprusside-treated samples to that of the control. For each of the six independent experiments, three male mice per replicate were considered for the final results.
Detection of intracellular ATP generation ATP in cells was quantitatively detected using an ATP Bioluminescence Assay Kit (CLS II; Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol (Shukla et al., 2013). Briefly, the cells were diluted to 105–108 cells/lL, and 25 lL of the diluted cells was placed in a 96-well plate. An equal volume of cell lysis reagent was added to each well and incubated at RT for 5 min. Finally, ATP dilutions in a 50-lL volume were added to 50 lL of luciferase reagent in a 96well plate, and luminescence was detected using a Microplate Multimode Reader (GloMaxâ-Multi Microplate Multimode Reader; Promega, Madison, WI, USA). For each of the four independent experiments, three male mice per replicate were considered for the final results. Detection of intracellular calcium ion concentration ([Ca2+]i) [Ca2+]i was measured according to a previously described method (Herrick et al., 2005; Kwon et al., 2013a; Shukla et al., 2013). Briefly, after incubating the spermatozoa in capacitation medium containing sodium nitroprusside for 60 min, 5 lM Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) was added. Dye was added to the spermatozoa for the last 30 min of the capacitation induction to passively load the probe. The sample was centrifuged at 200 g for 10 min, and then resuspended in DPBS. Two excitation wavelengths (340 and 380 nm) were used and emitted fluorescence was detected at 510 nm (Pinto et al., 2009). [Ca2+]i was calculated as the ratio of the fluorescence at 340 to that at 380 nm (F340/F380). A microplate fluorometer (Gemini Em) was utilized to detect the fluorescence, and SoftMax Pro 5 software (Molecular Devices) was used for the calculations. All the treatments and the control were calculated as the ratio of F340/F380, and the data are reported as the ratio of sodium nitroprusside-treated fluorescence to that of the © 2014 American Society of Andrology and European Academy of Andrology
Combined Hoechst 33258/chlortetracycline fluorescence assessment of capacitation status (H33258/CTC) The capacitation status of the spermatozoa was determined by a dual-staining method (Kwon et al., 2013a; Shukla et al., 2013). Briefly, 135 lL of treated spermatozoa was added to 15 lL of H33258 solution (10 lg H33258/mL DPBS) and incubated for 2 min at RT. Excess dye was removed by layering the mixture over 250 lL of 2% (w/v) polyvinylpyrrolidone in Dulbecco’s phosphate-buffered saline (DPBS). After centrifugation at 100 g for 2.5 min, the excess supernatant was removed, and the pellet was resuspended in 100 lL of DPBS, and then 100 lL of a freshly prepared chlortetracycline fluorescence (CTC) solution (750 mM CTC in 5 lL buffer: 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4) was added. Samples were observed under a Microphot-FXA microscope (Nikon, Tokyo, Japan) with epifluorescence illumination using ultraviolet BP 340–380/LP 425 and BP 450– 490/LP 515 excitation/emission filters for H33258 and CTC, respectively. This analysis revealed four separate patterns of capacitation status: dead (D pattern, blue fluorescence distributed uniformly over the entire sperm head), live non-capacitated (F pattern, bright green fluorescence distributed uniformly over the entire sperm head), live capacitated (B pattern, bright green fluorescence over the acrosomal region and a dark post-acrosomal region), or live acrosome-reacted (AR pattern, no fluorescence over the head, or green fluorescence only in the postacrosomal region) according to the criteria of Maxwell and Johnson (Maxwell & Johnson, 1999). At least 400 spermatozoa were evaluated per slide, under each condition. For each of the six independent experiments, three male mice per replicate were considered for the final results. Western blot analysis of phospho-PKA substrates and tyrosine phosphorylation of sperm proteins Western blot analysis of PKA activity and tyrosine phosphorylation in mouse spermatozoa was carried out according to a previously described method (Kwon et al., 2013b). Briefly, after treatment, each sample was washed twice with DPBS and centrifuged at 10 000 g for 10 min. Sperm pellets were resuspended in Laemmli sample buffer (63 mM Tris, 10% glycerol, 10% sodium dodecyl sulfate, and 5% bromophenol blue) containing Andrology, 1–11
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5% 2-mercaptoethanol and incubated for 10 min at RT (final concentration 500 9 106 cells/mL). Finally, the supernatants were separated by centrifugation at 10 000 g for 10 min. Samples were then subjected to SDS-PAGE using a 12% mini-gel system (Amersham, Piscataway, NJ, USA), and then the separated proteins were transferred to a polyvinylidene fluoride membrane (Amersham). The membrane was blocked by incubating it in a blocking agent (3%; Amersham) for 1 h at RT. To detect phospho-PKA substrates, the membrane was incubated with an antiphospho-PKA substrate rabbit monoclonal antibody [1 : 10 000 (diluted in blocking solution); Cell Signaling Technology, Danvers, MA, USA] overnight at 4 °C. The membrane was then incubated with an horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG [1 : 5000 (diluted in blocking solution); Abcam] for 1 h at RT. Tyrosine phosphorylation was detected using an HRP-conjugated mouse monoclonal anti-phosphotyrosine antibody [PY20, 1 : 2500 (diluted in blocking solution); Abcam] overnight at 4 °C. a-Tubulin was used as an internal control [detected with a monoclonal anti a-tubulin mouse antibody, 1 : 10 000 (diluted in blocking solution); Abcam] for 2 h at RT. Finally, the membranes were washed three times with PBST. Protein–antibody complexes on the membrane were visualized using an enhanced chemiluminescence technique. The bands were scanned with a GS-800-calibrated imaging densitometer (Bio-Rad, Hercules, CA, USA) and analyzed using Quantity One software (Bio-Rad). Finally, the ratios of phospho-PKA substrate/a-tubulin and phosphotyrosine/a-tubulin were calculated. For each of the four independent experiments, three male mice per replicate were considered for western blot analysis of phospho-PKA substrates and tyrosine phosphorylation. In vitro fertilization Eight- to twelve-week-old B6D2F1/CrljOri hybrid female mice were purchased from Nara Biotech and superovulated by an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin (hCG) 48 h later (Kwon et al., 2013a,b; Shukla et al., 2013). Cumulus–oocyte complexes (COCs) were collected from oviducts into DPBS after 15 h of hCG treatment. COCs were placed in 50 lL of BM supplemented with 10% FBS under mineral oil, and then incubated at 37 °C with 5% CO2 in air for 90 min before insemination. After capacitation, treated spermatozoa were washed with BM supplemented with 0.4% BSA, and 1 9 106/mL spermatozoa were
gently inseminated into the incubated COCs and further incubated for 6 h at 37 °C under the same conditions. After fertilization, normal zygotes were collected, and then incubated in 50 lL of BM supplemented with 0.4% BSA. Eighteen hours after insemination, the fertilization rate was assessed by determining the number of two-cell embryos. All two-cell embryos were separated and cultured in 50 lL of BM supplemented with 0.4% BSA for 4 days at 37 °C in 5% CO2. All embryos that developed to the blastocyst stage were counted. The final results are representative of three independent experiments. Statistical analysis Data were analyzed using one-way ANOVA in SPSS (Version 12.0; SPSS Inc., Chicago, IL, USA), and Tukey’s test was used to locate differences. All numerical data are presented as mean SEM. Tests were interpreted at a confidence level of 95% (p < 0.05).
RESULTS Effect of sodium nitroprusside on sperm motility and motion kinematics To explore the effect of sodium nitroprusside on the motility and motion kinematics of spermatozoa, CASA was performed following a 90-min incubation. The motility and motion kinematic parameters of spermatozoa treated with sodium nitroprusside (1, 10, and 100 lM) and the untreated control are shown in Table 1. The percentage of motile spermatozoa decreased significantly in the presence of higher sodium nitroprusside concentrations. A variety of motion parameters, including hyperactivated motility (%), curvilinear velocity, velocity straight line, velocity average path, mean angular displacement, dance, dance mean, and mean lateral head displacement, were decreased by sodium nitroprusside in a dose-dependent manner, and particularly noticeable effects were observed at the highest concentration (100 lM) of sodium nitroprusside (Table 1). The effect of sodium nitroprusside on the intracellular iron and ROS level in spermatozoa To evaluate the effect of sodium nitroprusside on intracellular iron and ROS levels in spermatozoa, both were quantitatively detected as described in the Materials and methods.
Table 1 Effect of sodium nitroprusside on sperm motility and motion kinematics Parameter
Control
1 MOT (%) HYP (%) VCL (lm/sec) VSL (lm/sec) VAP (lm/sec) MAD (degree) DNC DNM (lm) ALH
65.18 9.68 127.87 55.11 61.97 14.91 704.90 12.53 5.36
p-value
Sodium nitroprusside (lM)
3.00a 1.56a 8.0a 4.52a 4.08a 1.50a 77.50a 0.55a 0.32a
52.99 5.95 106.74 41.80 48.26 10.97 459.73 11.37 4.36
10
2.08a 2.04a,b 8.18a,b 4.16a,b 4.36a,b 1.65a,b 64.17a,b 0.57a 0.30a,b
39.04 3.18 76.33 26.68 33.87 5.78 258.19 9.62 3.25
100
2.31a,b 1.05a,b 6.88b,c 2.68b,c 3.01b,c 1.0b,c 42.19b,c 0.86a 0.29b,c
17.27 0c 40.79 15.17 19.19 2.50 87.66 4.60 1.68
5.28c 11.40c 4.32c 5.43c 0.86c 24.71c 1.30b 0.47c
0.001 0.006 0.001 0.001 0.001 0.008 0.002 0.01 0.007
Spermatozoa were incubated at 37 °C with 5% CO2 in air for 90 min in BM supplemented with sodium nitroprusside (1, 10, and 100 lM). Sperm motility (%) and kinematic parameters, including % hyperactivated motility (HYP), curvilinear velocity (VCL), velocity straight line (VSL), velocity average path (VAP), mean angular displacement (MAD), dance (DNC), dance mean (DNM), and mean lateral head displacement (ALH), were measured using the CASA system. Data are the mean of six replicates SEM. Different superscripts letters within the same row indicate significant differences (p < 0.05) by one-way ANOVA.
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Spermatozoa intracellular iron levels were significantly (p = 0.001) increased by treatment with higher concentrations (10 and 100 lM) of sodium nitroprusside (Fig. 1A). In different cells, extracellular iron catalyzes the production of ROS through the Fenton reaction (Borkowska et al., 2011); therefore, we evaluated the effect of sodium nitroprusside on ROS production in spermatozoa using fluorescent dye assays. Significant increases in ROS production were observed in spermatozoa treated with all concentrations of sodium nitroprusside compared with the control (p = 0.001) (Fig. 1B). Effect of sodium nitroprusside on ATP and [Ca2+]i levels in spermatozoa It has been demonstrated that the fertilizing capability of spermatozoa depends on the appropriate and time-dependent occurrence of several fundamental events in spermatozoa, with [Ca2+]i and ATP extensively involved in almost every step (Tateno et al., 2013; Rahman et al., 2014; Krapf et al., 2014). Therefore, we evaluated the effect of sodium nitroprusside treatment on ATP and [Ca2+]i levels in spermatozoa. A significant decrease in ATP production was noted in spermatozoa treated with the highest concentration of sodium nitroprusside compared with the control (p = 0.001; Fig. 2A). Sodium nitroprusside treatment also decreased [Ca2+]i levels in a dose-dependent manner (p = 0.001; Fig. 2B). Effect of sodium nitroprusside on LDH levels in spermatozoa A literature search demonstrated that sodium nitroprusside as a NO donor has been reported to play an important role in cellular signal transduction as well as a cytotoxic effector molecule (Buzadzic et al., 2014). Therefore, next, we evaluated the effect of sodium nitroprusside treatment on LDH production as a measure of cytotoxicity. Significantly increased LDH levels were observed in spermatozoa treated with the highest concentration of sodium nitroprusside (p = 0.002) (Fig. 3).
Involvement of PKA-dependent phosphorylation of tyrosine proteins in the sodium nitroprusside-induced AR in spermatozoa To measure the effect of sodium nitroprusside on sperm capacitation, we performed combined Hoechst 33258/chlortetracycline fluorescence staining. Increased numbers of spontaneous acrosome-reacted (AR pattern, Fig. 4A) spermatozoa were noted after treatment with all tested concentrations of sodium nitroprusside (p = 0.001) (Fig. 4E). In contrast, the numbers of capacitated (B pattern, Fig. 4B) spermatozoa in the 1 and 100 lM sodium nitroprusside-treated groups did not differ from that of the control group; however, the 10 lM sodium nitroprusside-treated group did differ from the control group (p = 0.048) (Fig. 4F). Sodium nitroprusside treatment resulted in a dose-dependent decrease in numbers of non-capacitated (F pattern, Fig. 4C) spermatozoa (p = 0.001) (Fig. 4G). In addition, to evaluate the regulatory mechanism underlying the sodium nitroprusside-induced AR, we measured spermatozoa PKA activity and phosphotyrosine levels (Kwon et al., 2013a) by western blotting (Fig. 5A and B). We observed dose-dependent increases in the levels of five different PKA substrate species (~16, ~21, ~22, ~55, and ~65 kDa) compared with their levels in the control (p < 0.05), with particularly noticeable increases in the ~21 and ~65 kDa species (Fig. 5A, B). Similarly, we observed significant increases in the ~20 and ~56 kDa tyrosine-phosphorylated species in sodium nitroprusside-treated spermatozoa (p < 0.05) (Fig. 5C, D). Effect of sodium nitroprusside on fertilization and embryonic development Next, we used an IVF system to evaluate the effect of sodium nitroprusside on fertilization and embryonic development. The rate of fertilization was significantly decreased in spermatozoa treated with higher concentrations of sodium nitroprusside (10 and 100 lM; p = 0.001) (Fig. 6A). Similarly, the rate of blastocyst formation was significantly decreased by the treatment of spermatozoa with sodium nitroprusside in a dose-dependent manner (p = 0.001) (Fig. 6B).
Figure 1 Effect of sodium nitroprusside on intracellular iron ([Fe2+]i) and reactive oxygen species (ROS) levels in spermatozoa. (A) Differences in [Fe2+]i levels in spermatozoa after incubation with different concentrations of sodium nitroprusside. The [Fe2+]i level of the control was set to one, and the levels in the sodium nitroprusside-treated samples are expressed relative to the control. Data represent the mean of six replicates SEM. Data are the means of six replicates SEM. Values with different lower case letters are significantly different between the control and treatment samples by one-way ANOVA (p = 0.001). (B) Differences in ROS levels in control and sodium nitroprusside-treated samples. Data are the means of six replicates SEM. Values with different superscripts are significantly different between the control and treatment samples by one-way ANOVA (p = 0.001).
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Figure 2 Effect of sodium nitroprusside on ATP and [Ca2+]i levels in spermatozoa. (A) Differences in ATP levels between control and sodium nitroprussidetreated samples. Data are the means of four replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001). (B) Differences in [Ca2+]i in control and sodium nitroprusside-treated samples. Data are the means of four replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001).
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DISCUSSION A variety of biological and pharmacological agents can regulate sperm function (Thundathil et al., 2002). The presence of NO in diverse cells of the male reproductive organs (Rosselli, 1997) suggests a major role in the regulation of male fertility/ reproduction. Among NO donors, sodium nitroprusside is regarded as the most effective for the study of the action of NO (Joo et al., 1999). It has been demonstrated that sodium nitroprusside-induced low and controlled concentrations of NO play an important role in sperm physiology (Herrero et al., 2003; Rodriguez et al., 2005; Zalazar et al., 2012). However, opposite effects have been reported following exposure to higher concentrations of sodium nitroprusside (Rosselli et al., 1995; Nobunaga et al., 1996). Our findings revealed that the higher concentrations of sodium nitroprusside increase ROS production and premature AR may alter overall sperm function, leading to poor fertilization and compromised early embryonic development.
Figure 3 Effect of sodium nitroprusside on lactate dehydrogenase (LDH) levels in spermatozoa. Differences in LDH levels in control and sodium nitroprusside-treated samples. Data are the mean of six replicates SEM. Values with different superscripts are significantly different between control and treatment groups by one-way ANOVA (p = 0.002).
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In this study, sodium nitroprusside dose dependently decreased the motility and hyperactivation of spermatozoa, which were significantly decreased at the highest concentration of sodium nitroprusside tested (100 lM) (Table 1). Similar findings were reported in several previous studies (Rosselli et al., 1995; Weinberg et al., 1995; Nobunaga et al., 1996; Joo et al., 1999). Interestingly, in another study on bovine spermatozoa, no decrease in motility or hyperactivation was noted after incubation with 1–200 lM sodium nitroprusside (Rodriguez et al., 2005). The differences in these results may be because of the different culture conditions or different species used. In contrast, low doses of sodium nitroprusside have been reported to increase sperm motility in hamster (Yeoman et al., 1998), ram (Hassanpour et al., 2007), mice (Sliwa & Stochmal, 2000), and human (Zhang & Zheng, 1996). Consistent results were also reported for the effect of sodium nitroprusside on sperm kinematic parameters assessed in this study (Table 1). Clinically, higher levels of NO have been reported in infertile men with decreased sperm motility, whereas inhibition of NO synthase improved sperm motility (Nobunaga et al., 1996; Perera et al., 2002; Wu et al., 2004). According to our data, the sodium nitroprusside-mediated decreases in sperm motility and motion kinematics were associated with increased intracellular iron/ROS and decreased Ca2+/ ATP levels (Figs 1 & 2). The mammalian spermatozoa must be progressively motile for a certain period of time to fertilize an oocyte (Mukai & Okuno, 2004). It has been proposed that the energy required to regulate sperm motility is provided by ATP, which is predominantly produced via mitochondrial respiration nchez et al., 2003; Miki et al., 2004; Mukai & Okuno, (Dıez-Sa 2004). Therefore, if mitochondrial respiration is unable to synthesize ATP, it could decrease the sperm motility. Sodium nitroprusside produces NO and iron (Kim et al., 2006). Excess iron has a harmful effect as a catalyst for the production of ROS which is responsible for oxidative stress (Cozzi et al., 2000). Spermatozoa, which have a relatively low antioxidant capacity, are accordingly more susceptible to oxidative stress (Sawyer et al., 2003). In clinical cases, iron overload in b-thalassemia patients predisposes spermatozoa to oxidative injury, DNA damage, and motility loss (Perera et al., 2002). Loss of motility in © 2014 American Society of Andrology and European Academy of Andrology
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Figure 4 Effect of sodium nitroprusside on the capacitation status of spermatozoa. (A) AR pattern (live acrosome-reacted spermatozoa, no fluorescence over the head, or green fluorescence only in the post-acrosomal region). (B) B pattern (live capacitated spermatozoa, bright green fluorescence over the acrosomal region, and a dark post-acrosomal region). (C) F pattern (live non-capacitated spermatozoa, bright green fluorescence distributed uniformly over the entire sperm head). (D) D pattern spermatozoa (dead spermatozoa, blue fluorescence distributed over the head). (E) Acrosome-reacted (AR pattern) spermatozoa in the control and sodium nitroprusside-treated samples. Data are the mean of six replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001). (F) Differences in the number of capacitated (B pattern) spermatozoa in response to sodium nitroprusside treatment. Data are the mean of six replicates SEM. Values with different superscripts (a,b) were significantly different by one-way ANOVA (p = 0.048). (G) Differences in the number of non-capacitated (F pattern) spermatozoa in response to sodium nitroprusside. Data are the mean of six replicates SEM. Values with different superscripts are significantly different (between the control and treatment groups) by one-way ANOVA (p = 0.001).
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human spermatozoa exposed to extracellular iron and ROS was directly correlated with lipid peroxidation (Gomez et al., 1998; Wennemuth et al., 2003; Buzadzic et al., 2014). Indeed, the mechanism by which lipid peroxidation leads to sperm motility loss involves change in the membrane fluidity, alteration of the plasma membrane integrity, and failure to maintain optimal flagella movement. Recent publications have suggested that the ROS can damage sperm axoneme, impair mitochondrial function, DNA, RNA, and proteins, subsequently decreases the sperm motility (de Lamirande & Gagnon, 1992; Agarwal & Prabakaran, 2005). Exposure of ROS causes ATP depletion and inhibition of the motility of intact human spermatozoa (de Lamirande & Gagnon, 1992). In addition, it has been reported that incubation of spermatozoa with an extracellular Ca2+ source induces motility and hyperactivation in mammalian spermatozoa (Xia et al., 2007; Marquez & Suarez, 2008). That said, measuring [Ca2+]i levels with the fluorescent Ca2+ indicator indo-1 suggested that spermatozoa motility and hyperactivation are regulated by Ca2+ influx. Therefore, the decreased sperm motility observed in this study might also be the direct result of reduced [Ca2+]i in spermatozoa. Based on the results of several published studies and our own, it is tempting to hypothesize that higher concentrations of sodium nitroprusside decrease sperm motility © 2014 American Society of Andrology and European Academy of Andrology
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parameters through multiple signaling cascades that are regulated via not only increased intracellular iron and ROS but also decreased Ca2+ and ATP production. Successful fertilization requires that spermatozoa complete capacitation and the AR (Rahman et al., 2013, 2014). Capacitation adjusts the ability of a spermatozoon to gain hyperactive motility to interact with the oocyte ZP, undergo the AR, and finally initiate oocyte plasma membrane fusion (Yanagimachi, 1994). Both changes are associated with alterations in sperm membrane fluidity and intracellular levels of, ions, cAMP, tyrosine phosphorylation, and PKA activity (Gomez et al., 1998; Baldi et al., 2000). We showed that the addition of sodium nitroprusside to spermatozoa leads to a significant dose-dependent induction of the AR (Fig. 4). Although similar findings have been reported in several studies on human (Joo et al., 1999; Revelli et al., 2001), bovine (Rodriguez et al., 2005), and mouse (Herrero et al., 1997; Zalazar et al., 2012) spermatozoa, the underlining molecular basis has not been clearly elucidated. We demonstrated herein that decrease in ATP/[Ca2+]i and induced corresponding increase in PKA/tyrosine phosphorylation activity accompany the AR in spermatozoa (Figs 2 & 5). Rogers & Morton (1973) reported that ATP levels in capacitated/AR hamster spermatozoa are ~20–35% of Andrology, 1–11
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M. S. Rahman et al.
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Figure 5 Effect of sodium nitroprusside on PKA activity and tyrosine phosphorylation in spermatozoa. (A) Density of PKA substrates (open bar, ~65 kDa, p = 0.022; striped bar, ~55 kDa, p = 0.001; dotted bar, ~22 kDa, p = 0.001; gray bar, ~21 kDa, p = 0.006; and black bar, ~16 kDa, p = 0.001). Data are the mean of four replicates SEM. Values with different indicators (A,B,a,b,c,d,a,b,I,II,m,n,o) are significantly different between the control and treatment groups by one-way ANOVA. (B) Phospho-PKA substrates were probed with an anti-phospho-PKA antibody. Lane 1, Control; lane 2, 1 lM sodium nitroprusside; lane 3, 10 lM sodium nitroprusside; lane 4, 100 lM sodium nitroprusside. (C) Density of tyrosine-phosphorylated proteins (open bar, ~20 kDa, p = 0.001; black bar, ~56 kDa, p = 0.001) in control and sodium nitroprusside-treated samples. Data are the mean of four replicates SEM. Values with different upper and lower case letters (A,B,C,a,b) are significantly different between control and treatment groups by one-way ANOVA. (D) Tyrosinephosphorylated proteins were probed with PY 20. Lane 1, Control; lane 2, 1 lM sodium nitroprusside; lane 3, 10 lM sodium nitroprusside; lane 4, 100 lM sodium nitroprusside.
Figure 6 Effect of sodium nitroprusside on fertilization and embryonic development. (A) Cleavage rate in control and sodium nitroprusside-treated samples. Data represent the mean of three replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001). (B) Blastocyst formation rate in control and sodium nitroprusside-treated samples. Data represent the mean of three replicates SEM. Values with different superscripts are significantly different between the control and treatment groups by one-way ANOVA (p = 0.001).
(A)
those in non-capacitated spermatozoa. ATP depletion during capacitation has been attributed to the loss of ATP needed to maintain sperm motility and activation of cAMP (Rogers & Morton, 1973). In contrast, for capacitation, extracellular Ca2+ influx into spermatozoa through the calcium-specific CatSper channel increases [Ca2+]i (Kwon et al., 2013a,b; Rahman et al., 2014). However, AR spermatozoa discharge Ca2+ from the cell interior, resulting in decreased [Ca2+]i (Kwon et al., 2013a,b). In addition, tyrosine phosphorylation also has an important role in the regulation of various physiological processes in spermatozoa, including the AR (Kwon et al., 2013a; Shukla 8
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et al., 2013). In fact, PKA activity directly affects the tyrosine phosphorylation status of sperm proteins during capacitation and the AR (Kwon et al., 2013a). The capacitation- and the AR-associated increases in the levels of phosphorylated Ser and Thr residues provide a linkage between the early increases in PKA activity and the increases in tyrosine phosphorylation (Jha & Shivaji, 2002). Based on our data and data from other published reports, we hypothesize that sodium nitroprusside modulates cellular tyrosine phosphorylation by controlling ATP and [Ca2+]i levels via a PKA-dependent pathway, culminating in the AR in spermatozoa. © 2014 American Society of Andrology and European Academy of Andrology
SODIUM NITROPRUSSIDE DECREASES MALE FERTILITY
Another novel finding is the significant inhibitory effect of sodium nitroprusside on fertilization and early embryonic development (Fig. 6), which might be mediated by a robust AR in these spermatozoa. The AR is important for fertilization (Kawano et al., 2014); however, studies have demonstrated that a premature AR may result in altered mitochondrial function and it can lead to a reduction in motility and failure of chromatin decondensation which have a tragedy consequences for sperm viability and fertility (Chaveiro et al., 2006; Wongtawan et al., 2006). Under normal circumstances, a component of the ZP of the unfertilized egg induces the AR in spermatozoa following binding. However, spermatozoa in which the AR has taken place prematurely prior to the spermatozoa reaching the oocyte are unable to penetrate the ZP resulting in a loss in fertilizing capacity (Florman & Ducibella, 2006). Similar conclusions regarding the effect of premature AR on spermatozoa-fertilizing ability have been drawn in two previous studies (Joo et al., 1999; Wu et al., 2004). In contrast, it has recently been documented that fertilizing mouse spermatozoa begins the AR before contacting the ZP during IVF (Inoue et al., 2011; Jin et al., 2011). Therefore, the increased ROS (Fig. 1) and LDH (Fig. 3) production because of sodium nitroprusside exposure might lead directly to low fertility. Increased ROS is associated with lipid peroxidation and mitochondrial damage (Chaveiro et al., 2006; Wongtawan et al., 2006), which ultimately decrease fertilization. Alternatively, the germ cell-specific disruption of LDH leads to male infertility through a rapid decline in cellular ATP levels, progressive motility, and hyperactivated motility (Odet et al., 2011). Collectively, our findings support the notion that compromised fertilization and embryonic development following exposure of spermatozoa to high concentrations of sodium nitroprusside are related to increased ROS, LDH, and a premature AR together with decreased sperm motility and hyperactivation. Moreover, we suggest that sodium nitroprusside induces increases in cellular tyrosine phosphorylation through the control of ATP and [Ca2+]i via a PKA-dependent pathway, ultimately resulting in a robust AR in spermatozoa. In summary, a high dose of sodium nitroprusside has a profound effect on critical sperm functions and fertilization, and has considerable implications for male fertility.
FUNDING This work was supported by the “Cooperative Research Program for Agricultural Science and Technology Development” (project no. PJ008415) of the Rural Development Administration, Korea. Jin Kim was supported through the “Chung-Ang University Excellent Student Scholarship”, Chung-Ang University, Korea.
AUTHOR CONTRIBUTIONS M.S.R., W.S.K., J.S.L., J.K., S.J.Y., Y.J.P., Y.A.Y., and S.H. performed experiments, analyzed data, and drafted the manuscript. M.G.P. supervised the design of study and the data analysis, and revised the manuscript. All authors critically reviewed the manuscript for intellectual content and gave final approval for the version to be published.
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