ORIGINAL ARTICLE
Effect of mitochondrial calcium uniporter blocking on human spermatozoa A. Bravo1, F. Treulen1, P. Uribe1, R. Boguen1, R. Felmer1,2 & J. V. Villegas1,3 1 Scientific and Technological Bioresources Nucleus-Centre of Reproductive Biotechnology (BIOREN-CEBIOR), Universidad de La Frontera, Temuco, Chile; 2 Department of Agronomic Sciences and Natural Resources, Faculty of Agriculture and Forest Sciences, Universidad de La Frontera, Temuco, Chile; 3 Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Keywords Calcium—human sperm function—mitochondrial calcium uniporter—Ru360 Correspondence Juana V. Villegas, Universidad de La Frontera, Francisco Salazar 01145, Temuco, Chile. Tel.: +56 045 2325592; Fax: +56 045 2325600; E-mail: [email protected] Accepted: May 26, 2014 doi: 10.1111/and.12314
Summary Calcium (Ca2+) regulates a number of essential processes in spermatozoa. Ca2+ is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 lM Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer-aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence-based method, mitochondrial membrane potential (DΨm) using JC-1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, DΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.
Introduction The calcium ion (Ca2+) is a second messenger that regulates various cellular processes in somatic cells, including proliferation, post-translational protein modification and aerobic metabolism (Clapham, 2007). Intracellular Ca2+ concentration ([Ca2+]i) also plays a major role in all the important post-ejaculatory sperm functions (JimenezGonzalez et al., 2006). In spermatozoa, Ca2+ regulates a number of processes, such as the acrosome reaction, flagellar beat mode (including hyperactivation) and chemotaxis, and has a significant role in capacitation as has been reviewed (Publicover et al., 2007). The sperm plasma membrane, acrosome and mitochondria are places where the spermatozoa regulate [Ca2+]i (Rodriguez et al., 2012). The mitochondria have been recognised as organelles that impact on many different signalling pathways (Duchen, 2004; Soubannier & McBride, 2009), and their © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
relationship with Ca2+ is intimate and dynamic (Walsh et al., 2009). Mitochondrial Ca2+ uptake was described in the early 1960s in somatic cells (Deluca & Engstrom, 1961; Vasington & Murphy, 1962). Increases in mitochondrial Ca2+ concentrations ([Ca2+]-mt) contribute to the regulation of many cell functions, stimulation of the tricarboxylic acid cycle, coupling energy demand to adenosine triphosphate (ATP) production (McCormack et al., 1990) and to cell death (Patergnani et al., 2011). Ca2+ uptake across the inner mitochondrial membrane (IMM) is mediated by different channels, such as the rapid uptake mode (RaM) (Gunter et al., 2004; Rasola & Bernardi, 2011), mitochondrial ryanodine receptor (mRyR) (Beutner et al., 2001; Michels et al., 2009) and mitochondrial Ca2+ uniporter (mCU) (Carafoli, 2010; Rasola & Bernardi, 2011). The mCU is the main transporter involved in the uptake of Ca2+ into mitochondria (Patergnani et al., 2011). It is characterised by a highly 1
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selective ion channel, mediating Ca2+ influx across the IMM driven by negative DΨm (Kirichok et al., 2004). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been established as the most potent and specific inhibitor of the mCU in vitro (Matlib et al., 1998). Ru360 is a compound isolated from commercial ruthenium red (RR) (Ying et al., 1991). Blocking mCU with Ru360 has revealed a protective role against ischaemia– reperfusion in cardiac cells (Zhang et al., 2006), and a negative effect on the capacitation of bovine cryopreserved spermatozoa (Rodriguez et al., 2012). In human spermatozoa, Ru360 has been shown to decrease caspase3 activation and phosphatidylserine exposure induced by either H2O2 or progesterone (Bejarano et al., 2008). However, the contribution of the mCU to normal human sperm function has not been studied to date. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Materials and methods
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re-suspended in spermatozoa-free seminal plasma and kept at 37 °C (World Health Organization, 2010). Threehundred microlitres aliquots were exposed to 5 and 10 lM of Ru360 at 37 °C, and an untreated control group was also included in the analysis. Sperm motility was measured every 1 h for 4 h. Effect of mCU blocking on sperm viability Sperm viability was evaluated using propidium iodide (PI) exclusion staining. Briefly, 2 9 106 spermatozoa ml1 were incubated with 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control was also included. After incubation, the spermatozoa were washed twice by centrifugation at 500 g for 5 min, re-suspended with HTF, and 1 lM of PI (Sigma-Aldrich Inc., St Louis, MO, USA) was added. After 5 min, the results were analysed by flow cytometry (see Analysis by flow cytometry below), and sperm viability was measured as the percentage of PI-negative spermatozoa.
Semen collection, preparation and analysis
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The semen samples were obtained by masturbation from four apparently healthy donors along the time study. Donors previously agreed to participate and signed an informed consent, which was approved by Scientific Ethics Committee at the Universidad de La Frontera. Semen collection and analysis were performed according to the guidelines of the World Health Organization (2010). Human spermatozoa were selected by direct swim-up method and re-suspended in human tubal fluid medium (HTF) (Quinn et al., 1985).
The ATP levels were assessed using an ATP Assay kit (Merck KGaA, Darmstadt, Germany) based on a luminescence method. The assay utilises luciferase to catalyse the formation of light from ATP, luciferin and oxygen, whereby the amount of ATP is quantified by the amount of light produced. Briefly, spermatozoa were adjusted to 5 9 106 spermatozoa ml1 and exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control group was also included. After incubation, the spermatozoa were washed once, re-suspended in 1 ml of HTF, and 10 ll of each suspension were deposited into a white-walled 96well luminometer plate. Then, 100 ll of nucleotide-releasing buffer and 1 ll of ATP-monitoring enzyme were added to each well, incubated for 60 s at 25 °C and finally measured with a luminometer (Luminoskan; Thermo Scientific, Atlanta, GA, USA).
Mitochondrial calcium uniporter blocking The mCU blocking was performed with Ru360 (Merck KGaA, Darmstadt, Germany), which is a potent and specific inhibitor of mCU. In isolated mitochondria, Ru360 has IC50 of 184 pM; however, in intact cells, a concentration of 10 lM of Ru360 is required for 30 min to effectively block Ca2+ uptake into the mitochondria (Matlib et al., 1998). For this reason, 5 and 10 lM of Ru360 were used to assess a concentration-dependent effect. Effect of mCU blocking on sperm motility Sperm motility was evaluated using a computer-aided sperm analysis (CASA) with INTEGRATED SPERM ANALYSIS SYSTEM software (ISAS; Proiser, Valencia, Spain). The percentage of total sperm motility was assessed for each experiment. Neat semen samples were adjusted to 15 9 106 spermatozoa ml1. For this, spermatozoa were 2
Effect of mCU blocking on DΨm The DΨm was assessed using a M^ıt-E-ΨTM mitochondrial permeability detection kit (Enzo Life Sciences Inc., Farmingdale, NY, USA), which uses the reagent 5,50 6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-benzamidazolocarbocyanin iodide (JC-1), a cationic dye that exhibits potential-dependent accumulation in mitochondria, which is indicated by a change in orange fluorescence intensity (590 nm). Briefly, 2 9 106 spermatozoa ml1 were exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control group was also included in the analysis. After incubation, the spermatozoa were washed once and re-suspended in 1 ml of HTF, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
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and then 1 ll of JC-1 1009 was added and incubated for 15 min at 37 °C. Finally, the spermatozoa were washed once, re-suspended in 500 ll of HTF and analysed by flow cytometry (see Analysis by flow cytometry below). Effect of mCU blocking on reactive oxygen species production The dihydroethidium dye (DHE; Molecular probes, Life Technologies, Carlsbad, CA, USA) was used to evaluate the effect of Ru360 on reactive oxygen species (ROS) production. DHE enters the cells and reacts with the superoxide anion to form 2-hydroxyethidium, a fluorescent product which binds strongly to DNA. SYTOXâ Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA), a nucleic acid staining with high affinity that readily enters to cells with damaged plasma membrane but cannot penetrate the membranes of living cells, was used to evaluate cell viability. Briefly, spermatozoa were adjusted to 2 9 106 spermatozoa ml1 and exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control group was also included. After incubation, the spermatozoa were washed once and re-suspended in 1 ml of HTF, then 1 ll of DHE (2 mM) and 1 ll of SYTOX green (0.5 mM) were added. Spermatozoa were incubated for 15 min at 37 °C. Finally, spermatozoa were washed once, re-suspended in 500 ll of HTF and analysed by flow cytometry (see Analysis by flow cytometry below). Analysis by flow cytometry
test. Data from all parameters analysed were transformed to a logarithmic scale prior to analysis because they did not pass the normality test. Sperm viability, ATP levels, DΨm and ROS production were evaluated by one-way analysis of variance (ANOVA). Sperm motility was assessed using a two-way ANOVA with a Bonferroni’s post-test. A P value below 0.05 was considered statistically significant. Results Effect of mCU blocking on sperm motility Sperm motility is one of the most important functions of the male gamete, and Ca2+ plays a pivotal role in its regulation (Darszon et al., 2011). For this reason, the effect of mCU blocking on total sperm motility was evaluated. Figure 1 shows a decrease in total sperm motility, which is significant after 4 h of incubation with 10 lM of Ru360, compared to the untreated control. However, 5 lM of Ru360 showed no significant effect on total sperm motility (Fig. 1). Due to motility decreased with 10 lM of Ru360 at 4 h, this incubation time was used for subsequent evaluations. Effect of mCU blocking on sperm viability Our results showed that treatment of human spermatozoa with Ru360 did not affect sperm viability after exposure to concentrations of 5 and 10 lM, as compared with the untreated control group after 4 h of incubation at 37 °C (Fig. 2). Because the Ru360 concentrations used did not
Fluorescence analysis was performed in a BD FACSCanto II flow cytometer (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA), controlled by FACSDiva 6.1.3 software (Becton, Dickinson and Company). A total of 10 000 sperm events were analysed for each test. JC-1 orange aggregates and DHE fluorescence were read in the orange PE channel (585/42 nm filter). The SYTOX greenpositive stain was read with the green FITC channel (530/ 30 nm filter). The far red PerCP channel (LP670 nm) was used to read PI fluorescence. Statistical analysis The treatment of spermatozoa with Ru360 at each dose was carried out in duplicate; that is, the experimental groups and its control were made each one in two test tubes at the same day and with the same cell sample and experiments were repeated at least four times on different days. Results were expressed as mean standard deviation. Statistical evaluation was performed with the GRAPHPAD PRISM 5 software package (GraphPad, La Jolla, CA, USA). Data normality was checked using D’Agostino’s © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Fig. 1 Evaluation of mCU blocking on total sperm motility. Spermatozoa were incubated with 5 and 10 lM of Ru360 for 4 h at 37 °C, and an untreated control was included. Results correspond to the mean standard deviation of four different experiments. mCU, mitochondrial calcium uniporter; (*): P < 0.05 compared to untreated control. 156 9 82 mm (300 9 300 DPI).
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alter sperm viability, these concentrations were also used to evaluate other parameters. Effect of mCU blocking on ATP levels Considering that the mCU blocking resulted in a decreased total sperm motility, the decision was made to assess whether this reduction was associated with decreased ATP levels, which are essential to maintaining motility (Ferramosca et al., 2012). The results demonstrated a significant decrease in ATP levels of spermatozoa exposed to both concentrations of Ru360, compared to the untreated control group (Fig. 3a). This result suggests that mCU blocking affects the ability of spermatozoa to maintain the ATP levels required for their normal function. Effect of mCU blocking on DΨm DΨm is an important indicator of the functional integrity of spermatozoa (Wang et al., 2003) because it is required for mitochondrial ATP production and sperm motility
Fig. 2 Evaluation of mCU blocking on sperm viability. Human spermatozoa were exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C, and an untreated control was included. The results correspond to the mean standard deviation of six different experiments. mCU, mitochondrial calcium uniporter. 95 9 76 mm (300 9 300 DPI).
(a)
(b)
(Guthrie & Welch, 2012). The results do not demonstrate a reduction of mean intensity of fluorescence (MFI) of JC-1 aggregated in human spermatozoa exposed to 5 and 10 lM Ru360 compared to the untreated control (Fig. 3b), demonstrating that the DΨm was not affected after mCU blocking. Effect of mCU blocking on ROS production Mitochondria are a major site of intracellular ROS formation in spermatozoa (Guthrie & Welch, 2012), and the oxidative stress caused by an increase in ROS production alters sperm function (Makker et al., 2009). It was observed that exposure of spermatozoa to 5 and 10 lM Ru360 did not significantly increase the MFI of DHE, indicating that mCU blocking did not induce an increase in ROS production in our experimental conditions (Fig. 3c). Discussion The mCU is the main mechanism of Ca2+ transport from the cytosol to the mitochondria to increase the Ca2+ level in the matrix (Zhang et al., 2006). In this study, we evaluated the effect of mCU blocking on human sperm function. We used Ru360, which has been used in different cell types (Zhang et al., 2006; Delmotte et al., 2012; Sanganahalli et al., 2013; de la Fuente et al., 2014) as well as in bovine (Rodriguez et al., 2012) and human spermatozoa (Bejarano et al., 2008). Our results showed that the percentage of sperm motility was decreased by the addition of 10 lM of Ru360 without affecting the sperm viability, suggesting a relationship between mCU and the maintenance of human sperm motility. It has been reported that Ca2+ accumulated in the mitochondrial matrix stimulates multiple energy-producing reactions, thereby increasing intramitochondrial ATP (c)
Fig. 3 Evaluation of mCU blocking on adenosine triphosphate (ATP) levels (a), DΨm (b) and intracellular ROS (c) on human spermatozoa. Spermatozoa were exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C, and an untreated control was added. The results correspond to the mean standard deviation of four different experiments for ATP levels, five different experiments for DΨm and four different experiments for intracellular ROS. ROS, reactive oxygen species; mCU, mitochondrial calcium uniporter; MFI, mean fluorescence intensity; AU, arbitrary units; RLU, relative light units; (*): P < 0.05 and (**): P < 0.01 both compared to untreated control. 249 9 92 mm (300 9 300 DPI).
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levels, which can be used as fuel for sperm-activated or hyperactivated motility (reviewed by Piomboni et al., 2012). Thus, the decrease in sperm motility after Ru360 exposure is likely due to the decrease in ATP levels as a result of a decrease in mitochondrial Ca2+ by mCU blocking. Therefore, we subsequently measured ATP levels in terms of a mCU blockade. We observed a decrease in ATP levels in spermatozoa exposed to Ru360, indicating that mCU blocking alters the ability of spermatozoa to maintain ATP levels. We propose that a decrease in ATP levels is due to mCU blocking, causing a decrease in mitochondrial Ca2+, and affecting ATP production. This is supported by studies conducted on somatic cells, which have reported that Ca2+ activates mitochondrial oxidative phosphorylation (OXPHOS), stimulating several dehydrogenases of the Krebs cycle, the electron transport chain (ETC) and the Fo–F1 ATP synthase (reviewed by Piomboni et al., 2012). Thus, when mCU blocking occurs, mitochondrial Ca2+ also decreases, reducing ATP production, which finally results in impaired sperm motility. In this study, sperm motility was only decreased at 4 h of mCU blocking. The maintenance of sperm motility for the first 3 h after mCU blocking could be explained by the fact that creatine phosphate produced by creatine kinase, is an energy reservoir for the rapid regeneration of ATP, playing an important role in sperm motility as yet has been affirmed (Ghaffari & Rostami, 2013). Thus, these ATP reserves could have been utilised by the spermatozoa for the first 3 h of incubation with Ru360. Moreover, both sperm motility and ATP levels were decreased, but they did not fall completely. This may be because in human spermatozoa there are other ways to produce ATP, such as glycolysis, which has been shown to produce ATP to contribute to sperm motility (Nascimento et al., 2008). Also, it has recently been described that spermatozoa have enzymatic tools to obtain energy from endogenous substrates like fatty acids, ketone bodies and glycogen (Amaral et al., 2013). These metabolic strategies on the part of spermatozoa could explain why ATP levels and sperm motility did not decrease completely after mCU blocking. In addition to the ATP level, sperm motility has been positively related to intact DΨm (Marchetti et al., 2002); therefore, this variable was also evaluated. Under our experimental conditions, this association was not observed, because the sperm motility was decreased but DΨm was not affected after mCU blocking. The maintenance of DΨm under mCU blocking conditions is supported by previous studies on other cell types. In rats, the perfusion of Ru360 partially inhibited the mCU, maintained OXPHOS and prevented the dissipation of DΨm (Cheng et al., 2013). Likewise, Ru360 decreased © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Ca2+-induced mitochondrial depolarisation in synaptic and nonsynaptic mitochondria (Yarana et al., 2012). An increase in the concentration of mitochondrial Ca2+ when this is incorporated by mCU can uncouple the ETC. and thus decrease DΨm (reviewed by Rodriguez et al., 2012). Therefore, our results are in agreement with this because mCU blocking prevents the decrease of DΨm by inhibiting Ca2+ uptake and its adverse effects on ETC. Sperm motility has been negatively related to high levels of ROS (reviewed by Makker et al., 2009); therefore, ROS production by spermatozoa was measured. We did not observe any differences in ROS production after mCU blocking. These results indicate that the decrease in sperm motility under our experimental conditions was not a result of increased ROS production. The nonincreased ROS production under our experimental conditions is supported by investigations into other cells types, where it has been described that mitochondria take up and buffer large cytosolic Ca2+ loads, resulting in the release of ROS from the mitochondria to the cytosol (Dugan et al., 1995; Reynolds & Hastings, 1995; Bindokas et al., 1996). In this vein, mCU blocking, preventing entry of Ca2+ to the mitochondria, could explain the low levels of ROS production observed in our results. In summary, mCU blocking, in conditions that maintain viability, reduces sperm motility and ATP levels without affecting DΨm and ROS production. This indicates that the entry of Ca2+ into the mitochondrial matrix through this channel has a major role in the maintenance of sperm motility and ATP levels. Acknowledgements This work was supported by grant DI12-0102 (JV) from the Universidad de La Frontera and by doctoral thesis scholarship No. 24120981 from CONICYT (FT), Chile. References Amaral A, Castillo J, Estanyol JM, Ballesca JL, Ramalho-Santos J, Oliva R (2013) Human sperm tail proteome suggests new endogenous metabolic pathways. Mol Cell Proteomics 12:330–342. Bejarano I, Lozano GM, Ortiz A, Garcia JF, Paredes SD, Rodriguez AB, Pariente JA (2008) Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertil Steril 90:1340–1347. Beutner G, Sharma VK, Giovannucci DR, Yule DI, Sheu SS (2001) Identification of a ryanodine receptor in rat heart mitochondria. J Biol Chem 276:21482–21488. Bindokas VP, Jordan J, Lee CC, Miller RJ (1996) Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J Neurosci 16:1324–1336.
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Effect of mitochondrial calcium uniporter blocking on human spermatozoa A. Bravo1, F. Treulen1, P. Uribe1, R. Boguen1, R. Felmer1,2 & J. V. Villegas1,3 1 Scientific and Technological Bioresources Nucleus-Centre of Reproductive Biotechnology (BIOREN-CEBIOR), Universidad de La Frontera, Temuco, Chile; 2 Department of Agronomic Sciences and Natural Resources, Faculty of Agriculture and Forest Sciences, Universidad de La Frontera, Temuco, Chile; 3 Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Keywords Calcium—human sperm function—mitochondrial calcium uniporter—Ru360 Correspondence Juana V. Villegas, Universidad de La Frontera, Francisco Salazar 01145, Temuco, Chile. Tel.: +56 045 2325592; Fax: +56 045 2325600; E-mail: [email protected] Accepted: May 26, 2014 doi: 10.1111/and.12314
Summary Calcium (Ca2+) regulates a number of essential processes in spermatozoa. Ca2+ is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 lM Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer-aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence-based method, mitochondrial membrane potential (DΨm) using JC-1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, DΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.
Introduction The calcium ion (Ca2+) is a second messenger that regulates various cellular processes in somatic cells, including proliferation, post-translational protein modification and aerobic metabolism (Clapham, 2007). Intracellular Ca2+ concentration ([Ca2+]i) also plays a major role in all the important post-ejaculatory sperm functions (JimenezGonzalez et al., 2006). In spermatozoa, Ca2+ regulates a number of processes, such as the acrosome reaction, flagellar beat mode (including hyperactivation) and chemotaxis, and has a significant role in capacitation as has been reviewed (Publicover et al., 2007). The sperm plasma membrane, acrosome and mitochondria are places where the spermatozoa regulate [Ca2+]i (Rodriguez et al., 2012). The mitochondria have been recognised as organelles that impact on many different signalling pathways (Duchen, 2004; Soubannier & McBride, 2009), and their © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
relationship with Ca2+ is intimate and dynamic (Walsh et al., 2009). Mitochondrial Ca2+ uptake was described in the early 1960s in somatic cells (Deluca & Engstrom, 1961; Vasington & Murphy, 1962). Increases in mitochondrial Ca2+ concentrations ([Ca2+]-mt) contribute to the regulation of many cell functions, stimulation of the tricarboxylic acid cycle, coupling energy demand to adenosine triphosphate (ATP) production (McCormack et al., 1990) and to cell death (Patergnani et al., 2011). Ca2+ uptake across the inner mitochondrial membrane (IMM) is mediated by different channels, such as the rapid uptake mode (RaM) (Gunter et al., 2004; Rasola & Bernardi, 2011), mitochondrial ryanodine receptor (mRyR) (Beutner et al., 2001; Michels et al., 2009) and mitochondrial Ca2+ uniporter (mCU) (Carafoli, 2010; Rasola & Bernardi, 2011). The mCU is the main transporter involved in the uptake of Ca2+ into mitochondria (Patergnani et al., 2011). It is characterised by a highly 1
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selective ion channel, mediating Ca2+ influx across the IMM driven by negative DΨm (Kirichok et al., 2004). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been established as the most potent and specific inhibitor of the mCU in vitro (Matlib et al., 1998). Ru360 is a compound isolated from commercial ruthenium red (RR) (Ying et al., 1991). Blocking mCU with Ru360 has revealed a protective role against ischaemia– reperfusion in cardiac cells (Zhang et al., 2006), and a negative effect on the capacitation of bovine cryopreserved spermatozoa (Rodriguez et al., 2012). In human spermatozoa, Ru360 has been shown to decrease caspase3 activation and phosphatidylserine exposure induced by either H2O2 or progesterone (Bejarano et al., 2008). However, the contribution of the mCU to normal human sperm function has not been studied to date. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Materials and methods
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re-suspended in spermatozoa-free seminal plasma and kept at 37 °C (World Health Organization, 2010). Threehundred microlitres aliquots were exposed to 5 and 10 lM of Ru360 at 37 °C, and an untreated control group was also included in the analysis. Sperm motility was measured every 1 h for 4 h. Effect of mCU blocking on sperm viability Sperm viability was evaluated using propidium iodide (PI) exclusion staining. Briefly, 2 9 106 spermatozoa ml1 were incubated with 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control was also included. After incubation, the spermatozoa were washed twice by centrifugation at 500 g for 5 min, re-suspended with HTF, and 1 lM of PI (Sigma-Aldrich Inc., St Louis, MO, USA) was added. After 5 min, the results were analysed by flow cytometry (see Analysis by flow cytometry below), and sperm viability was measured as the percentage of PI-negative spermatozoa.
Semen collection, preparation and analysis
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The semen samples were obtained by masturbation from four apparently healthy donors along the time study. Donors previously agreed to participate and signed an informed consent, which was approved by Scientific Ethics Committee at the Universidad de La Frontera. Semen collection and analysis were performed according to the guidelines of the World Health Organization (2010). Human spermatozoa were selected by direct swim-up method and re-suspended in human tubal fluid medium (HTF) (Quinn et al., 1985).
The ATP levels were assessed using an ATP Assay kit (Merck KGaA, Darmstadt, Germany) based on a luminescence method. The assay utilises luciferase to catalyse the formation of light from ATP, luciferin and oxygen, whereby the amount of ATP is quantified by the amount of light produced. Briefly, spermatozoa were adjusted to 5 9 106 spermatozoa ml1 and exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control group was also included. After incubation, the spermatozoa were washed once, re-suspended in 1 ml of HTF, and 10 ll of each suspension were deposited into a white-walled 96well luminometer plate. Then, 100 ll of nucleotide-releasing buffer and 1 ll of ATP-monitoring enzyme were added to each well, incubated for 60 s at 25 °C and finally measured with a luminometer (Luminoskan; Thermo Scientific, Atlanta, GA, USA).
Mitochondrial calcium uniporter blocking The mCU blocking was performed with Ru360 (Merck KGaA, Darmstadt, Germany), which is a potent and specific inhibitor of mCU. In isolated mitochondria, Ru360 has IC50 of 184 pM; however, in intact cells, a concentration of 10 lM of Ru360 is required for 30 min to effectively block Ca2+ uptake into the mitochondria (Matlib et al., 1998). For this reason, 5 and 10 lM of Ru360 were used to assess a concentration-dependent effect. Effect of mCU blocking on sperm motility Sperm motility was evaluated using a computer-aided sperm analysis (CASA) with INTEGRATED SPERM ANALYSIS SYSTEM software (ISAS; Proiser, Valencia, Spain). The percentage of total sperm motility was assessed for each experiment. Neat semen samples were adjusted to 15 9 106 spermatozoa ml1. For this, spermatozoa were 2
Effect of mCU blocking on DΨm The DΨm was assessed using a M^ıt-E-ΨTM mitochondrial permeability detection kit (Enzo Life Sciences Inc., Farmingdale, NY, USA), which uses the reagent 5,50 6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-benzamidazolocarbocyanin iodide (JC-1), a cationic dye that exhibits potential-dependent accumulation in mitochondria, which is indicated by a change in orange fluorescence intensity (590 nm). Briefly, 2 9 106 spermatozoa ml1 were exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control group was also included in the analysis. After incubation, the spermatozoa were washed once and re-suspended in 1 ml of HTF, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
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and then 1 ll of JC-1 1009 was added and incubated for 15 min at 37 °C. Finally, the spermatozoa were washed once, re-suspended in 500 ll of HTF and analysed by flow cytometry (see Analysis by flow cytometry below). Effect of mCU blocking on reactive oxygen species production The dihydroethidium dye (DHE; Molecular probes, Life Technologies, Carlsbad, CA, USA) was used to evaluate the effect of Ru360 on reactive oxygen species (ROS) production. DHE enters the cells and reacts with the superoxide anion to form 2-hydroxyethidium, a fluorescent product which binds strongly to DNA. SYTOXâ Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA), a nucleic acid staining with high affinity that readily enters to cells with damaged plasma membrane but cannot penetrate the membranes of living cells, was used to evaluate cell viability. Briefly, spermatozoa were adjusted to 2 9 106 spermatozoa ml1 and exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C. An untreated control group was also included. After incubation, the spermatozoa were washed once and re-suspended in 1 ml of HTF, then 1 ll of DHE (2 mM) and 1 ll of SYTOX green (0.5 mM) were added. Spermatozoa were incubated for 15 min at 37 °C. Finally, spermatozoa were washed once, re-suspended in 500 ll of HTF and analysed by flow cytometry (see Analysis by flow cytometry below). Analysis by flow cytometry
test. Data from all parameters analysed were transformed to a logarithmic scale prior to analysis because they did not pass the normality test. Sperm viability, ATP levels, DΨm and ROS production were evaluated by one-way analysis of variance (ANOVA). Sperm motility was assessed using a two-way ANOVA with a Bonferroni’s post-test. A P value below 0.05 was considered statistically significant. Results Effect of mCU blocking on sperm motility Sperm motility is one of the most important functions of the male gamete, and Ca2+ plays a pivotal role in its regulation (Darszon et al., 2011). For this reason, the effect of mCU blocking on total sperm motility was evaluated. Figure 1 shows a decrease in total sperm motility, which is significant after 4 h of incubation with 10 lM of Ru360, compared to the untreated control. However, 5 lM of Ru360 showed no significant effect on total sperm motility (Fig. 1). Due to motility decreased with 10 lM of Ru360 at 4 h, this incubation time was used for subsequent evaluations. Effect of mCU blocking on sperm viability Our results showed that treatment of human spermatozoa with Ru360 did not affect sperm viability after exposure to concentrations of 5 and 10 lM, as compared with the untreated control group after 4 h of incubation at 37 °C (Fig. 2). Because the Ru360 concentrations used did not
Fluorescence analysis was performed in a BD FACSCanto II flow cytometer (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA), controlled by FACSDiva 6.1.3 software (Becton, Dickinson and Company). A total of 10 000 sperm events were analysed for each test. JC-1 orange aggregates and DHE fluorescence were read in the orange PE channel (585/42 nm filter). The SYTOX greenpositive stain was read with the green FITC channel (530/ 30 nm filter). The far red PerCP channel (LP670 nm) was used to read PI fluorescence. Statistical analysis The treatment of spermatozoa with Ru360 at each dose was carried out in duplicate; that is, the experimental groups and its control were made each one in two test tubes at the same day and with the same cell sample and experiments were repeated at least four times on different days. Results were expressed as mean standard deviation. Statistical evaluation was performed with the GRAPHPAD PRISM 5 software package (GraphPad, La Jolla, CA, USA). Data normality was checked using D’Agostino’s © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Fig. 1 Evaluation of mCU blocking on total sperm motility. Spermatozoa were incubated with 5 and 10 lM of Ru360 for 4 h at 37 °C, and an untreated control was included. Results correspond to the mean standard deviation of four different experiments. mCU, mitochondrial calcium uniporter; (*): P < 0.05 compared to untreated control. 156 9 82 mm (300 9 300 DPI).
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alter sperm viability, these concentrations were also used to evaluate other parameters. Effect of mCU blocking on ATP levels Considering that the mCU blocking resulted in a decreased total sperm motility, the decision was made to assess whether this reduction was associated with decreased ATP levels, which are essential to maintaining motility (Ferramosca et al., 2012). The results demonstrated a significant decrease in ATP levels of spermatozoa exposed to both concentrations of Ru360, compared to the untreated control group (Fig. 3a). This result suggests that mCU blocking affects the ability of spermatozoa to maintain the ATP levels required for their normal function. Effect of mCU blocking on DΨm DΨm is an important indicator of the functional integrity of spermatozoa (Wang et al., 2003) because it is required for mitochondrial ATP production and sperm motility
Fig. 2 Evaluation of mCU blocking on sperm viability. Human spermatozoa were exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C, and an untreated control was included. The results correspond to the mean standard deviation of six different experiments. mCU, mitochondrial calcium uniporter. 95 9 76 mm (300 9 300 DPI).
(a)
(b)
(Guthrie & Welch, 2012). The results do not demonstrate a reduction of mean intensity of fluorescence (MFI) of JC-1 aggregated in human spermatozoa exposed to 5 and 10 lM Ru360 compared to the untreated control (Fig. 3b), demonstrating that the DΨm was not affected after mCU blocking. Effect of mCU blocking on ROS production Mitochondria are a major site of intracellular ROS formation in spermatozoa (Guthrie & Welch, 2012), and the oxidative stress caused by an increase in ROS production alters sperm function (Makker et al., 2009). It was observed that exposure of spermatozoa to 5 and 10 lM Ru360 did not significantly increase the MFI of DHE, indicating that mCU blocking did not induce an increase in ROS production in our experimental conditions (Fig. 3c). Discussion The mCU is the main mechanism of Ca2+ transport from the cytosol to the mitochondria to increase the Ca2+ level in the matrix (Zhang et al., 2006). In this study, we evaluated the effect of mCU blocking on human sperm function. We used Ru360, which has been used in different cell types (Zhang et al., 2006; Delmotte et al., 2012; Sanganahalli et al., 2013; de la Fuente et al., 2014) as well as in bovine (Rodriguez et al., 2012) and human spermatozoa (Bejarano et al., 2008). Our results showed that the percentage of sperm motility was decreased by the addition of 10 lM of Ru360 without affecting the sperm viability, suggesting a relationship between mCU and the maintenance of human sperm motility. It has been reported that Ca2+ accumulated in the mitochondrial matrix stimulates multiple energy-producing reactions, thereby increasing intramitochondrial ATP (c)
Fig. 3 Evaluation of mCU blocking on adenosine triphosphate (ATP) levels (a), DΨm (b) and intracellular ROS (c) on human spermatozoa. Spermatozoa were exposed to 5 and 10 lM of Ru360 for 4 h at 37 °C, and an untreated control was added. The results correspond to the mean standard deviation of four different experiments for ATP levels, five different experiments for DΨm and four different experiments for intracellular ROS. ROS, reactive oxygen species; mCU, mitochondrial calcium uniporter; MFI, mean fluorescence intensity; AU, arbitrary units; RLU, relative light units; (*): P < 0.05 and (**): P < 0.01 both compared to untreated control. 249 9 92 mm (300 9 300 DPI).
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levels, which can be used as fuel for sperm-activated or hyperactivated motility (reviewed by Piomboni et al., 2012). Thus, the decrease in sperm motility after Ru360 exposure is likely due to the decrease in ATP levels as a result of a decrease in mitochondrial Ca2+ by mCU blocking. Therefore, we subsequently measured ATP levels in terms of a mCU blockade. We observed a decrease in ATP levels in spermatozoa exposed to Ru360, indicating that mCU blocking alters the ability of spermatozoa to maintain ATP levels. We propose that a decrease in ATP levels is due to mCU blocking, causing a decrease in mitochondrial Ca2+, and affecting ATP production. This is supported by studies conducted on somatic cells, which have reported that Ca2+ activates mitochondrial oxidative phosphorylation (OXPHOS), stimulating several dehydrogenases of the Krebs cycle, the electron transport chain (ETC) and the Fo–F1 ATP synthase (reviewed by Piomboni et al., 2012). Thus, when mCU blocking occurs, mitochondrial Ca2+ also decreases, reducing ATP production, which finally results in impaired sperm motility. In this study, sperm motility was only decreased at 4 h of mCU blocking. The maintenance of sperm motility for the first 3 h after mCU blocking could be explained by the fact that creatine phosphate produced by creatine kinase, is an energy reservoir for the rapid regeneration of ATP, playing an important role in sperm motility as yet has been affirmed (Ghaffari & Rostami, 2013). Thus, these ATP reserves could have been utilised by the spermatozoa for the first 3 h of incubation with Ru360. Moreover, both sperm motility and ATP levels were decreased, but they did not fall completely. This may be because in human spermatozoa there are other ways to produce ATP, such as glycolysis, which has been shown to produce ATP to contribute to sperm motility (Nascimento et al., 2008). Also, it has recently been described that spermatozoa have enzymatic tools to obtain energy from endogenous substrates like fatty acids, ketone bodies and glycogen (Amaral et al., 2013). These metabolic strategies on the part of spermatozoa could explain why ATP levels and sperm motility did not decrease completely after mCU blocking. In addition to the ATP level, sperm motility has been positively related to intact DΨm (Marchetti et al., 2002); therefore, this variable was also evaluated. Under our experimental conditions, this association was not observed, because the sperm motility was decreased but DΨm was not affected after mCU blocking. The maintenance of DΨm under mCU blocking conditions is supported by previous studies on other cell types. In rats, the perfusion of Ru360 partially inhibited the mCU, maintained OXPHOS and prevented the dissipation of DΨm (Cheng et al., 2013). Likewise, Ru360 decreased © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Ca2+-induced mitochondrial depolarisation in synaptic and nonsynaptic mitochondria (Yarana et al., 2012). An increase in the concentration of mitochondrial Ca2+ when this is incorporated by mCU can uncouple the ETC. and thus decrease DΨm (reviewed by Rodriguez et al., 2012). Therefore, our results are in agreement with this because mCU blocking prevents the decrease of DΨm by inhibiting Ca2+ uptake and its adverse effects on ETC. Sperm motility has been negatively related to high levels of ROS (reviewed by Makker et al., 2009); therefore, ROS production by spermatozoa was measured. We did not observe any differences in ROS production after mCU blocking. These results indicate that the decrease in sperm motility under our experimental conditions was not a result of increased ROS production. The nonincreased ROS production under our experimental conditions is supported by investigations into other cells types, where it has been described that mitochondria take up and buffer large cytosolic Ca2+ loads, resulting in the release of ROS from the mitochondria to the cytosol (Dugan et al., 1995; Reynolds & Hastings, 1995; Bindokas et al., 1996). In this vein, mCU blocking, preventing entry of Ca2+ to the mitochondria, could explain the low levels of ROS production observed in our results. In summary, mCU blocking, in conditions that maintain viability, reduces sperm motility and ATP levels without affecting DΨm and ROS production. This indicates that the entry of Ca2+ into the mitochondrial matrix through this channel has a major role in the maintenance of sperm motility and ATP levels. Acknowledgements This work was supported by grant DI12-0102 (JV) from the Universidad de La Frontera and by doctoral thesis scholarship No. 24120981 from CONICYT (FT), Chile. References Amaral A, Castillo J, Estanyol JM, Ballesca JL, Ramalho-Santos J, Oliva R (2013) Human sperm tail proteome suggests new endogenous metabolic pathways. Mol Cell Proteomics 12:330–342. Bejarano I, Lozano GM, Ortiz A, Garcia JF, Paredes SD, Rodriguez AB, Pariente JA (2008) Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertil Steril 90:1340–1347. Beutner G, Sharma VK, Giovannucci DR, Yule DI, Sheu SS (2001) Identification of a ryanodine receptor in rat heart mitochondria. J Biol Chem 276:21482–21488. Bindokas VP, Jordan J, Lee CC, Miller RJ (1996) Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J Neurosci 16:1324–1336.
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