ANDROLOGY
ISSN: 2047-2919
ORIGINAL ARTICLE
Correspondence: Anna Maria Lobascio, Reproductive Medicine European Hospital, via Portuense 700, 00149 Rome, Italy. E-mail: [email protected]
Keywords: DNA fragmentation, MMP, ROS, leukocyte, human spermatozoa Received: 31-Mar-2014 Revised: 30-Sep-2014 Accepted: 1-Oct-2014 doi: 10.1111/andr.302
Involvement of seminal leukocytes, reactive oxygen species, and sperm mitochondrial membrane potential in the DNA damage of the human spermatozoa 1 1
A. M. Lobascio, 2M. De Felici, 3M. Anibaldi, 1P. Greco, 1M. G. Minasi and E. Greco
1
Reproductive Medicine European Hospital, 2Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, and 3Department of Research and Development, Chemi SpA, Patrica, Italy
SUMMARY Measurement of reactive oxygen species (ROS) producing leukocytes in semen has been a standard component of the semen analysis, but its true significance remains still unknown. In this study, we have correlated the number of seminal leukocytes to various semen parameters. We found a negative correlation between the leukocyte number and sperm concentration (rs = 0.22; p = 0.01) and motility (rs = 0.20; p = 0.02). In contrast, a positive correlation between the number of leukocytes and both seminal ROS (rs = 0.70, p < 0.001; n = 125) and the number of spermatozoa with DNA fragmentation (rs = 0.43, p = 0.032; n = 25) was found. However, only a trend of positive correlation between ROS and the number of spermatozoa with TUNEL-detected DNA fragmentation was observed. Moreover, this latter was not correlated with loss of sperm mitochondrial membrane potential (MMP) (10% vs 35%, rs = 0.25, p = 0.08; n = 50). Overall these results indicate that the presence of high number of leukocytes in the ejaculate negatively affects key semen parameters, as sperm concentration and motility, associated with infertility conditions. Moreover, they suggest that leukocytes are the major source of the seminal ROS and cause of sperm DNA fragmentation. However, the absence of a clear correlation between ROS and sperm DNA fragmentation, and spermatozoa with damaged DNA and MMP loss, suggest that ROS produced by leukocytes might be not the only cause of DNA damage in spermatozoa and that intrinsic mitochondrial-dependent apoptotic pathways might not have a major impact on sperm DNA fragmentation.
INTRODUCTION Spermatozoa from infertile men have been shown to contain various nuclear alterations, including abnormal chromatin structure, microdeletions, chromosomal rearrangements, aneuploidy, and DNA strand breaks (Kodama et al., 1997; Aitken et al., 1998; Bungum et al., 2007; Collins et al., 2008; Zini et al., 2008). Sperm DNA damage is believed to depend on several factors such as aberrant protamines expression, reactive oxygen species (ROS) production, and apoptotic process during spermatogenesis (de Yebra et al., 1993; Sakkas et al., 2003). It is estimated that almost 25% of infertile men have high levels of ROS in seminal plasma and spermatozoa (Iwasaki & Gagnon, 1992). When ROS production is not balanced by antioxidant compounds, an oxidative state occurs. ROS are highly reactive agents that belong to free radicals class. These compounds, in appropriate concentration, are needed for the regulation of normal sperm functions such as sperm capacitation, acrosome reaction, and spermatozoa–oocyte fusion (Griveau & Le Lannou, 1997; Wang © 2015 American Society of Andrology and European Academy of Andrology
et al., 2003a; Sigman et al., 2009). These functions require the presence of sperm plasmalemma unsaturated fatty acids providing the membrane fluidity, necessary for sperm motility and membrane fusion events. However, excessive ROS production leads to peroxidation of unsaturated fatty acids and consequently causes loss of sperm motility and impairs acrosome reaction and/or oocytes–sperm fusion capability. Moreover, ROS induce a variety of DNA damages. The two main semen ROS sources are defective or immature spermatozoa (intrinsic source) and polymorph nucleated leukocytes (extrinsic source) (Aitken & Fisher, 1994; Gomez et al., 1996; Barroso et al., 2000; Muratori et al., 2000; Sati et al., 2008). Leukocytes produce ROS (Aitken & West, 1990; Ochsendorf, 1999) as a consequence of inflammation and infection (Pasqualotto et al., 2000b). Recently, some studies have demonstrated that in infertile men, sperm mitochondria produce an excess of ROS that is correlated with reduced motility and DNA damage (Shi et al., 2012). It has been found that defective human spermatozoa are characterized by Andrology, 1–6
1
ANDROLOGY
A. M. Lobascio et al.
an abnormally high content of unsaturated fatty acids that promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence. (Koppers et al., 2008; du Plessis et al., 2010). The sperm ROS production is linked to NADPH oxidase system localized in sperm plasma membrane and to NADH oxidoreductase system contained in sperm mitochondrial respiratory chain. These enzymes, in particular conditions, produce H2O2, considered a highly reactive substrate in chemical reactions with metals (e.g. Fe²+), bringing to radical OH hydroxyl formation. ·OH is considered the most reactive and dangerous radical able to react with membrane lipids, proteins, and DNA/RNA when its concentration exceeds that of the scavengers (Kemal Duru et al., 2000). Two factors protect sperm DNA from free radicals effects: packaging of DNA by histone protamines and antioxidant compounds (Greco et al., 2005a,b). The main purpose of this study was to evidence correlation between the presence of seminal leukocytes and several semen parameters and to elucidate the possible source of DNA fragmentation in human spermatozoa.
METHODS Patients and standard semen analyses Standard semen parameters of 125 patients undergoing semen analyses for infertility conditions were used; the presence of inflammatory conditions in the patients was not investigated. Samples were analyzed for sperm viability (eosin test) and sperm number and motility through Computer-Assisted Sperm Analysis (CASA), according to WHO criteria 2010. In particular, about sperm motility, A and B types were considered: A and B types are spermatozoa with rapid and average progressive motility, respectively. Seminal ROS and leukocyte concentration After standard semen analyses, samples were divided into two aliquots: one was used to measure total ROS concentration by chemiluminescence assay with luminol as probe (5-amino-2,3dihydro-1,4-phthalazinedione; Sigma Chemical, Milan, Italy) (Sharma et al., 1999) and using a Berthold luminometer (Autolumat LB 953, Bad Wildbad, Germany); the second aliquot was used for the histochemistry detection of the leukocyte numbers showing peroxidase activity by Leucoscreen (FertiPro, Origio, Italy), according to the manufacturer’s instructions. TUNEL staining Of the 125 samples, 25 were examined for the presence of spermatozoa with nuclear DNA fragmentation by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay (In situ Cell Detection Kit Fluorescein, Roche, Germany). Briefly, fresh sperm samples were centrifuged in PBS for 10 min at 380 g. The pellet was resuspended in PBS at final concentration of 10 9 106/mL and smeared on microscope slides. Cells were fixed with 4% paraformaldehyde solution for 15 min. Each samples was permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min. The remaining procedure was carried out according to the manufacturer’s instruction. Sperm samples were incubated in TUNEL reaction mixture, in the dark and at the temperature of 37 °C for 1 h followed by evaluation under a 2
Andrology, 1–6
fluorescence microscopy; 200 spermatozoa per slide were randomly analyzed. Evaluation of MMP To evaluate the sperm mitochondrial membrane potential (MMP), semen samples of other 50 patients obtained and subjected to standard analyses as above reported, were evaluated by the immunofluorescence assay MitoCapture TM Mitochondrial Apoptosis kit from Vinci Biochem. Briefly, each semen sample was rinsed and divided into two aliquots with a final sperm concentration of 10 9 106/mL. The first aliquot was used to evaluate MMP, the other to detect spermatozoa with DNA fragmentation by TUNEL staining (see above). For the MMP assay, spermatozoa were rinsed and centrifuged in the kit incubation buffer for 5 min at 270 g. They were then resuspended in MitoCapture solution (2 lL of MitoCapture solution in 1 mL of pre-warmed buffer) and left 30 min at 37 °C in a 5% CO2 incubator. Finally, spermatozoa were centrifuged for 5 min at 270 g, rinsed in the incubation buffer, resuspended in 1 mL of pre-warmed buffer, and quickly observed by fluorescence microscopy. Statistical analyses For each samples, the median and the interquartile range or the mean and the standard deviation were calculated. The Shapiro–Wilk test was used to evaluate the data distribution. The Spearman coefficient (rs) was calculated to correlate semen parameters. p < 0.05 was considered a significant value.
RESULTS In a first series of experiments, we correlated the number of peroxidase-positive leukocytes with semen parameters in the ejaculates of 125 patients. Namely, in each sample, leukocyte concentration was correlated with sperm motility, concentration, DNA fragmentation, and ROS amount in the whole semen (Table 1). We found a negative correlation between the leukocyte number and both the percentage of sperm motility (rs = 0.20; p = 0.02) and sperm number (rs = 0.22; p = 0.01) (Figs 1 & 2). On the contrary, a positive correlation was detected between the leukocyte number and both total ROS concentration (rs = 0.70, p < 0.001) and the number of spermatozoa showing TUNEL detected DNA fragmentation (rs = 0.43; p = 0.032) (Figs 3 & 4). In addition, a trend of positive correlation between total seminal ROS and the number of spermatozoa with damaged DNA was observed (rs = 0.37; p = 0.07) (Fig. 5). In the second part of our study, semen parameters of other 50 different patients were correlated with sperm MMP. None
Table 1 Summary of the correlation analyses among leukocyte concentration, sperm motility, concentration, seminal ROS, and the number of spermatozoa showing DNA fragmentation referred to the semen samples analyzed. The Spearman coefficient (rs) was calculated to correlate these semen parameters Leukocyte concentration Sperm motility Sperm concentration Log (ROS + 1) DNA fragmentation
n n n n
= = = =
125 125 125 25
Spearman coefficient (rs) 0.20 0.22 0.70 0.43
p value (
ISSN: 2047-2919
ORIGINAL ARTICLE
Correspondence: Anna Maria Lobascio, Reproductive Medicine European Hospital, via Portuense 700, 00149 Rome, Italy. E-mail: [email protected]
Keywords: DNA fragmentation, MMP, ROS, leukocyte, human spermatozoa Received: 31-Mar-2014 Revised: 30-Sep-2014 Accepted: 1-Oct-2014 doi: 10.1111/andr.302
Involvement of seminal leukocytes, reactive oxygen species, and sperm mitochondrial membrane potential in the DNA damage of the human spermatozoa 1 1
A. M. Lobascio, 2M. De Felici, 3M. Anibaldi, 1P. Greco, 1M. G. Minasi and E. Greco
1
Reproductive Medicine European Hospital, 2Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, and 3Department of Research and Development, Chemi SpA, Patrica, Italy
SUMMARY Measurement of reactive oxygen species (ROS) producing leukocytes in semen has been a standard component of the semen analysis, but its true significance remains still unknown. In this study, we have correlated the number of seminal leukocytes to various semen parameters. We found a negative correlation between the leukocyte number and sperm concentration (rs = 0.22; p = 0.01) and motility (rs = 0.20; p = 0.02). In contrast, a positive correlation between the number of leukocytes and both seminal ROS (rs = 0.70, p < 0.001; n = 125) and the number of spermatozoa with DNA fragmentation (rs = 0.43, p = 0.032; n = 25) was found. However, only a trend of positive correlation between ROS and the number of spermatozoa with TUNEL-detected DNA fragmentation was observed. Moreover, this latter was not correlated with loss of sperm mitochondrial membrane potential (MMP) (10% vs 35%, rs = 0.25, p = 0.08; n = 50). Overall these results indicate that the presence of high number of leukocytes in the ejaculate negatively affects key semen parameters, as sperm concentration and motility, associated with infertility conditions. Moreover, they suggest that leukocytes are the major source of the seminal ROS and cause of sperm DNA fragmentation. However, the absence of a clear correlation between ROS and sperm DNA fragmentation, and spermatozoa with damaged DNA and MMP loss, suggest that ROS produced by leukocytes might be not the only cause of DNA damage in spermatozoa and that intrinsic mitochondrial-dependent apoptotic pathways might not have a major impact on sperm DNA fragmentation.
INTRODUCTION Spermatozoa from infertile men have been shown to contain various nuclear alterations, including abnormal chromatin structure, microdeletions, chromosomal rearrangements, aneuploidy, and DNA strand breaks (Kodama et al., 1997; Aitken et al., 1998; Bungum et al., 2007; Collins et al., 2008; Zini et al., 2008). Sperm DNA damage is believed to depend on several factors such as aberrant protamines expression, reactive oxygen species (ROS) production, and apoptotic process during spermatogenesis (de Yebra et al., 1993; Sakkas et al., 2003). It is estimated that almost 25% of infertile men have high levels of ROS in seminal plasma and spermatozoa (Iwasaki & Gagnon, 1992). When ROS production is not balanced by antioxidant compounds, an oxidative state occurs. ROS are highly reactive agents that belong to free radicals class. These compounds, in appropriate concentration, are needed for the regulation of normal sperm functions such as sperm capacitation, acrosome reaction, and spermatozoa–oocyte fusion (Griveau & Le Lannou, 1997; Wang © 2015 American Society of Andrology and European Academy of Andrology
et al., 2003a; Sigman et al., 2009). These functions require the presence of sperm plasmalemma unsaturated fatty acids providing the membrane fluidity, necessary for sperm motility and membrane fusion events. However, excessive ROS production leads to peroxidation of unsaturated fatty acids and consequently causes loss of sperm motility and impairs acrosome reaction and/or oocytes–sperm fusion capability. Moreover, ROS induce a variety of DNA damages. The two main semen ROS sources are defective or immature spermatozoa (intrinsic source) and polymorph nucleated leukocytes (extrinsic source) (Aitken & Fisher, 1994; Gomez et al., 1996; Barroso et al., 2000; Muratori et al., 2000; Sati et al., 2008). Leukocytes produce ROS (Aitken & West, 1990; Ochsendorf, 1999) as a consequence of inflammation and infection (Pasqualotto et al., 2000b). Recently, some studies have demonstrated that in infertile men, sperm mitochondria produce an excess of ROS that is correlated with reduced motility and DNA damage (Shi et al., 2012). It has been found that defective human spermatozoa are characterized by Andrology, 1–6
1
ANDROLOGY
A. M. Lobascio et al.
an abnormally high content of unsaturated fatty acids that promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence. (Koppers et al., 2008; du Plessis et al., 2010). The sperm ROS production is linked to NADPH oxidase system localized in sperm plasma membrane and to NADH oxidoreductase system contained in sperm mitochondrial respiratory chain. These enzymes, in particular conditions, produce H2O2, considered a highly reactive substrate in chemical reactions with metals (e.g. Fe²+), bringing to radical OH hydroxyl formation. ·OH is considered the most reactive and dangerous radical able to react with membrane lipids, proteins, and DNA/RNA when its concentration exceeds that of the scavengers (Kemal Duru et al., 2000). Two factors protect sperm DNA from free radicals effects: packaging of DNA by histone protamines and antioxidant compounds (Greco et al., 2005a,b). The main purpose of this study was to evidence correlation between the presence of seminal leukocytes and several semen parameters and to elucidate the possible source of DNA fragmentation in human spermatozoa.
METHODS Patients and standard semen analyses Standard semen parameters of 125 patients undergoing semen analyses for infertility conditions were used; the presence of inflammatory conditions in the patients was not investigated. Samples were analyzed for sperm viability (eosin test) and sperm number and motility through Computer-Assisted Sperm Analysis (CASA), according to WHO criteria 2010. In particular, about sperm motility, A and B types were considered: A and B types are spermatozoa with rapid and average progressive motility, respectively. Seminal ROS and leukocyte concentration After standard semen analyses, samples were divided into two aliquots: one was used to measure total ROS concentration by chemiluminescence assay with luminol as probe (5-amino-2,3dihydro-1,4-phthalazinedione; Sigma Chemical, Milan, Italy) (Sharma et al., 1999) and using a Berthold luminometer (Autolumat LB 953, Bad Wildbad, Germany); the second aliquot was used for the histochemistry detection of the leukocyte numbers showing peroxidase activity by Leucoscreen (FertiPro, Origio, Italy), according to the manufacturer’s instructions. TUNEL staining Of the 125 samples, 25 were examined for the presence of spermatozoa with nuclear DNA fragmentation by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay (In situ Cell Detection Kit Fluorescein, Roche, Germany). Briefly, fresh sperm samples were centrifuged in PBS for 10 min at 380 g. The pellet was resuspended in PBS at final concentration of 10 9 106/mL and smeared on microscope slides. Cells were fixed with 4% paraformaldehyde solution for 15 min. Each samples was permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min. The remaining procedure was carried out according to the manufacturer’s instruction. Sperm samples were incubated in TUNEL reaction mixture, in the dark and at the temperature of 37 °C for 1 h followed by evaluation under a 2
Andrology, 1–6
fluorescence microscopy; 200 spermatozoa per slide were randomly analyzed. Evaluation of MMP To evaluate the sperm mitochondrial membrane potential (MMP), semen samples of other 50 patients obtained and subjected to standard analyses as above reported, were evaluated by the immunofluorescence assay MitoCapture TM Mitochondrial Apoptosis kit from Vinci Biochem. Briefly, each semen sample was rinsed and divided into two aliquots with a final sperm concentration of 10 9 106/mL. The first aliquot was used to evaluate MMP, the other to detect spermatozoa with DNA fragmentation by TUNEL staining (see above). For the MMP assay, spermatozoa were rinsed and centrifuged in the kit incubation buffer for 5 min at 270 g. They were then resuspended in MitoCapture solution (2 lL of MitoCapture solution in 1 mL of pre-warmed buffer) and left 30 min at 37 °C in a 5% CO2 incubator. Finally, spermatozoa were centrifuged for 5 min at 270 g, rinsed in the incubation buffer, resuspended in 1 mL of pre-warmed buffer, and quickly observed by fluorescence microscopy. Statistical analyses For each samples, the median and the interquartile range or the mean and the standard deviation were calculated. The Shapiro–Wilk test was used to evaluate the data distribution. The Spearman coefficient (rs) was calculated to correlate semen parameters. p < 0.05 was considered a significant value.
RESULTS In a first series of experiments, we correlated the number of peroxidase-positive leukocytes with semen parameters in the ejaculates of 125 patients. Namely, in each sample, leukocyte concentration was correlated with sperm motility, concentration, DNA fragmentation, and ROS amount in the whole semen (Table 1). We found a negative correlation between the leukocyte number and both the percentage of sperm motility (rs = 0.20; p = 0.02) and sperm number (rs = 0.22; p = 0.01) (Figs 1 & 2). On the contrary, a positive correlation was detected between the leukocyte number and both total ROS concentration (rs = 0.70, p < 0.001) and the number of spermatozoa showing TUNEL detected DNA fragmentation (rs = 0.43; p = 0.032) (Figs 3 & 4). In addition, a trend of positive correlation between total seminal ROS and the number of spermatozoa with damaged DNA was observed (rs = 0.37; p = 0.07) (Fig. 5). In the second part of our study, semen parameters of other 50 different patients were correlated with sperm MMP. None
Table 1 Summary of the correlation analyses among leukocyte concentration, sperm motility, concentration, seminal ROS, and the number of spermatozoa showing DNA fragmentation referred to the semen samples analyzed. The Spearman coefficient (rs) was calculated to correlate these semen parameters Leukocyte concentration Sperm motility Sperm concentration Log (ROS + 1) DNA fragmentation
n n n n
= = = =
125 125 125 25
Spearman coefficient (rs) 0.20 0.22 0.70 0.43
p value (
