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The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators such as white blood cells (WBC), two combinations of proinflammatory cytokines (interleukin (IL)-6+IL-8 and IL-12+IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples of normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). WBC were isolated from the whole heparinized blood using a density gradient centrifugation. DNA fragmentation in sperm fractions was evaluated using a flow cytometry with TUNEL labeling. Bacteria significantly increased the percentage of TUNEL-positive spermatozoa compared to untreated cells (p < 0.05). In the case of B. ureolyticus, these changes were accompanied by significant increase in the mean fluorescence intensity (MFI) levels of TUNEL-positive spermatozoa from the 90% Percoll fraction. Proinflammatory cytokines significantly increased MFI levels in the swim-up and the 90% Percoll sperm fractions, respectively for combinations of IL-6+IL-8 or IL-12+IL-18 (p < 0.01 and p < 0.05 when compared to sperm incubated alone). The results indicate that bacteria or their toxins may directly induce DNA fragmentation in ejaculated spermatozoa. Proinflammatory cytokines may be additional factor to cause DNA breaks in spermatozoa. Contrary to the swim-up technique, the selection of spermatozoa by gradient procedures increases the risk of using sperm with damaged DNA for assisted reproduction.
In analyzing the influence of the male genitourinary tract inflammation on oxygen metabolism, and its effect on sperm structure and function, we should also consider the issue of sperm DNA integrity. There have been numerous reports indicating that integrity of sperm genetic material is of great importance for fertilization and embryo quality (Comhaire et al. 1999; Sergerie et al. 2005; Carrell et al. 2006; Aitken et al. 2009). The 'poor quality' of sperm DNA appears to be one of important factors affecting male reproductive ability (Hughes et al. 1996). This has been confirmed by some reports in which a higher percentage of spermatozoa with fragmented DNA has been found in infertile men when compared to the fertile individuals (Baccetti et al. 1996; Lopes et al. 1998). There are three mechanisms described in the literature which can disrupt sperm DNA integrity, such as defective chromatin packaging, apoptosis and oxidative stress (Aitken and De Iuliis 2007). In case of inflammation of the genitourinary tract, the redox imbalance is probably the main etiological factor responsible for destructive effects of the inflammatory process on male gametes, which is mainly associated with peroxidation of sperm macromolecules (Comhaire et al. 1999, Aitken and Baker 2006, Fraczek and Kurpisz 2007).
Taking into consideration the fact that DNA fragmentation may result from reactive oxygen intermediates (ROI)-dependent activity, and the inflammatory process is inseparably connected with oxidative stress, we decided to reconstruct the inflammatory reaction in semen and to analyze the effect of selected inflammatory mediators on an in vitro DNA fragmentation of different sperm subpopulations. Out of many factors taking part in the inflammatory process we chose white blood cells (WBC), two combinations of proinflammatory cytokines (interleukin (IL)-6+IL-8 and IL-12+IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus) for the study. These factors were selected on the basis of our earlier results regarding lipid sperm membrane peroxidation (Fraczek et al. 2007; 2008). Namely, we used samples in which the highest malondialdehyde (MDA) levels in sperm lysates were previously observed.
Effect of inflammatory factors on the percentage of TUNEL-positive cells in different sperm fractions
The effect of selected inflammatory mediators on the percentage of TUNEL-positive cells in different sperm fractions is summarized in Table 1. Untreated control spermatozoa isolated by the swim-up technique had the lowest percentage of the TUNEL-positive cells. When compared with the swim-up sperm fraction, approximately 2-3-fold elevated TUNEL-positive cells were observed for the analyzed Percoll sperm fractions (90% and 47%). In general, the presence of leukocytes was associated with the decrease in the number of TUNEL-positive cells. However, this effect was not statistically significant when compared to sperm incubated alone (controls). Incubation of spermatozoa with IL-6 combined with IL-8 was connected with increased percentage of TUNEL-positive cells, especially in the 90% Percoll sperm fraction. However, this increase was also statistically insignificant, as compared to the respective control. Combination of IL-12 with IL-18 caused an increase in the percentage of TUNEL-positive cells, especially in sperm fractions isolated by Percoll gradient, although this increase was statistically insignificant when compared to untreated cells. When spermatozoa from the 47% Percoll fraction were exposed to E. coli, a much higher percentage of TUNEL-positive cells was noted only in case of spermatozoa recovered from the 47% Percoll fraction (p < 0.05). Anaerobic bacteria represented by B. ureolyticus had a statistically significant influence on the percentage of the TUNEL-positive cells in sperm from the 90% Percoll fraction (p < 0.05). Moreover, this increase was the highest among all the inflammatory factors applied in this study.
Using flow cytometry we have also detected some propidium iodide (PI) positive cells in all analyzed sperm fractions after their in vitro incubation in 37°C. The sperm fractions contained approximately 12, 26 and 45% of PI positive cells for the swim-up, 90% and 47% Percoll sperm fractions, respectively.
Effect of inflammatory factors on MFI in different sperm fractions
The results for MFI levels in all the examined sperm fractions incubated with selected inflammatory mediators are presented in Table 2. The incubation of spermatozoa with leukocytes resulted in elevated, but not statistically significant (compared to spermatozoa alone) MFI levels, irrespective of sperm fraction. For spermatozoa isolated by the swim-up technique, a significant increase in MFI in the presence of combination of IL-6 with IL-8 was observed (p < 0.01). As for the combination of IL-12 with IL-18, it significantly increased MFI level in sperm recovered from the 90% Percoll fraction (p < 0.05). From the two selected bacterial strains, anaerobes (B. ureolyticus) had a greater influence on MFI level, especially in sperm from the 90% Percoll fraction (p < 0.05). Moreover, this increase turned out to be the highest among all the inflammatory factors used in this study.
Representative sperm samples observed in the TUNEL-assay are presented in Figure 1. Apart from typical TUNEL-positive cells and characteristic features of apoptosis, we also observed spermatozoa with strong fluorescence within the midpiece.
The presented study is a continuation of our previous reports concerning the influence of oxidative stress associated with the inflammatory process on structural, metabolic and functional disorders of three various sperm subpopulations using an in vitro system (Fraczek et al. 2004; Fraczek et al. 2007; Fraczek et al. 2008). As for the percentage of spermatozoa with the DNA breaks in the population of physiological gametes (selected by the swim-up procedure), we expected to show results similar to those obtained by other authors (Ramos and Wetzels, 2001; Muratori et al. 2003; Aziz et al. 2007). Indeed, we observed the lowest number of TUNEL-positive cells in this fraction. Besides, the highest percentage of cells with spontaneously fragmented DNA in the fraction of spermatozoa with the poorest seminological parameters (47% Percoll fraction) corresponded with our previous observations indicating the high ROI secretion within such a sperm subpopulation (Fraczek et al. 2004; Fraczek et al. 2007). These findings are also in agreement with the other reports showing the significantly higher proportion of sperm with DNA damage (and ROI generation) in the fraction of sperm with low motility, as compared to the fraction with high sperm motility (Barroso et al. 2000).
In our previous study (Fraczek et al. 2004) in which the in vitro model of semen inflammation was created, we demonstrated that in normozoospermic ejaculates may exist sperm subpopulations differing in their oxidoreductive potential, which influences their function and the response to the environmental stress. As expected, normal spermatozoa isolated by swim-up technique turned out to be the most resistant to inflammatory agent-induced DNA fragmentation in contrast to spermatozoa obtained from the 90% Percoll sperm fraction which appeared to be the most susceptible ones. The results obtained have confirmed that the structural and functional differences between the studied spermatozoal fractions may influence the frequency and intensity of sperm DNA fragmentation, when subjected to various inflammatory mediators. In the light of data presented, the selection of spermatozoa by gradient procedures increases the risk of using sperm with DNA alterations for assisted reproduction.
The main potential mechanism in which genital tract inflammation/infection might affect male germ cells can be the impact of leukocytes (infiltrating the inflammatory site) especially phagocytic cells being associated with the production and release of large amounts of ROI and biologically active substances, such as proteases and proinflammatory cytokines (Ochsendorf, 1999). However, there is an ongoing controversy concerning the relationship between sperm DNA integrity and leukocytospermia (Alvarez et al. 2002; Erenpreiss et al. 2002; Henkel et al. 2005; Moskovtsev et al. 2007). In our study we observed lower percentage of TUNEL-positive cells after the incubation of spermatozoa with leukocytes, whereas the fluorescence intensity in the TUNEL-positive population was clearly (although not statistically significant) increased after the same co-incubation with leukocytes, as compared to spermatozoa alone. One potential hypothesis for this paradox is the rapid removal of apoptotic germ cells by leukocytes due to phagocytosis. This mechanism of sperm selection is an important part of the elimination of non-viable or damaged spermatozoa. Thus, the observed decrease in the number of germ cells with fragmented DNA, especially in the 47% Percoll sperm fraction with altered morphology and motility as well as with increased proportion of dead spermatozoa, partially provides an evidence for the positive role of leukocytes in ejaculate, which has already been mentioned in the literature (Tomlinson et al. 1992; Ricci et al. 2002). On the other hand, it cannot be excluded that the increase in the MFI parameter was a natural consequence of ROS excess in co-incubated mixtures of leukocytes and spermatozoa due to e.g. the release of cytokines from activated white blood cells.
One of the mechanisms by which oxidative stress connected with the inflammatory process might lead to sperm DNA damage is the activation of endogenous endonucleases reported by some authors (Sotolongo et al. 2005, Aitken and De Iuliis 2007). On the other hand, the most critical mechanism to explain the origin of DNA degeneration occurring in ejaculated spermatozoa indicated in the present study is apoptosis. In this study, we applied the flow cytometric TUNEL assay which seems to be quite useful for the detection of sperm DNA fragmentation. However, the latest reports suggest that all frequently used methods of identifying sperm DNA fragmentation are not specific enough since they detect DNA breaks which may occur through both apoptosis and/or necrosis (Aitken et al. 2009). It cannot be excluded that differences among sperm fractions as for a number of dead and/or dying sperm (PI-positive cells) have influenced the results here obtained, particularly in the situation when spermatozoa were further exposed to the inflammatory mediators. However, we have to remember that PI positive subpopulations may include not only necrotic cells but also the cells in a later stage of apoptosis as well as the cells with damaged membrane integrity (Ricci et al. 2002; Lachaud et al. 2004). Most probably, the use of additional techniques for detecting apoptosis, such as annexin V/PI binding assay or electron microscopy technique, could dispel doubts upon the nature of detected DNA breaks during the male genital inflammation/infection.
The involvement of some proinflammatory cytokines in the deepening of harmful influence of oxidative stress on spermatozoa is being examined in the recent literature. The expression of genes responsible for redox system in semen or the modulation of the activities within prooxidative and antioxidative systems leading to the ROI burst are the most frequent mechanisms that could be revealed during the male genital tract inflammation (Shimoya et al. 1993; Rajasekaran et al. 1995; Sanocka et al. 2003). It is known that another mechanism by which proinflammatory cytokines facilitate the development of inflammatory reactions can be the induction of apoptosis (Feldmannn and Saklatvala 2001; Perdichizzi et al. 2007). In our previous report, however (Fraczek et al, 2008), we analyzed a large set of proinflammatory cytokines, and we did not observe a direct significant in vitro effect of proinflammatory cytokines on malondialdehyde (MDA) content in sperm membranes. Moreover, only in the presence of some combinations of cytokines (IL-6+IL-8 or IL-12+IL-18) used together with leukocytes we observed a significant increase in MDA concentration when compared to sperm incubated with leukocytes alone. Previously reported results regarding sperm membrane peroxidation were the main reason to choose these cytokine combinations and to examine influence on DNA fragmentation. An additional cause of choosing these factors was the fact that from a large group of proinflammatory cytokines, IL-6, IL-8 and IL-18 have been most frequently mentioned in the literature as diagnostic markers for male genitourinary tract infections (Depuydt et al. 1996; Eggert-Krusse et al. 2001, Sanocka et al. 2003, Matalliotakis et al. 2006). Significant increase in MFI levels disproportionate to the number of TUNEL-positive sperm, observed in the presence of cytokines, has suggested that these proinflammatory factors may be additional agents causing DNA breaks in ejaculated spermatozoa with already initiated DNA damage. It cannot be excluded that the 90% Percoll fraction, in which we observed a higher percentage of TUNEL-positive cells as well as a significant increase in fluorescence intensity when using the cytokine combinations, contained spermatozoa rich in Fas which earlier 'avoided' undergoing apoptosis.
Interestingly, we have observed that bacteria may directly induce DNA fragmentation in ejaculated spermatozoa. Based on our previous chemiluminometric observations we can state that during the inflammatory process, bacteria are important inducers of ROI overproduction in semen, and their participation in oxidative stress vastly depends on the type of the pathogen infecting, colonizing or contaminating the male reproductive system (Fraczek et al. 2007). In analyzing the in vitro influence of infectious factors on sperm DNA damage, one should also take into consideration the induction of apoptosis. A few years ago, the first work appeared which dealt with the induction of apoptosis in human ejaculated spermatozoa following in vitro incubation with bacteria (Villegas et al. 2005). The authors observed an increase in the percentage of spermatozoa with phosphatidylserine (PS) externalization in the presence of E. coli and S. aureus. Our data are in agreement with these results which are the evidence of the induction of male germ cells apoptosis as a consequence of direct contact of spermatozoa with bacteria or their toxins, perhaps even without the ROI mediation.
In vivo studies also demonstrated that deficiency in the mitochondrial respiration can be a cause of male infertility (Burkman 1995). The analysis of TUNEL-positive cells in the fluorescent microscope showed the presence spermatozoa with increased DNA fragmentation within the midpiece (Figure 1). This intriguing observation may suggest the increased sensitivity of sperm mitochondria to degradation and mtDNA to fragmentation, which has already been the subject of discussion (Folgero et al. 1993). Recently, Cohen-Bacrie and co-workers have shown for the first time a positive correlation between sperm DNA fragmentation and some deformities of the spermatozoal midpiece (Cohen-Bacrie et al. 2009). Although these interesting results shed some light on this problem, an attempt at combining sperm DNA and/or mtDNA fragmentation with clinically observed teratozoospermia requires further studies.
The data obtained in this study reveal that the co-incubation of spermatozoa with inflammatory factors results in elevated DNA fragmentation. An increase of DNA damage observed in the ejaculated spermatozoa in the presence of mediators of the inflammatory process in vitro seems to be in line with the observed DNA fragmentation during the inflammation in vivo (Aitken and De Iuliis 2007). The present study supports the view that during the male urogenital inflammation the microbial pathogens are the most prominent agents responsible for injuries to both sperm membranes and DNA with potential consequences for sperm function. Although this study provides a better understanding for the harmful effects of particular inflammatory factors on DNA status in specific sperm subpopulations with different fertilizing potential, further investigations using morphological and molecular tests determining sperm membrane status, mitochondrial function, and DNA integrity should be applied to achieve a clear picture of subcellular changes in ejaculated spermatozoa (also in the native, unprocessed sperm fraction) occurring in the course of semen inflammation/infection.
MATERIALS AND METHODS
Sample collection and preparation
Semen specimens were obtained from 10 healthy volunteers with no sperm abnormalities attending the Outpatient Clinic for Andrology, Poznan, Poland after 4 days of sexual abstinence. Only semen samples whose semen parameters corresponded with the World Health Organization norm (WHO, 1999) and Kruger's strict criteria (Kruger at al. 1986) for morphology were utilized for further experiments. Semen samples qualified for the study were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). The cells from all three sperm fractions were finally washed in phosphate-buffered saline (PBS) and adjusted to a density of 1Ã-107 spermatozoa/ml. Specimens of heparinized venous blood were collected from 10 healthy adults donating to the Regional Blood Centre, Poznan, Poland. WBC were isolated by density gradient centrifugation technique with Histopaque-1.077 (Sigma) as described in detail elsewhere (Fraczek et al, 2004). The WBC suspensions were diluted to concentration of 1Ã-107 cells/ml for further use. The bacterial isolates used in this study were obtained from the Outpatient Clinic of the Poznan Hospital Medical College. They represented strains commonly isolated from semen of our infertile patients. Suspensions of all isolates were prepared containing approximately 3,000 bacteria per ml in a sterile 0.85% saline no more than three hours before the experiment in which they were to be used.
Incubation of sperm fractions with inflammatory mediators
Cells of all the three sperm fractions were then incubated with WBC (1Ã-106 per ml of sperm suspension), human recombinant proinflammatory cytokines (500 pg, 50 pg and 500 pg per ml of sperm suspension, respectively for IL-6, IL-8, IL-12 and IL-18) or bacteria (1Ã-103 cells per ml of sperm cells, for both E. coli and B. ureolyticus) for 1 hour at 37°C. The co-incubated mixtures of spermatozoa and leukocytes were then selectively depleted using a Dynal MPC-1 immunomagnetic cell isolation system (Fraczek et al, 2004, 2007, 2008).
DNA fragmentation in sperm fractions was evaluated using the TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assay (FlowTACS Apoptosis Detection Kit, R&D Systems, Minneapolis, MN, USA) following the manufacturer's instruction. The results were analyzed using FacsDiva software (Becton Dickinson). The percentage of TUNEL-positive cells as well as the mean fluorescence intensity (MFI) of TUNEL positive population were determined. The background fluorescence was assessed in comparison to both negative (sperm exposed to the reaction mixture without TdT) as well as positive (sperm pre-treated with DNase I) controls. Besides, the TUNEL-FITC-labeled spermatozoa were observed in a fluorescent microscope (BX41, Olympus, Tokyo, Japan) to see morphological forms of TUNEL-positive spermatozoa.
Statistical analysis was performed using STATISTICA, version 7.0 (StatSoft, Tulsa, OK, USA) by the non-parametric analysis of variances (Kruskal-Wallis test) followed by the Dunnett and Dunn multiple comparisons tests. All data was presented as mean ± standard deviation (SD). Differences were regarded as significant if p < 0.05.
LEGENDS TO FIGURES
Figure 1. Representative sperm samples observed with TUNEL assay (histogram in the left panel and the same sperm as observed under fluorescent microscope in the right panel) showing: (A) the negative control (sperm from the 90% Percoll fraction exposed to the reaction mixture without TdT); (B) the positive control (sperm from the 90% Percoll fraction pretreated with DNase I); (C) sperm from the 90% Percoll fraction incubated alone; (D) sperm from the 90% Percoll fraction incubated with B. ureolyticus