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ABSTRACT: The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38°C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + × (0.6 mM)-XO (0.05 U/mL); and 4) sperm + × (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38°C) with × (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment 3, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P < .005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by SOD, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment 3, cryopreservation of equine spermatozoa was associated with an increase (P < .01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.
Human (Aitken and Clarkson, 1987; Aitken et al, 1997) and equine spermatozoa (Ball et al, 2001) are capable of generating reactive oxygen species (ROS), and this generation is believed to play a physiological role in the signaling events controlling sperm capacitation (de Lamirande and Gagnon, 1993a, 1993b; Griveau et al, 1994; Leclerc et al, 1997); acrosome reaction (de Lamirande et al, 1993, 1998; Griveau et al, 1995b); hyperactivation (de Lamirande and Gagnon, 1993a, 1993b); and sperm-oocyte fusion (Aitken et al, 1995b, 1998b). In contrast, excessive generation of ROS by defective spermatozoa (Aitken et al, 1989b, 1994a; Rao et al, 1989; Iwasaki and Gagnon, 1992; Ball et al, 2001) or contaminating leukocytes (Aitken and West, 1990; Wolff et al, 1990; Aitken et al, 1994b, 1995a; Baumber et al, 2002) can have a detrimental effect on sperm function.
Generation of ROS in vitro by the xanthine—xanthine oxidase (X-XO) system results in a reduction in sperm motility (de Lamirande and Gagnon, 1992a, 1992b; Aitken et al, 1993a; Baumber et al, 2001); viability (de Lamirande and Gagnon, 1992a; Baiardi et al, 1997); ionophore-induced acrosome reaction (Aitken et al, 1993a; Griveau et al, 1995a); and sperm-oocyte fusion (Aitken et al, 1993a; Blondin et al, 1997). Hydrogen peroxide appears to be the primary ROS responsible for these changes (Alvarez and Storey, 1989; de Lamirande and Gagnon, 1992a; Aitken et al, 1993a; Griveau et al, 1995a; Blondin et al, 1997; Baumber et al, 2001), and membrane lipid peroxidation is believed to be an important mechanism of action (Aitken et al, 1989a, 1993b; Storey, 1997).
In addition to membrane effects, lipid peroxidation can also damage DNA. Peroxidation of DNA can lead to chromatin cross-linking, base changes, and DNA strand breaks (Hughes et al, 1996; Kodama et al, 1997; Twigg et al, 1998c). Several researchers have reported DNA damage in human spermatozoa associated with membrane lipid peroxidation (Chen et al, 1997; Twigg et al, 1998b; Potts et al, 2000) and oxidative stress (Hughes et al, 1996; McKelvey-Martin et al, 1997; Aitken et al, 1998a; Lopes et al, 1998; Twigg et al, 1998c; Donnelly et al, 1999). Previous work in our laboratory has demonstrated that ROS can promote lipid peroxidation in equine spermatozoa with an associated loss of motility (Baumber et al, 2001; Ball and Vo, 2002). However, the influence of ROS on equine sperm DNA has not been reported.
Oxidative DNA damage in human sperm suspensions can be counteracted by the addition of seminal plasma (Twigg et al, 1998a; Potts et al, 2000). Seminal plasma contains a variety of antioxidants that counteract the damaging effects of ROS, and 3 major enzyme systems have been described: the glutathione peroxidase/reductase system (Li, 1975; Alvarez and Storey, 1989); superoxide dismutase (Nissen and Kreysel, 1983; Alvarez et al, 1987); and catalase (Jeulin et al, 1989). Work in our laboratory has demonstrated that equine seminal plasma contains a high activity of catalase (Ball et al, 2000) and that this antioxidant can protect equine sperm motility from the detrimental effect of ROS (Baumber et al, 2001).
Sperm preparation for cryopreservation involves the removal of seminal plasma and consequently the predominant source of antioxidant protection. Freeze-thawing of equine spermatozoa is also associated with an increase in ROS generation (Ball et al, 2001); consequently, cryopreservation of equine spermatozoa may subject sperm cells to oxidative stress and potential DNA damage. Cooled storage of equine spermatozoa has been associated with an increase in lipid peroxidation (Ball and Vo, 2002) and DNA fragmentation (Linfor and Meyers, 2002); however, the effect of cryopreservation on equine sperm DNA fragmentation has not been reported.
The objective of the study reported here was to evaluate the effect of cryopreservation and exogenously generated ROS on DNA fragmentation of equine spermatozoa and to compare the ability of catalase, superoxide dismutase (SOD), and reduced glutathione (GSH) to preserve DNA integrity after ROS challenge.
Materials and Methods
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All reagents were obtained from Sigma Chemical (St. Louis, Mo) unless otherwise indicated. Glutathione (reduced free acid) and xanthine were obtained from Calbiochem (San Diego, Calif). For the comet assay, proteinase K and RNase A were purchased from Amresco (Solon, Ohio), and fully frosted microscope slides were obtained from Fisher Scientific (Pittsburgh, Penn).
Three experiments were conducted within this study. Experiment 1 investigated the effect of ROS, generated by the X-XO system, on DNA fragmentation of equine spermatozoa. Experiment 2 examined the potential of catalase, SOD, and GSH to counteract ROS-induced DNA fragmentation. Experiment 3 determined the effect of cryopreservation on DNA fragmentation of equine spermatozoa. The concentrations of X-XO and enzyme scavengers used in experiments 1 and 2 were based on those described by Baumber et al (2001). Experiments 1 and 2 were replicated across 2 ejaculates from each of 3 stallions, and experiment 3 was replicated across 2 ejaculates from each of 4 stallions.
Experiment 1: The Effect of Reactive Oxygen Species on Equine Sperm DNA Fragmentation
Semen was collected by artificial vagina from mature stallions of light horse breed, filtered and diluted 1:1 in a modified Tyrode's albumin-lactate-pyruvate medium supplemented with 0.1% polyvinyl alcohol (TALP; Padilla and Foote, 1991). A sperm-rich supernatant was obtained by centrifugation at 50 × g for 10 minutes (Meyers et al, 1995). Two milliliters of this supernatant was placed over an 80%/40% Percoll gradient (Drobnis et al, 1991) and centrifuged for 20 minutes at 300 × g. The sperm pellets were aspirated and resuspended in 5 mL of TALP, then centrifuged again for 10 minutes at 300 × g. Spermatozoa were then resuspended in TALP to a final concentration of 25 × 106 cells/mL and incubated at 38°C (5% CO2 in air) for 1 hour according to the following treatments: 1) control; 2) sperm + × (0.3 mM)-XO (0.025 U/mL); 3) sperm + × (0.6 mM)-XO (0.05 U/mL); and 4) sperm + × (1 mM)-XO (0.1 U/mL). After incubation, treatments were subjected to the comet assay as described below.
Experiment 2: Effect of Antioxidants on DNA Fragmentation Induced by Reactive Oxygen Species
Semen was collected and processed as described in experiment 1; however, spermatozoa were incubated according to the following treatments: 1) control; 2) sperm + × (1 mM)-XO (0.1 U/mL); 3) sperm + X-XO + catalase (from Aspergillus niger, 200 U/mL); 4) sperm + X-XO + SOD (from bovine erythrocytes, 200 U/mL); 5) sperm + X-XO + GSH (10 mM). Following incubation, treatments were subjected to the comet assay as described below.
Experiment 3: The Effect of Cryopreservation on Equine Sperm DNA Fragmentation
Semen was collected as described above, filtered, and extended to 50 × 106 cells/mL in EZ Mixin-BF (Animal Reproduction Systems; Chino, Calif). All ejaculates contained at least 5 billion spermatozoa and had an initial progressive motility of more than 50%. Following centrifugation (400 × g, 15 minutes), sperm pellets were resuspended in cryopreservation extender (INRA 82 + 2.5% glycerol; Vidament et al, 2000) at a concentration of 400 × 10 cells/mL. The extended semen was loaded into 0.5-mL polyvinylchloride straws (IMV; Minneapolis, Minn) that were then sealed with polyvinyl chloride sealing powder (IMV). Straws were frozen with a programmable freezer according to the following freezing curve: from 20°C to 5°C, −0.5°C/min; from 5°C to −15°C, −10°C/min; and from −15°C to −150°C, −25°C/min. Straws were thawed (30 seconds at 37°C) for postthaw analysis following at least 1 week of storage at −196°C. Sperm motility and DNA fragmentation were determined by computer-assisted sperm analysis and the comet assay, respectively, in fresh (extended in EZ Mixin; Animal Reproduction Systems), processed (loaded into straws) and frozen (postthaw) sperm samples. For postthaw motility analysis, spermatozoa were diluted to 25 × 106 cells/mL in prewarmed EZ Mixin-BF and incubated at 38°C; motility was determined after 15 and 30 minutes of incubation.
Comet Assay Protocol
The DNA status of individual cells was determined by the neutral single cell gel electrophoresis (comet) assay, which was modified to measure DNA damage in equine spermatozoa (Linfor and Meyers, 2002). For this assay, spermatozoa were embedded in agarose, followed by cell lysis, DNA decondensation, electrophoresis, neutralization, and DNA staining with ethidium bromide. The cells were then visualized by fluorescent microscopy. Intact nuclei in the comet assay appeared to have compact and brightly fluorescent heads; in contrast, strand breaks in damaged cells allow DNA migration during electrophoresis, and a trail of DNA could be seen behind the head, giving the appearance of a comet (McKelvey-Martin et al, 1993; Hughes et al, 1996).
After subjecting spermatozoa to the comet assay protocol as described by Linfor and Meyers (2002), sperm nuclei were imaged with epifluorescent microscopy (ex = 510; em = 595; Olympus BX60, Melville, NY). Images of sperm nuclei were digitized with an MTI CCD-72 camera (Dage-MTI, Michigan City, Ind) and NIH Image 1.61 (NIH, Bethesda, Md). Digitized images of sperm nuclei (n = 100 per slide) were subsequently evaluated for each treatment by an examiner that was unaware of treatment groups. Sperm comets were scored (Collins et al, 1995) from grade 0 (no comet, ie, no damage) to grade 4 (large comet, ie, extensive damage) as illustrated in Figure 1. The individual grade scores for 100 spermatozoa from each treatment were converted into a composite score by multiplying the number of sperm nuclei by the corresponding numerical score. Thus the composite score could range from 0 (all undamaged) to 400 (all maximally damaged).
Figure 1. . Epifluorescent photomicrographs of equine spermatozoa after the single cell gel electrophoresis (comet) assay and staining by ethidium bromide. Relative changes in DNA fragmentation are represented by an increasing amount of DNA present in the comet tail and are scored from grade 0 (no comet tail) to grade 4. Magnification 400×; scale bar represents 20 μM.
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Data were analyzed by analysis of variance (ANOVA), and comparisons between individual means were performed with Fisher's protected least significant difference test. In experiment 3, correlations between motility and composite score were determined by Fisher r to z test. Differences with values of P < .05 were considered to be statistically significant.
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In the present study, we report that exogenous generation of ROS increases DNA fragmentation in equine spermatozoa. Our results agree with those of Lopes et al (1998) in that treatment with the X-XO system promotes DNA fragmentation in sperm cells. Given the detrimental effect of ROS on sperm DNA, antioxidants might be expected to prevent the loss of DNA integrity. In the current study, the antioxidants, catalase, and GSH reduced equine sperm DNA damage subsequent to X-XO treatment. In addition to catalase and glutathione (Donnelly et al, 2000; Lopes et al, 1998), several other antioxidants have been shown to reduce human sperm DNA fragmentation resulting from oxidative stress, including ascorbic acid (Hughes et al, 1998; Donelly et al, 1999); hypotaurine (Lopes et al, 1998; Donelly et al, 2000); N-acetylcholine (Lopes et al, 1998); α-tocopherol (Hughes et al, 1998; Donnelly et al, 1999); and urate (Hughes et al, 1998). The protective action of catalase and not SOD in this study suggests that hydrogen peroxide (H2O2) and not superoxide (O2−·) is the species responsible for DNA damage. Direct addition of exogenous H2O2 also promotes DNA fragmentation in human spermatozoa (Hughes et al, 1996; McKelvey-Martin, 1997; Aitken et al, 1998a; Twigg et al, 1998b, 1998c; Donnelly et al, 1999, 2000; Duru et al, 2000). Hydrogen peroxide, in contrast to O2−, is more stable and readily crosses the plasma membrane (Halliwell, 1991), it has therefore been determined to be the predominant ROS responsible for oxidative damage to spermatozoa in vitro (Alvarez and Storey, 1989; de Lamirande and Gagnon, 1992a, 1992b; Aitken et al, 1993a; Griveau et al, 1995a; Baumber et al, 2001). It is important to note that the detrimental action of H2O2 is predominantly due to transition metal ion-catalyzed production of the hydroxyl radical (OH•) via the Fenton reaction. The hydroxyl radical is a highly reactive species and a powerful initiator of lipid peroxidation. Although OH• can react directly with DNA bases, it must be generated immediately adjacent to the nucleic acid molecule; the reactivity of OH• is so great that it can only diffuse short distances before reacting with a cellular component (Marnett, 2000). Peroxidation of DNA by cytotoxic products of lipid peroxidation (eg, peroxyl radicals, malondialdehyde and 4-hydroxynonenol) is an important mechanism in the loss of DNA integrity observed following oxidative stress.
The generation of ROS by sperm cells is believed to involve an NADPH oxidase similar to that reported in leukocytes (Aitken et al, 1992, 1997; Ball et al, 2001; Banfi et al, 2001). Incubation with NADPH promotes endogenous ROS generation by equine (Ball et al, 2001) and human (Aitken et al, 1997) spermatozoa. Spermatozoa primarily generate O2−·; however, it rapidly dismutates, either spontaneously or catalyzed by SOD to H2O2. Consequently, incubation with NADPH can also decrease sperm motility, increase lipid peroxidation, and induce DNA damage in human spermatozoa (Aitken et al, 1998a; Twigg et al, 1998b, 1998c), demonstrating that endogenous as well as exogenous ROS can be detrimental to spermatozoa.
The endogenous generation of ROS can also be increased by freeze-thawing equine spermatozoa (Ball et al, 2001), and DNA fragmentation was reported to increase in a linear fashion with the number of freeze-thaw cycles (Linfor and Meyers, 2002). With this in mind, we hypothesized that cryopreservation of equine spermatozoa would result in a significant increase in DNA fragmentation; the results of the current study support our hypothesis. An increase in DNA fragmentation, subsequent to cryopreservation, was also reported in trout (Labbe et al, 2001) and human spermatozoa (Donnelly et al, 2001a). In contrast, Duty et al (2002) did not find an increase in DNA fragmentation following cryopreservation of human spermatozoa, and Donnelly et al (2001b) reported that only spermatozoa from infertile males and not from fertile males demonstrated a significant increase in DNA fragmentation following cryopreservation. Cryopreservation protocols and extender formulations vary between laboratories and between species and may account for the differences observed. Additionally, it is possible that equine spermatozoa are more susceptible to cryopreservation-induced DNA damage than human spermatozoa.
There is a burgeoning interest in the implications of DNA-damaged spermatozoa to fertility. DNA fragmentation of human spermatozoa was negatively correlated with fertilization rates after in vitro fertilization (IVF; Sun et al, 1997; Host et al, 2000). In contrast, sperm DNA damage did not preclude pronucleus formation after intracytoplasmic sperm injection (ICSI; Twigg et al, 1998c; Host et al, 2000; Morris et al, 2002). In the case of in vivo fertilization and IVF, it is believed that sperm DNA damage induced by oxidative stress will be associated with collateral peroxidative damage to the sperm plasma membrane, consequently limiting fertilization. However, Aitken et al (1998a) report that ROS-induced sperm DNA damage occurred prior to any measurable changes in sperm-oocyte fusion or motility. Furthermore, in IVF, sperm suffering from a moderate degree of DNA damage were more likely to fuse with the oocyte than normal cells because of the positive effects of low-level oxidative stress on sperm capacitation (Aitken et al, 1998a). Consequently, fertilization by spermatozoa with damaged DNA is a possibility that requires consideration, particularly with regard to assisted reproductive techniques. Fertilization with DNA-damaged spermatozoa may lead to paternal transmission of defective genetic material with adverse consequences for embryonic development (Aitken and Krausz, 2001). Spermatozoa are unable to repair DNA damage, as they do not contain functional repair enzymes (Van Loon et al, 1991; Drost and Lee, 1995). After fertilization, the oocyte can repair a certain level of sperm DNA damage (Genesca et al, 1992); however, Ahmadi and Ng (1999a) report that damage beyond a certain level results in a low rate of embryonic development and early pregnancy loss. Several researchers report a reduction in embryonic development after fertilization with DNA-damaged spermatozoa (Sun et al, 1997; Ahmadi and Ng, 1999a, 1999b; Morris et al, 2002). The clinical consequence of sperm DNA damage to equine fertility in vivo or in vitro requires investigation.
In summary, ROS and cryopreservation promote DNA fragmentation in equine spermatozoa. DNA fragmentation induced by exogenous ROS, generated by the X-XO system, can be counteracted by the addition of antioxidants, catalase, and reduced glutathione. The involvement of ROS in cryopreservation-induced DNA damage remains to be determined.