| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Departments of * Population Health and
Reproduction and Anatomy, Physiology, and Cell
Biology, University of California, Davis.
| Correspondence to: B. A. Ball, Department of Population Health and Reproduction, 1114 Tupper Hall, University of California, Davis, CA 95616 (e-mail: baball{at}ucdavis.edu). |
| Received for publication December 13, 2002; accepted for publication February 5, 2003. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: DNA damage, oxidative stress, antioxidants, horse, sperm
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).
The lipid peroxidation cascade is initiated when reactive oxygen species attack polyunsaturated fatty acids (PUFA) in the sperm cell membrane (Storey, 1997; Jones et al, 1979). Spermatozoa are particularly susceptible to oxidative attack because they contain high concentrations of unsaturated fatty acids (Jones et al, 1979) and have limited repair mechanisms (Aitken and Clarkson, 1987; Van Loon et al, 1991). As a consequence of lipid peroxidation, the plasma membrane loses the fluidity and integrity it requires for participation in the membrane fusion events associated with fertilization (Ohyashiki et al, 1988; Block, 1991; 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 |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
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 x 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 x g. The sperm pellets were
aspirated and resuspended in 5 mL of TALP, then centrifuged again for 10
minutes at 300 x g. Spermatozoa were then resuspended in TALP
to a final concentration of 25 x 106 cells/mL and incubated
at 38°C (5% CO2 in air) for 1 hour according to the following
treatments: 1) control; 2) sperm + X (0.3 mM)-XO (0.025 U/mL); 3) sperm + X
(0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (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 + X (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
x 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 x g, 15 minutes), sperm pellets were resuspended in
cryopreservation extender (INRA 82 + 2.5% glycerol;
Vidament et al, 2000) at a
concentration of 400 x 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 x 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).
|
Statistical Analyses
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.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Experiment 2: Effect of Antioxidants on DNA Fragmentation Induced by
Reactive Oxygen Species
Treatment with X (1 mM)-XO (0.1 U/mL) resulted in a significant (P
< .01) increase in composite score when compared with the control. The
increase in composite score associated with X-XO treatment was reduced
(P < .01) by the addition of catalase and GSH, but not by the
addition of SOD (P = .98; Figure
3).
|
Experiment 3: The Effect of Cryopreservation on Equine Sperm DNA
Fragmentation
Cryopreservation of equine spermatozoa resulted in a significant decrease
in total (P < .0001) and progressive (P < .0001)
motility (Figure 4) and a
significant increase (P < .01) in DNA fragmentation
(Figure 5) when compared with
both fresh and processed sperm samples. Composite score was significantly
correlated with total motility at time 15 (r = –0.453;
P < .05) and time 30 (r = –0.413; P <
.05) minutes postthaw. There was no significant difference in sperm motility
or DNA fragmentation between fresh and processed spermatozoa.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-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.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa.
Hum Reprod.1999b; 14:2279
–2285.
Aitken RJ, Buckingham DW, Brindle J, Gomez E, Baker HW, Irvine DS.
Analysis of sperm movement in relation to the oxidative stress created by
leukocytes in washed sperm preparations and seminal plasma. Hum
Reprod. 1995a;10:2061
–2071.
Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil.1993a; 97:441 –450.
Aitken RJ, Buckingham DW, West KM. Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Phys. 1992;151:466 –477.[Medline]
Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil.1987; 81:459 –469.
Aitken RJ, Clarkson JS, Fisher S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod. 1989a;41:183 –197.[Abstract]
Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of
the relationship between defective sperm function and the generation of
reactive oxygen species in cases of oligozoospermia. J
Androl. 1989b;10:214
–220.
Aitken RJ, Fisher HM, Fulton N, Gomez E, Knox W, Lewis B, Irvine S. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol Reprod Dev.1997; 47:468 –482.[Medline]
Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z,
Irvine DS. Relative impact of oxidative stress on the functional competence
and genomic integrity of human spermatozoa. Biol
Reprod. 1998a;59:1037
–1046.
Aitken RJ, Harkiss D, Buckingham D. Relationship between iron-catalyzed lipid peroxidation potential and human sperm function. J Reprod Fertil.1993b; 98:257 –265.
Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS. A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. J Cell Sci.1998b; 111:645 –656.[Abstract]
Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction.2001; 122:497 –506.[Abstract]
Aitken RJ, Krausz C, Buckingham D. Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol Reprod Dev.1994a; 39:268 –279.[Medline]
Aitken RJ, Paterson M, Fisher H, Buckingham DW, Van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci.1995b; 108:2017 –2025.[Abstract]
Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl.1990;13:433 –451.[Medline]
Aitken RJ, West K, Buckingham D. Leukocytic infiltration into the
human ejaculate and its association with semen quality, oxidative stress, and
sperm function. J Androl.1994b; 15:343
–352.
Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gam Res.1989;23:77 –90.
Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid
peroxidation and production of hydrogen peroxide and superoxide in human
spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen
toxicity. J Androl.1987; 8:338
–348.
Baiardi G, Ruiz RD, Fiol de Cuneo M, Ponce AA, Lacuara JL, Vincenti L. Differential effects of pharmacologically generated reactive oxygen species upon functional activity of epididymal mouse spermatozoa. Can J Phys Pharm. 1997;75:173 –178.[Medline]
Ball BA, Gravance CG, Medina V, Baumber J, Liu IKM. Catalase activity in equine semen. Am J Vet Res.2000; 61:1026 –1030.[Medline]
Ball BA, Vo AT. Detection of lipid peroxidation in equine spermatozoa based upon the lipophilic fluorescent dye C1l-BODIPY581/591. J Androl. 2002;23:259 –269.[Abstract]
Ball BA, Vo AT, Baumber J. Reactive oxygen species generation by equine spermatozoa. Am J Vet Res.2001; 62:5508 –5515.
Banfi B, Molnar G, Maturana A, Steger K, Hegedus B, Demaurex N,
Krause KH. A Ca2+-activated NADPH oxidase in testis,
spleen and lymph nodes. J Biol Chem.2001; 276:37594
–37601.
Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl.2001; 21:895 –902.
Baumber J, Vo A, Sabeur K, Ball BA. Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa. Theriogenology2002; 57:1005 –1198.[Medline]
Block ER. Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells. J Cell Phys. 1991;146:362 –369.[Medline]
Blondin P, Coenen K, Sirard MA. The impact of reactive oxygen
species on bovine sperm fertilizing ability and oocyte maturation.
J Androl. 1997;18:454
–460.
Chen CS, Chao HT, Pan RL, Wei YH. Hydroxyl radical-induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm. Biochem Mol Biol Int.1997; 43:291 –303.[Medline]
Collins AR, Ma AG, Duthie SJ. The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mut Res. 1995;336:69 –77.[Medline]
de Lamirande E, Eiley D, Gagnon C. Inverse relationship between the induction of human sperm capacitation and spontaneous acrosome reaction by various biological fluids and the superoxide scavenging capacity of these fluids. Int J Androl.1993; 16:258 –266.[Medline]
de Lamirande E, Gagnon C. Reactive oxygen species and human
spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm
axonemes. J Androl.1992a; 13:368
–378.
de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J Androl.1992b; 13:379 –386.[Abstract]
de Lamirande E, Gagnon C. A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa. Int J Androl.1993a; 16:21 –25.[Medline]
de Lamirande E, Gagnon C. Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Rad Biol Med. 1993b;14:157 –166.[Medline]
de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of
reactive oxygen species in human sperm arcosome reaction induced by A23187,
lysophosphatidylcholine, and biological fluid ultrafiltrates. J
Androl. 1998;19:585
–594.
Donnelly ET, McClure N, Lewis SE. The effect of ascorbate and
alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen
peroxide-induced DNA damage in human spermatozoa.
Mutagenesis.1999; 14:505
–512.
Donnelly ET, McClure N, Lewis SE. Glutathione and hypotaurine in
vitro: effects on human sperm motility, DNA integrity and production of
reactive oxygen species. Mutagenesis.2000; 15:61
–68.
Donnelly ET, McClure N, Lewis SE. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil Steril.2001a; 76:892 –900.[Medline]
Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA
integrity and morphology of ejaculated spermatozoa from fertile and infertile
men before and after cryopreservation. Hum Reprod.2001b; 16:1191
–1199.
Drobnis EZ, Zhong CQ, Overstreet JW. Separation of cryopreserved
human semen using Sephadex columns, washing, or Percoll gradients.
J Androl. 1991;12:201
–208.
Drost JB, Lee WR. Biological basis of germline mutation: comparisons of spontaneous germline mutation rates among Drosophila, mouse, and human. Environ Mol Mutagen.1995; 25(suppl 26):48 –64.
Duru NK, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertil Steril. 2000;74:1200 –1207.[Medline]
Duty SM, Singh NP, Ryan L, Chen Z, Lewis C, Huang T, Hauser R.
Reliability of the comet assay in cryopreserved human sperm. Hum
Reprod. 2002;17:1274
–1280.
Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromosome aberrations in the hamster egg. Hum Genet.1992; 89:181 –186.[Medline]
Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defense systems in human spermatozoa. J Reprod Fertil.1995a; 103:17 –26.
Griveau JF, Renard P, Le Lannou D. An in vitro promoting role for hydrogen peroxide in human sperm capacitation. Int J Androl. 1994;17:300 –307.[Medline]
Griveau JF, Renard P, Le Lannou D. Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process. Int J Androl.1995b; 18:67 –74.
Halliwell B. Reactive oxygen species in living systems: source, biochemistry and role in human disease. Am J Med.1991; 91(suppl 3c):14s –22s.
Host E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand. 2000;79:559 –563.[Medline]
Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. A comparison
of baseline and induced DNA damage in human spermatozoa from fertile and
infertile men, using a modified comet assay. Mol Human
Reprod. 1996;2:613
–619.
Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. The effects of
antioxidant supplementation during Percoll preparation on human sperm DNA
integrity. Human Reprod.1998; 13:1240
–1247.
Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril.1992; 57:409 –416.[Medline]
Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Res. 1989;24:185 –196.[Medline]
Jones R, Mann T, Sherins R. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil Steril.1979;31:531 –537.[Medline]
Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril.1997; 68:519 –524.[Medline]
Labbe C, Martoriati A, Devaux A, Maisse G. Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout. Mol Reprod Dev.2001; 60:397 –404.[Medline]
Leclerc P, de Lamirande E, Gagnon C. Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. Free Rad Biol Med.1997; 22:643 –656.[Medline]
Li TK. The glutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol Reprod.1975; 12:641 –646.[Abstract]
Linfor JJ, Meyers SA. Detection of DNA damage in response to cooling injury in equine spermatozoa using single cell gel electrophoresis. J Androl. 2002;23:107 –113.[Abstract]
Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species:
potential cause for DNA fragmentation in human spermatozoa. Human
Reprod. 1998;13:896
–900.
Marnett LJ. Oxyradicals and DNA damage.
Carcinogenesis.2000; 21:361
–370.
McKelvey-Martin VJ, Green MH, Schmezer P, Pool-Zobel BL, De Meo MP, Collins A. The single cell gel electrophoresis assay (comet assay): a European review. Mut Res.1993; 288:47 –63.[Medline]
McKelvey-Martin VJ, Melia N, Walsh IK, Johnston SR, Hughes CM, Lewis SE, Thompson W. Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1). Human bladder washings and transitional cell carcinoma of the bladder; and (2). human sperm and male infertility. Mut Res.1997; 375:93 –104.[Medline]
Meyers SA, Overstreet JW, Liu IKM, Drobnis EZ. Capacitation in
vitro of stallion spermatozoa: comparison of progesterone-induced acrosome
reactions in fertile and subfertile males. J Androl.1995; 16:47
–54.
Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage
in human sperm assessed by single cell gel electrophoresis (comet assay) and
its relationship to fertilization and embryo development. Hum
Reprod. 2002;17:990
–998.
Nissen HP, Kreysel HW. Superoxide dismutase in human semen. Klinische Wochenschrift.1983; 61:63 –65.[Medline]
Ohyashiki T, Ohtsuka T, Mohri T. Increase of the molecular rigidity of the protein conformation in the intestinal brush-border membranes by lipid peroxidation. Biochim Biophys Acta.1988; 939:383 –392.[Medline]
Padilla AW, Foote RH. Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa. J Anim Sci. 1991;69:3308 –3313.[Abstract]
Potts RJ, Notarianni LJ, Jefferies TM. Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mut Res.2000; 447:249 –256.[Medline]
Rao B, Soufir JC, Martin M, David G. Lipid peroxidation in human spermatozoa as related to mid-piece abnormalities and motility. Gamete Res.1989; 24:127 –134.[Medline]
Storey BT. Biochemistry of the induction and prevention of
lipoperoxidative damage in human spermatozoa. Mol Human
Reprod. 1997;3:203
–213.
Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod.1997; 56:602 –607.[Abstract]
Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ.
Iatrogenic DNA damage induced in human spermatozoa during sperm preparation:
protective significance of seminal plasma. Mol Human
Reprod. 1998a;4:439
–445.
Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Analysis of the
impact of intracellular reactive oxygen species generation on the structural
and functional integrity of human spermatozoa: lipid peroxidation, DNA
fragmentation and effectiveness of antioxidants. Human
Reprod. 1998b;13:1429
–1436.
Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human
spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm
injection. Human Reprod.1998c; 13:1864
–1871.
Van Loon AA, Den Boer PJ, Van der Schans GP, Mackenbach P, Grootegoed JA, Baan RA, Lohman PH. Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation. Exp Cell Res.1991; 193:303 –309.[Medline]
Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E. Centrifugation and addition of glycerol at 22°C instead of 4°C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology2000; 54:907 –919.[Medline]
Wolff H, Politch JA, Martinez A, Haimovici F, Hill JA, Anderson DJ. Leukocytospermia is associated with poor semen quality. Fertil Steril. 1990;53:528 –536.[Medline]
This article has been cited by other articles:
|
|
 |
|
  L.K. Thomson, S.D. Fleming, R.J. Aitken, G.N. De Iuliis, J.-A. Zieschang, and A.M. Clark Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis Hum. Reprod., June 12, 2009; (2009) dep214v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-W. Li, S. Meyers, T. L. Tollner, and J. W. Overstreet Damage to Chromosomes and DNA of Rhesus Monkey Sperm Following Cryopreservation J Androl, July 1, 2007; 28(4): 493 - 501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Rosello, C. Matas, S. Canovas, P. N. Moreira, J. Gadea, and P. Coy Influence of Sperm Pretreatment on the Efficiency of Intracytoplasmic Sperm Injection in Pigs J Androl, March 1, 2006; 27(2): 268 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Roca, M. J. Rodriguez, M. A. Gil, G. Carvajal, E. M. Garcia, C. Cuello, J. M. Vazquez, and E. A. Martinez Survival and In Vitro Fertility of Boar Spermatozoa Frozen in the Presence of Superoxide Dismutase and/or Catalase J Androl, January 1, 2005; 26(1): 15 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Dobrzynska, A. Baumgartner, and D. Anderson Antioxidants modulate thyroid hormone- and noradrenaline-induced DNA damage in human sperm Mutagenesis, July 1, 2004; 19(4): 325 - 330. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
