| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Department of * Anatomy and
Biochemistry, Faculty of Medicine, The
University of Hong Kong, and Department of
Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong
SAR, P. R. China.
| Correspondence to: Dr Wai-sum O, Department of Anatomy, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, P. R. China (e-mail: owaisum{at}hkucc.hku.hk). |
| Received for publication December 2, 2002; accepted for publication March 20, 2003. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Superoxide dismutase, glutathione peroxidase, catalase, sperm DNA damage, oxidative stress, Syrian hamster
Our previous investigations demonstrated that removal of all or some of the male accessory sex glands (ASGs) in the golden hamster lowered fertility and increased embryonic wastage during implantation and the postimplantation period (Chow et al, 1986; O et al, 1988), structural abnormalities in implanted embryos were more extensive (Chan et al, 2001; Jiang et al, 2001), whereas sperm chromatin decondensation and DNA replication in the first cell cycle were delayed (Ying et al, 1998, 1999). These could be attributed to sperm DNA damage. Recently, we demonstrated that DNA damage was more extensive in sperm not exposed to male ASG secretions. Mature epididymal spermatozoa were less susceptible to NADPH treatment and male ASG secretions could protect uterine sperm DNA from breakage after NADPH treatment, indicating that male ASG secretions might have a role in preserving sperm genomic integrity in the female genital tract (Chen et al, 2002). In the golden hamster, the major male ASGs include ampullary glands, ventral prostate, dorsolateral prostates, coagulating glands, and seminal vesicles. Their secretions make up the bulk of seminal plasma. In humans, seminal plasma has been reported to contain antioxidant enzymes, such as SOD, catalase (CAT), and glutathione peroxidase (GPx), and free radical scavengers, such as vitamins C and E, hypotaurine, taurine, uric acid, and albumin (Lewis et al, 1997; Yeung et al, 1998; Zini et al, 2002). Rodent epididymis also secretes antioxidant enzymes and free radical scavengers (Aumüller et al, 1990).
In this study, we address 3 questions: 1) Do male ASGs of golden hamster secrete antioxidant enzymes, such as SOD, GPx, and CAT? 2) What are the main antioxidant enzymes present in postcoital uterine fluid? and 3) Can SOD protect sperm that are not exposed to male ASG secretions against NADPH-induced oxidative stress? Comet assay, also known as single-cell gel electrophoresis, is used to assess sperm DNA integrity. It is rapid, simple, visual, and sensitive for detecting alkali labile sites and DNA strand breaks in individual mammalian cells (Ostling and Johanson, 1984; Singh et al, 1988; Olive et al, 1990). The introduction of alkaline (pH >13) condition to unwind, denature, and separate DNA by electrophoresis makes the revelation of single-strand DNA (ssDNA) breakage more obvious.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Animal Model
Animals were maintained and handled in compliance with a protocol approved
by the Committee on the Use of Life Animals for Teaching and Research of The
University of Hong Kong. Randomly bred Syrian hamsters (Mesocricetus
auratus) were supplied by and housed in the Laboratory Animal Unit of the
Faculty of Medicine, The University of Hong Kong, under a 14 hours light-10
hours dark lighting cycle, with lights on from 1100 to 0100 hours. Temperature
was maintained at 22°C, and food and tap water were available ad libitum.
Vaginal secretions of 6- to 8-week-old female hamsters were checked daily for
at least 2 normal consecutive cycles before mating.
Under general anesthesia with a 2:1 mixture of 10% ketamine and 2% xylazine (0.2 mL/100 g of body weight, intraperitoneal) (Alfasan, Woerden, Holland), major male ASGs were surgically ablated from 6- to 8-week-old male hamsters according to procedures described by Chow et al (1986) and placed in the following 2 groups: sham-operated control hamster (SH; n = 8) and hamsters that underwent bilateral excision of ampullary gland, ventral prostate, dorsolateral prostate, coagulating gland, and seminal vesicle (TX; n = 10). VX stood for bilaterally vasectomized hamsters (n = 5). The animals were allowed to recuperate for 1 month and success of surgery was confirmed post mortem.
Sample Preparation
Collection of ASG Secretion—
Fourteen- to 20-week-old intact male hamsters of proven fertility were
killed with an overdose of sodium pentobarbital injected intraperitoneally
(Boehringer Ingelheim, Artamon, New South Wales, Australia). Blood was
withdrawn by cardiac puncture. Male ASGs were individually removed and cleared
of connective tissues and blood. Secretions were drained from the seminal
vesicles and coagulating glands. Cuts were made on the dorsolateral prostate,
ventral prostate, and ampullary gland to release secretions. Secretions were
pooled, dispensed into microcentrifuge tubes, kept in ice, and centrifuged at
200 x g for 20 minutes. The supernatant was collected and kept
at 4°C for immediate assay of enzyme activity and protein content.
Collection of Postejaculatory Uterine Fluid— Each normally cycling female hamster was mated with one operated-on male for 15 minutes on the day of estrus and killed with an overdose of sodium pentobarbital (Boehringer Ingelheim) within 30 minutes after mating. Uterine horns were removed, cleared of blood and connective tissues, and flushed with 1 mL of 1x PBS (pH 7.4). Debris and cells were removed by 2 rounds of centrifugation at 10 036 x g for 5 minutes. The supernatant was collected and stored at -70°C for enzyme activity measurement and protein assay later. Uterine content from virgin females at estrus was collected in the same manner.
Collection of Uterine Sperm— Sperm was collected by flushing the uterine horns with TALP medium with or without SOD at 50 U/mL (Roche, Mannheim, Germany). Samples from 2 females were pooled, washed, and centrifuged twice at 300 x g for 10 minutes. The pellet was resuspended in m-TALP medium with or without SOD at 50 U/mL followed by NADPH (Roche) treatment.
Treatment of Sperm With ß-NADPH
Uterine sperm (1 x 106 cells/mL) ejaculated by SH and TX
males was incubated for 2 hours at 37°C under 5% CO2 in media
containing 0, 1.25, 2.5, 5, 10, or 20 mmol/L NADPH with or without 50 U/mL of
SOD. After incubation, the treated sperm were pelleted twice at 300 x
g for 10 minutes and resuspended in fresh medium for comet assay.
Assessment of Sperm DNA Damage by Comet Assay
Comet assay for sperm was adapted from Shen and Ong
(2000) and was performed in
darkness. Sperm were resuspended in PBS at 6 to 8 x 105
cells/mL. Ten microliters of sperm suspension was thoroughly mixed with 75
µL of 0.5% (wt/vol) agarose 3:1 (Ameresco, Solon, Ohio). Fifty microliters
of the sperm suspension was pipetted onto a CometSlide (Trevigen,
Gaithersburg, Md) and allowed to gel. The slides were carefully submersed in
cold lysis buffer made up of 2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L
Tris-HCl, 10% (vol/vol) DMSO, and 1% (vol/vol) Triton-X 100, pH 10, for 1 hour
at 4°C. The slides were then transferred to another buffer (2.5 mol/L
NaCl, 100 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.4) containing DN-ase-free
proteinase K (10 µg/mL; Amresco) and incubated at 37°C overnight.
After enzyme treatment, the slides were transferred into an electrophoresis chamber (Pharmacia Biotech, San Francisco, Calif) filled with alkaline buffer made up of 300 mmol/L NaOH (E. Merck), 1 mmol/L EDTA, 0.2% (vol/vol) DMSO, and 0.1% (wt/vol) 8-hydroxyquinoline (pH >13). The slides were left side by side in alkaline buffer for 20 minutes to allow unwinding of the DNA before being electrophoresed at 0.96 V/cm, 250 mA for 20 minutes at room temperature. After electrophoresis, the slides were equilibrated with 0.4 mol/L Tris-HCl (pH 7.4) for 15 minutes followed by fixation in 100% (vol/vol) ethanol for 15 minutes and air-dried. The dried slides were immersed in 10 mmol/L NaH2PO4 and 5% (wt/vol) sucrose for 10 minutes and finally stained with 0.25 µmol/L YOYO-1 (Molecular Probes, Eugene, Oregon) in 5% (vol/vol) DMSO and 5% (wt/vol) sucrose for 10 minutes.
CometSlides were examined at x250 with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) fitted with a cooled CCD camera (KX Series Imaging System, Apogee Instruments Inc, Tucson, Ariz). The incidence of comet formation was scored for all the sperm on each circle of the CometSlide (2 circles) and expressed as the number of comets per 10 000 sperm.
Enzyme Activity Assays
Activities of antioxidant enzymes in individual ASG secretions, expressed
per milligram of secretory protein, were assayed using Shimadzu UV-Visible
Recording Spectrophotometer UV 1601 (Shimadzu Company, Tokyo, Japan). Total
SOD activity was determined by the nitroblue tetrazolium (NBT) assay
(Oberley and Spitz, 1985). One
unit of activity was defined as the quantity of SOD required to produce 50%
reduction of NBT. Total GPx activity was detected by the standard indirect
coupled method using cumene hydroperoxide
(Glunzler and Flohe, 1985),
and one unit of activity was defined as 1 µmol NADPH oxidized at 37°C
per minute using [GSH]0 equal to 1 mmol/L. Catalase activity assay
based on rate of peroxidation of titanium oxysulfate as described by Sun et al
(1975) was used. Enzyme
activity in red blood cells was determined in the same way. Their contribution
to activity of each type of enzyme in the samples was found to be negligible
(data not shown).
The SOD, GPx, and CAT activities in postcoital sperm-free uterine flushing were measured and expressed per milliliter of uterine flushing. Spectrophotometer equipped with Cary WinUV software (Cary 300 Bio UV-Visible Spectrophotometer, Varian Australia Pty Ltd, Victoria, Australia) was used to measure SOD and GPx activity. SOD activity assessment was based on NADH oxidation (Paoletti and Mocali, 1990), and one unit was defined as the amount of enzyme that inhibited the NADH oxidation of the control by 50%. Total GPx activity was estimated by the same method used for ASG secretions. CAT activity was determined by the decrease in concentration of H2O2 after incubation with test samples. The horseradish peroxidase-dependent oxidation of phenol red to a blue derivative (absorbance, 630 nm) was used to estimate H2O2 (Yeung et al, 1996). One unit of activity was defined as 1 µmol H2O2 removed at 37°C per minute.
Protein Assay
The BCA (bicinchoninic acid) Kit (Pierce, Rockford, Ill) was used to
determine protein concentration. Manufacturer's instruction was followed and
absorbance was read with a SPECTRA-max 340 Microplate Reader (Molecular
Devices Corporation, Sunnyvale, Calif).
Statistical Analysis
All results were presented as mean ± SEM. The incidence of DNA
damage was arcsine transformed before applying statistical analyses. All
results were analyzed by 1-way analysis of variance (ANOVA) followed by
Dunnett's posttest. Two-way ANOVA was also applied to analyze the response of
uterine sperm to NADPH incubation. P < .05 was considered
statistically significant (Prism software version 3.0, GraphPad, San Diego,
Calif). Each experiment was repeated at least 5 times.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Antioxidant Enzymes in Postcoital Uterine Flushing
Activities of antioxidant enzymes in uterine flushing collected immediately
after mating with operated-on male hamsters are shown in
Figure 2A through C. SOD
activity (units per milliliter of uterine flushing) in samples from males with
all ASGs removed was higher than the control (P < .01, TX vs SH)
and vasectomized groups (P < .05, TX vs VX). Values for virgin
female were used as baseline. Similarly, CAT activity (milliunits per
milliliter of uterine flushing) from males with all ASGs removed was also
significantly lower compared with the control (P < .001, TX vs SH)
and vasectomized hamsters (P < .001, TX vs VX). However, total GPx
activity (milliunits per milliliter of uterine flushing) was statistically
lower only when compared with the control (P < .05, TX vs SH) but
not the vasectomized group (P > .05, TX vs VX).
|
Protective Effects of Antioxidant Enzymes In Vitro
The results are shown in Figure
3. Without SOD treatment, sperm not exposed to ASG secretions (TX)
presented a very significant dose-dependent increase in the incidence of ssDNA
damage compared with those from the SH group when incubated with increasing
dose of NADPH (P < .001, TX vs SH). However, addition of SOD at 50
U/mL into the flushing medium when sperm were collected, followed by
incubation with NADPH added to 50 U/mL of SOD before performing the comet
assay, induced a significant and reversible dose-dependent decrease in
incidence of ssDNA damage in sperm from hamsters with all ASGs removed
(P < .001, TX with SOD vs TX).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Antioxidant Enzyme Activity
ROS consists mainly of superoxide anions (O.-), hydroxyl
radicals (OH.-), and hydrogen peroxide
(H2O2), and they are toxic to sperm. Their harmful
effect is reduced by serial antioxidant actions of SOD, which catalyses the
rapid removal of (O.-) to form H2O2. If not
removed, H2O2 in turn forms highly toxic hydrogen
radicals (OH.-) that has been reported to induce DNA damage and
cause a rapid loss of sperm fertilizing potential by promoting lipid
peroxidation and loss of adenosine triphosphate
(de Lamirande and Gagnon,
1992; Aitken et al,
1993). CAT and/or GPx rapidly convert the
H2O2 to H2O. In the present study, we have
found that antioxidant enzymes such as SOD, GPx, and CAT are present in the
male reproductive tract of golden hamsters. GPx is abundant in the epididymis,
whereas SOD and CAT are secreted mainly by male ASGs. This may be related to
protecting the maturing sperm before ejaculation. GPx messenger RNA (mRNA),
including glutathione peroxidase (GPx3), phospholipid hydroperoxide
glutathione peroxidase (PH-GPx or GPx4), and secretory epididymal glutathione
peroxidase (E-GPx or GPx5), is highly expressed in epididymis
(Zini and Schlegel, 1997;
Jervis and Robaire, 2001),
whereas CAT mRNA expression is lower (Zini
and Schlegel, 1996). GPx, especially GPx4, is thought to regulate
the redox status of mammalian sperm during completion of sperm chromatin
compaction in the epididymis (Godeas et
al, 1997). Moreover, together with CuZn-SOD, it constitutes the
core antioxidant system of the epididymis defense. The relative paucity of
intracellular antioxidant enzymes and the high concentration of unsaturated
fatty acids in epididymal spermatozoa membrane
(Awano et al, 1993;
Aitken, 1999) make sperm
extremely vulnerable to oxidative stress
(de Lamirande et al, 1997). On
reaching the cauda epididymis, sperm are provided with a microenvironment full
of antioxidant enzyme and scavengers that keep the ROS at a low and defensible
level. These include antioxidant systems, such as glutathione synthetase,
GPx5, PH-GPx or GPx4, thioredoxin peroxidase, CuZu-SOD, and glutathione
S-transferases (Jervis and Robaire,
2001). A low intraluminal pH also helps to suppress NADPH-oxidase
activity (Breton et al, 1996; Aitken et al, 1998). Epididymis
can also synthesize lactoferrin to bind free ion
(Jin et al, 1997), secrete
albumin-like protein to sequester toxic lipid peroxides
(Carles et al, 1992), and
produce small-molecular-weight free radical scavengers such as vitamin C,
glutathione, and polyamines (Khan et al,
1992; Muscari et al,
1995).
The postcoital uterine fluid of the hamster has SOD and CAT mainly contributed from male ASGs and GPx from accessory sex glands and epididymis. These enzymes are there to protect sperm. The uterine environment contains many elements that are harmful to sperm. They include nitric oxide (Norman and Cameron, 1996) and amino acids (Fahning et al, 1967). The latter have been reported to stimulate production of H2O2 in bovine sperm through the action of an amino acid oxidase (Lapointe and Sirard, 1998). Following mating, leukocytes (Thompson et al, 1992; Williams et al, 1993), mainly eosinophils (Perez et al, 1996), are stimulated by seminal plasma to produce large quantities of H2O2 (Hansen et al, 1987). Moreover, estrogen has prooxidant and antioxidant effects. At estrus, under the influence of estrogen, endometrial epithelial NADPH oxidase produces NADPH (Moulton and Barker, 1971; Hilf et al, 1972; Swanson and Barker, 1983), superoxide anions (Laloraya et al, 1991; Jain et al, 1999, 2000), and hydrogen peroxide (Riley and Behrman, 1991). An estrogen-stimulated antioxidant system that maintains an optimal amount of these oxidants also regulates sperm capacitation. The estrogen-induced antioxidant system mainly includes SOD, peroxidase from eosinophils and endometrial epithelial cells (Hosoya and Saito, 1981; Anderson et al, 1986; Riley and Behrman, 1991), and glutathione peroxidase/reductase system (Ohwada et al, 1996; Diaz-Flores et al, 1999; Kaneko et al, 2001). Northern blot could only detect a low level of CAT mRNA in uterus (Lapointe et al, 1998). Taken together, GPx and SOD are key antioxidant enzymes for sperm maturation and postejaculatory function.
Protective Effects of SOD In Vitro
A mature sperm is particularly vulnerable to oxidative stress, because its
membrane is rich in PUFAs and NADPH oxidase but its scanty cytoplasm has
little anti-oxidant enzymes. During maturation, epididymal fluid provides them
with many antioxidant enzymes and free radical scavengers. At ejaculation,
male ASG secretions put in some more SOD, CAT, GPx, vitamins C and E,
hypotaurine, taurine, uric acid, and albumin. We have previously demonstrated
that in the total absence of ASG (TX) sperm, ssDNA damage is more extensive
and more frequent (Chen et al,
2002). In the present study, we found high SOD activity in
secretions of these glands. We proposed that SOD could counteract the
NADPH-induced oxidative stress in sperm, and we confirmed that the incidence
of ssDNA damage was significantly reduced in the presence of exogenous SOD.
Other reports also suggested that antioxidants such as ascorbate (vitamin C),
-tocopherol (vitamin E), and urate can guard ejaculated human sperm
against x-ray-induced and H2O2-induced DNA damage in
vitro (Hughes et al, 1998;
Donnelly et al, 1999).
In summary, we have demonstrated that SOD and CAT from the male ASGs are the main antioxidative enzymes in the postcoital uterine environment. GPx, equally contributed by ASGs and epididymis, is also present. SOD protects sperm against NADPH-induced DNA breakage in a dose-dependent manner.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aitken RJ. The Amoroso Lecture: the human spermatozoon-a cell in crisis? J Reprod Fertil. 1999; 115:1-7.
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. 1993; 97:441-450.
Aitken J, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays. 1994; 16:259-267.[Medline]
Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine S. On the cellular
mechanisms by which the bicarbonate ion mediates the extragenomic action of
progesterone on human spermatozoa. Biol Reprod. 1998; 58:186-196.
Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction. 2001; 122:497-506.[Abstract]
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.
Anderson WA, Gabriel BW, Nerurkar SG, Wyche JH, Yates PE, Hanker JS. Peroxidases induced in rat uterus by estrogen administration, II: cytochemical and biochemical heterogeneity. J Submicrosc Cytol. 1986;18:683-690.[Medline]
Aumüller G, Huntemann S, Larsch KP, Seitz J. Transglutaminase immunoreactivity in the male genital tract of the rat. Acta Histochem Suppl. 1990;38:209-212.[Medline]
Awano M, Kawaguchi A, Mohri H. Lipid composition of hamster epididymal spermatozoa. J Reprod Fertil. 1993; 99:375-383.
Breton S, Smith PJ, Lui B, Brown D. Acidification of the male reproductive tract by a proton pumping (H+)-ATPase. Nat Med. 1996;2:470-472.[Medline]
Carles C, Fournier-Delpech S, Ribadeau-Dumas B. Purification of an ovine, androgen-dependent epididymal protein: evidence for a strong amino acid sequence homology with serum albumin. Reprod Nutr Dev. 1992; 32:277-284.
Chan OC, Chow PH, O WS. Total ablation of paternal accessory sex glands curtails developmental potential in preimplantation embryos in the golden hamster. Anat Embryol (Berl). 2001; 204:117-122.[Medline]
Chen H, Cheung MPL, Chow PH, Cheung ALM, Liu W, O WS. Protection of sperm DNA against oxidative stress in vivo by accessory sex gland secretions in male hamster. Reproduction. 2002; 124:491-499.[Abstract]
Chow PH, Pang SF, Ng KW, Wong TM. Fertility, fecundity, sex ratio and the accessory sex glands in male golden hamsters. Int J Androl. 1986;9:312-320.[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. 1992; 13:368-378.
de Lamirande E, Gagnon C. Capacitation-associated production of superoxide anion by human spermatozoa. Free Radic Biol Med. 1995a;18:487-495.[Medline]
de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod. 1995b; 10(suppl 1):15-21.
de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm physiology. Rev Reprod. 1997; 2:48-54.[Abstract]
Diaz-Flores M, Baiza-Gutman LA, Pedron NN, Hicks JJ. Uterine glutathione reductase activity: modulation by estrogens and progesterone. Life Sci. 1999;65:2481-2488.[Medline]
Donnelly E, McClure N, Lewis SE. The effect of ascorbate and
-tocopherol supplementation in vitro on DNA integrity and
hydrogen peroxide-induced DNA damage in human spermatozoa.
Mutagenesis. 1999; 14:505-511.
Fahning ML, Schultz RH, Graham EF. The free amino acid content of uterine fluids and blood serum in the cow. J Reprod Fertil. 1967;13:229-236.
Glunzler WA, Flohe L. Glutathione peroxidase. In: Greenwald RA, ed. CRC Handbook of Methods for Oxygen Radical Research. Boca Raton, Fla: CRC Press; 1985:285-290.
Godeas C, Tramer F, Micali F, Soranzo M, Sandri G, Panfili E. Distribution and possible novel role of phospholipid hydroperoxide glutathione peroxidase in rat epididymal spermatozoa. Biol Reprod. 1997; 57:1502-1508.[Abstract]
Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. J Reprod Fertil. 1995; 103:17-26.
Hansen PJ, Hoggard MP, Rathwell AC. Effects of stallion seminal plasma on hydrogen peroxide release by leukocytes exposed to spermatozoa and bacteria. J Reprod Immunol. 1987; 10:157-166.[Medline]
Hilf R, McDonald E, Sartini J, Rector WD, Richards AH. Response of uterine glucose-6-phosphate dehydrogenase isoenzymes to estrogen. Endocrinology. 1972; 91:280-286.[Medline]
Hosoya T, Saito T. Comparative studies on estrogen-dependent
peroxidases contained in uterine microsomes and fluid of rats and pigs.
J Biochem (Tokyo). 1981; 89:203-215.
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 Hum
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. Hum 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]
Jain S, Saxena D, Kumar PG, Koide SS, Laloraya M. Effect of estradiol and selected antiestrogens on pro- and antioxidant pathways in mammalian uterus. Contraception. 1999; 60:111-118.[Medline]
Jain S, Saxena D, Kumar GP, Laloraya M. NADPH dependent superoxide generation in the ovary and uterus of mice during estrous cycle and early pregnancy. Life Sci. 2000; 66:1139-1146.[Medline]
Jervis KM, Robaire B. Dynamic changes in gene expression along the
rat epididymis. Biol Reprod. 2001; 65:696-703.
Jiang HY, Lee KH, Schneider C, O WS, Tang PL, Chow PH. The growth arrest specific gene (gas6) protein is expressed in abnormal embryos sired by male golden hamsters with accessory sex glands removed. Anat Embryol. 2001; 203:343-355.[Medline]
Jin YZ, Bannai S, Dacheux F, Dacheux JL, Okamura N. Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis. Mol Reprod Dev. 1997; 47:490-496.[Medline]
Jones R, Mann T. Lipid peroxides in spermatozoa; formation, role of plasmalogen, and physiological significance. Proc R Soc Lond B Biol Sci. 1976;193:317-333.[Medline]
Kaneko T, Iuchi Y, Kawachiya S, Fujii T, Saito H, Kurachi H, Fujii
J. Alteration of glutathione reductase expression in the female reproductive
organs during the estrous cycle. Biol Reprod. 2001; 65:1410-1416.
Khan AU, Mei YH, Wilson T. A proposed function for spermine and
spermidine: protection of replicating DNA against damage by singlet oxygen.
Proc Natl Acad Sci U S A. 1992; 89:11426-11427.
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]
Laloraya M, Kumar GP, Laloraya MM. Changes in the superoxide radical and superoxide dismutase levels in the uterus of Rattus norvegicus during the estrous cycle and a possible role for superoxide radical in uterine oedema and cell proliferation at proestrus. Biochem Cell Biol. 1991;69:313-316.[Medline]
Lapointe S, Legare C, Gaudreault C, Sullivan R, Sirard MA. cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase. Mol Reprod Dev. 1998; 51:265-273[Medline]
Lapointe S, Sirard MA. Catalase and oviductal fluid reverse the
decreased motility of bovine sperm in culture medium containing specific amino
acids. J Androl. 1998; 19:31-36.
Lewis SE, McKinney KA, Thompson W. Influence of pentoxifylline on human sperm motility in asthenozoospermic individuals using computer-assisted analysis. Arch Androl. 1994; 32:175-183.[Medline]
Lewis SEM, Sterling ESL, Young IS, Thompson W. Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men. Fertil Steril. 1997; 67:142-147.[Medline]
Moulton BC, Barker KL. Synthesis and degradation of glucose-6-phosphate dehydrogenase in the rat uterus. Endocrinology. 1971; 89:1131-1136.[Medline]
Muscari C, Guarnieri C, Stefanelli C, Giaccari A, Caldarera CM. Protective effect of spermine on DNA exposed to oxidative stress. Mol Cell Biochem. 1995; 144:125-129.[Medline]
Norman JE, Cameron IT. Nitric oxide in the human uterus. Rev Reprod. 1996; 1:61-68.[Abstract]
O WS, Chen HQ, Chow PH. Effects of male accessory sex gland secretions on early embryonic development in the golden hamster. J Reprod Fertil. 1988;84:341-344.
Oberley LW, Spitz DR. Nitroblue tetrazolium. In: Greenwald RA, ed. CRC Handbook of Methods for Oxygen Radical Research. Boca Raton, Fla: CRC Press; 1985:217-221.
Ohwada M, Suzuki M, Sato I, Tsukamoto H, Watanabe K. Glutathione peroxidase activity in endometrium: effects of sex hormones and cancer. Gynecol Oncol. 1996; 60:277-282.[Medline]
Olive PL, Banath JP, Durand RE. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res. 1990; 122:86-94.[Medline]
Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun. 1984; 123:291-298.[Medline]
Palan P, Naz R. Changes in various antioxidant levels in human seminal plasma related to immunoinfertility. Arch Androl. 1996;36:139-143.[Medline]
Paoletti F, Mocali A. Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. Methods Enzymol. 1990; 186:209-220.[Medline]
Perez MC, Furth EE, Matzumura PD, Lyttle CR. Role of eosinophils in uterine responses to estrogen. Biol Reprod. 1996; 54:249-254.[Abstract]
Perry AC, Jones R, Hall L. Isolation and characterization of a rat cDNA clone encoding a secreted superoxide dismutase reveals the epididymis to be a major site of its expression. Biochem J. 1993; 293(pt 1):21-25.
Riley JC, Behrman HR. Oxygen radicals and reactive oxygen species in reproduction. Proc Soc Exp Biol Med. 1991; 198:781-791.[Abstract]
Sailer BL, Jost LK, Erickson KR, Tajiran MA, Evenson DP. Effects of X-irradiation on mouse testicular cells and sperm chromatin structure. Environ Mol Mutagen. 1995; 25:23-30.[Medline]
Sailer BL, Sarkar LJ, Bjordahl JA, Jost LK, Evenson DP. Effects of
heat stress on mouse testicular cells and sperm chromatin structure.
J Androl. 1997;18:294-301.
Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999;4:31-37.[Abstract]
Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ Jr, Agarwal A. The
reactive oxygen species-total antioxidant capacity score is a new measure of
oxidative stress to predict male infertility. Hum
Reprod. 1999;14:2801-2807.
Shen H, Ong C. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med. 2000;28:529-536.[Medline]
Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175:184-191.[Medline]
Smith R, Vantman D, Ponce J, Escobar J, Lissi E. Total antioxidant
capacity of human seminal plasma. Hum Reprod. 1996; 11:1655-1660.
Sun AS, Aggarwal BB, Packer L. Enzymes level of normal human cells: aging in culture. Arch Biochem Biophys. 1975; 170:1-11.[Medline]
Swanson LV, Barker KL. Antagonistic effects of progesterone on estradiol-induced synthesis and degradation of uterine glucose-6-phosphate dehydrogenase. Endocrinology. 1983; 112:459-465.[Medline]
Thompson LA, Barratt CL, Bolton AE, Cooke ID. The leukocytic reaction of the human uterine cervix. Am J Reprod Immunol. 1992;28:85-89.
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 Hum
Reprod. 1998;4:439-445.
van Overveld FW, Haenen GR, Rhemrev J, Vermeiden JP, Bast A. Tyrosine as important contributor to the antioxidant capacity of seminal plasma. Chem Biol Interact. 2000; 127:151-161.[Medline]
Vernet P, Rigaudiere N, Ghyselinck N, Dufaure JP, Drevet JR. In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage. Biochem Cell Biol. 1996; 74:125-131.[Medline]
Williams M, Thompson LA, Li TC, Mackenna A, Barratt CL, Cooke ID.
Uterine flushing: a method to recover spermatozoa and leukocytes.
Hum Reprod. 1993; 8:925-928.
Yeung CH, Cooper TG, De Geyter M, De Geyter C, Rolf C, Kamischke A,
Nieschlag E. Studies on the origin of redox enzymes in seminal plasma and
their relationship with results of in-vitro fertilization. Mol Hum
Reprod. 1998;4:835-839.
Yeung CH, De Geyter C, De Geyter M, Nieschlag E. Production of reactive oxygen species by and hydrogen peroxide scavenging activity of spermatozoa in an IVF program. J Assist Reprod Genet. 1996; 13:495-500.[Medline]
Ying Y, Cheung MP, Chow PH, O WS. Effects of male accessory sex glands on sperm decondensation and oocyte activation during in vivo fertilization in golden hamsters. Int J Androl. 1999; 22:68-76.[Medline]
Ying Y, Chow PH, O WS. Effects of male accessory sex glands on
deoxyribonucleic acid synthesis in the first cell cycle of golden hamster
embryos. Biol Reprod. 1998; 58:659-663.
Zini A, Fisher MA, Mak V, Phang D, Jarvi K. Catalase-like and superoxide dismutase-like activities in human seminal plasma. Urol Res. 2002;30:321-323.[Medline]
Zini A, Schlegel PN. Catalase messenger RNA expression in the male
rat reproductive tract. J Androl. 1996; 17:473-480.
Zini A, Schlegel PN. Identification and characterization of antioxidant enzyme mRNA in the rat epididymis. Int J Androl. 1997;20:86-91.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. R. Fernandez-Santos, F. Martinez-Pastor, V. Garcia-Macias, M. C. Esteso, A. J. Soler, P. Paz, L. Anel, and J. J. Garde Sperm Characteristics and DNA Integrity of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa Frozen in the Presence of Enzymatic and Nonenzymatic Antioxidants J Androl, March 1, 2007; 28(2): 294 - 305. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). Addition of SOD in the flushing and incubation
media significantly decreased the incidence of NADPH-induced ssDNA damage in
uterine sperm ejaculated by males with all major accessory sex glands removed
(
; TX with SOD addition in vitro) compared with those without SOD
(•; TX without SOD addition in vitro) in a dose-dependent manner
(***P < .001).