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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We employed the xanthine-xanthine oxidase system to produce H2O2 or simply used commercially available H2O2 solution to investigate the effects of exogenous hydroxyl radicals on the motility characteristics and on lipid peroxidation and DNA modification of human sperm. The functional parameters of sperm motility declined concomitantly upon incubation of sperm with hydroxyl radicals. After incubation of freshly ejaculated human sperm with 0.23 mM H2O2 in the presence of 1.8 mM ADP and 2.7 mM FeSO4 for 1 hr at 37 degrees C, 90% reduction of motility was observed. Effect of hydroxyl radicals on sperm motility was dependent on the concentrations of FeSO4 and H2O2, respectively. The remaining motility of sperm after 1 hr incubation showed negative linear correlation with FeSO4 concentration. The response of sperm motility to FeSO4 was also dependent on the concentration of H2O2. Except for the amplitude of lateral head displacement, functional parameters of sperm declined with the increase of H2O2 concentration. Moreover, we found that lipid peroxidation measured as malondialdehyde (MDA) and accumulation of modified DNA indicated by 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in human sperm were significantly accelerated by exogenous hydroxyl radicals. The contents of lipid peroxides and 8-OH-dG in the spermatozoa were increased from 24.6 +/- 2.4 nmol MDA/1 x 10(7) sperm and 0.17 +/- 0.02% in the untreated group to 30.6 +/- 1.2 nmol MDA/1 x 10(7) sperm and 1.9 +/- 0.47%, respectively, in the sperm treated at 37 degrees C for 1 hr with 2.03 mM H2O2, 1.8 mM ADP and 4.5 mM FeSO4. Taken together, these results suggest that the detrimental effects of hydroxyl radicals on human sperm functions may be mediated, at least partly, through lipid peroxidation and DNA modification.
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PMID:Hydroxyl radical-induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm. 935 Mar 36

Reactive oxygen species (ROS) have a powerful cytotoxic effect on spermatozoa and have been implicated in spermatozoal dysfunction and male infertility. gamma-Glutamyl transpeptidase (GGT) is essential to the metabolism of the antioxidant glutathione and, as such, is believed to be important in protecting spermatozoa against oxidative stress. The aims of this study were 1) to establish in vitro conditions in which ROS were generated and 2) to determine whether oxidative stress regulated the expression of GGT mRNAs I-IV in the initial segment of the epididymis. Initial segments were collected from adult male rats and incubated in culture media to which ROS-generating compounds, hypoxanthine and xanthine oxidase, were added. By 6.5 hours, incubation of tissue in high-oxidative stress conditions caused a 56% decrease in reduced glutathione concentration, a concomitant 240% increase in oxidized glutathione concentration, and a 25% decrease in adenosine triphosphate concentration. RNase protection analyses demonstrated an approximate 70% up-regulation of GGT mRNAs II-IV in a differential manner, depending on the concentration of oxidizing agents and the type of ROS generated. gamma-Glutamyl transpeptidase mRNA I was not expressed. These results support the hypothesis that expression of GGT mRNAs is regulated by oxidative stress in the initial segment of the rat epididymis.
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PMID:Oxidative stress differentially regulates the expression of gamma-glutamyl transpeptidase mRNAs in the initial segment of the rat epididymis. 953 96

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.
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PMID:Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. 961 44

The lipid composition of the sperm membrane has been shown to exert a significant effect upon the functional quality of spermatozoa. We have studied the effect of induced peroxidation and of the presence of polymorphonuclear white blood cells (WBCs) on the fatty acid composition of the phospholipids of human spermatozoa. The spermatozoa were fractionated by a discontinuous Percoll gradient in two fractions (47% and 90% Percoll). Induced peroxidation of spermatozoa was assessed by determining the production of thiobarbituric acid reactive substances (TBARS), mostly malondialdehyde, after incubation with ferrous sulphate and sodium ascorbate as a promoter of peroxidation. TBARS production after induction of peroxidation was correlated with the abundance of polyunsaturated fatty acids (PUFA)(r = 0.68, p < 0.0001), with the double bond index (r = 0.72, p < 0.0001), and with the oxidative potential index (r = 0.73, p < 0.0001) of fatty acids of phospholipids. In comparison with samples containing > 1 x 10(6) WBCs/mL, those with < 1 x 10(6) WBCs/mL contained higher proportions of PUFA (90% Percoll, p < 0.05; 47% Percoll, p < 0.05), total omega 3 fatty acids (90% Percoll, p < 0.05; 47% Percoll, p < 0.001), docosahexaenoic acid (90% Percoll p < 0.05; 47% Percoll, p < 0.05), and double bond index (90% Percoll, p < 0.05; 47% Percoll, p < 0.001). In addition, mean melting point was significantly lower (90% Percoll, p < 0.05; 47% Percoll, p < 0.001) in samples with < 1 x 10(6) WBCs, indicating higher membrane fluidity. The increase of TBARS production by spermatozoa after incubation with the xanthine-xanthine oxidase system and/or ferrous sulphate as promoter of peroxidation was associated with a significant decrease of PUFA. Incubation of spermatozoa with WBCs, with or without activation by phorbol ester, decreased the PUFA (p < 0.05). Also, TBARS production was increased (p < 0.01) after activation of WBCs with phorbol ester. Our data provide evidence that oxidative stress induced by WBCs has a damaging effect on the polyunsaturated fatty acids of sperm phospholipids which may result, amongst other effects, in decreased membrane fluidity.
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PMID:White blood cells cause oxidative damage to the fatty acid composition of phospholipids of human spermatozoa. 966 99

A spectrophotometric assay for the measurement of malondialdehyde and 4 hydroxyalkenals (MA + 4HA) has been evaluated for the detection of sperm pathologies involving oxidative stress. In order to make sensitive measurements of MA + 4HA on human spermatozoa, the stimulation of a lipid peroxidation cascade with a ferrous ion promoter was found to be necessary. The optimal configuration for the promoter was defined (0.64 mM FeSO4 + 20 mM ascorbate for 2 h in Ca2+ and Mg2 free Hanks' balanced salt solution) and the assay used in a series of studies to elucidate the functional significance of MA + 4HA determinations. Such measurements were found to give highly significant correlations (p < 0.001) with the loss of motility induced by oxidative stress created either with a xanthine oxidase, free radical generating system or by prolonged incubation under aerobic conditions. Experiments involving the stimulation and suppression of lipid peroxide release from human sperm suspensions, in concert with a bioassay for cytotoxicity, confirmed the strength and causative nature of these associations. Measurements of lipid peroxidation potential in highly purified, leucocyte-free sperm suspensions revealed the presence of inverse correlations with the motility of the spermatozoa, their viability, their competence for sperm-oocyte fusion and, most significantly, the quality of sperm movement in the original semen samples. Similar negative correlations were observed between sperm function and phorbol ester-stimulated reactive oxygen species generation but, unlike the MA + 4HA determinations, these relationships were obfuscated by the presence of leucocytes. We conclude that the measurement of MA + 4HA in human spermatozoa provides important information on the underlying quality of spermatogenesis and should be of value in the clinical diagnosis of infertility involving oxidative stress and the selection of patients for antioxidant therapy.
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PMID:Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function. 967 17

Human serum albumin (HSA) is being considered as an alternate media for sperm enrichment in assisted reproductive technology (ART) because of recent concern with the use of Percoll. In this study, we compared HSA and Percoll for 1) sperm recovery, 2) reactive oxygen species scavenging potential, and 3) effects on total oxidative stress to spermatozoa. The spermatozoa-enriched fractions obtained from Percoll (80%:40%) and HSA (12%) were monitored for sperm motility, viability, hypoosmotic swelling test (HOST), and adenosine triphosphate (ATP) levels. The effect of superoxide anions (O2.-) on donor human spermatozoa was observed in the presence of either HSA or Percoll media. A combination of luminol and the Cypridina luciferin analog 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-alpha)pyraz in-3-one hydrochloride was used as a highly sensitive chemiluminescence probe in our hypoxanthine and xanthine oxidase-based assay for O2.-. Sperm membrane total oxidative stress was determined by measuring levels of the prostanoid 8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha). Significant differences in sperm parameters between the Percoll-enriched spermatozoa (motility 60%+/-4%, viability 56%+/-6%, and HOST 73%+/-7%) and those enriched with HSA (motility 84%+/-5%, viability 85%+/-4%, and HOST 84%+/-3%; P < 0.01) were observed. Adenosine triphosphate levels were significantly higher, by almost 50%, in samples processed with HSA than with Percoll (P=0.03). The dismutation rate of O2.- in HSA (slope -6.8) was significantly lower than in Percoll (slope -87.0; P < 0.01). Sperm motility and ATP levels decreased at a slower rate after treatment with O2.- in the presence of HSA when compared to Percoll; moreover, spermatozoa in HSA regained partial motility after 2 hours, whereas spermatozoa in Percoll were immobilized. No significant differences in 8-iso-PGF2alpha levels in spermatozoa enriched by either HSA or Percoll were observed. We conclude that the HSA sperm enrichment procedure improves the recovery of higher quality spermatozoa compared to Percoll and, because of its antioxidant properties, may be useful in processing high leukospermia semen samples for ART purposes.
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PMID:Antioxidant potential of human serum albumin: role in the recovery of high quality human spermatozoa for assisted reproductive technology. 973 43

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.
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PMID:Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. 979 19

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.
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PMID:Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. 1023 30

Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen.
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PMID:Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction. 1073 95

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.
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PMID:The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. 1110 16


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