Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa

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 degrees 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 + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (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.     

Sperm-immobilizing antibodies block capacitation in human spermatozoa

Sperm-immobilizing antibodies block human fertilization by interfering with the acrosome reaction (AR). To clarify the mechanism of blockage of AR by sperm-immobilizing antibodies, the authors examined their effects on the increase of intracellular free Ca2+ concentration induced by follicular fluids (Ca2+ influx) in spermatozoa and on their capacitation. Sperm-immobilizing antibodies did not suppress Ca2+ influx induced by follicular fluid, but they inhibited capacitation of human spermatozoa. Namely delta%AR (%AR after addition of an AR inducer--%AR before treatment) induced by progesterone was significantly (p < .0001) lower when spermatozoa were incubated in human tubal fluid medium cotaining antibody-positive serum (1.2%), compared to that when incubated in control medium (19.2%). Furthermore, the proportion of both spermatozoa that became capacitated and ones that had become capacitated decreased significantly (p < .0001) after 2, 4, and 6 h of incubation in medium containing antisperm antibody-positive serum, compared to those of spermatozoa incubated in control medium. In conclusion, sperm-immobilizing antibodies may be closely related to their blockage of capacitation.     

Electromechanical impairment of human auricle and rat myocardial strip subjected to exogenous oxidative stress.

OBJECTIVE: Animal myocardial dysfunction induced by remote ischemia-reperfusion (IR) was shown to be partly accomplished via a direct effect of the pro-oxidant xanthine oxidase (XO). This direct remote effect was not tested in humans. We now assessed the performance of human auricles in the presence of solutions containing XO and/or allopurinol and compared them to those of rat myocardial strips. METHODS: Human and rat specimens (n=64) were separately exposed for 2h to Krebs-Henseleit solution that either (1) exited from rat livers that were earlier perfused for 2h (control-human or control-rat), (2) exited from livers that were earlier made ischemic for 2h (IR-human, IR-rat), (3) contained xanthine (X) 3.8 microM + XO 3 mU ml(-1) (X+XO-human, X+XO-rat), or (4) exited from post 2h-ischemic livers and contained 100 microM allopurinol (human or rat IR + allopurinol groups). RESULTS: Unlike the unchanged electromechanical performance in the control and IR+allopurinol auricles and strips, the rates of contraction, maximal force of contraction and working index of either preparation were reduced by 75-98% (P<0.01) when exposed to the IR reperfusate or to the X+XO-enriched Krebs. The basal amplitudes of contraction in these four latter groups increased twofold (P<0.01). XO activity was similarly low in the control and in the IR+allopurinol groups, but four- to 45-fold (P<0.001) higher in the IR and the X+XO groups, both in the rat and human organs. The reduced glutathione was reduced by approximately 50% (P<0.01) in either preparation in the IR and the X+XO groups compared to the control and IR+allopurinol groups. CONCLUSIONS: Remotely and exogenously originated oxidative burst directly induces electromechanical dysfunction and disrupts oxidant/antioxidant balance in human auricles as it does in the rat myocardial strip.     

The role of edaravone on the impairment of endothelial barrier function induced by acute oxidative stress in cultured human umbilical vein endothelial cell monolayer

BACKGROUND: The aim was to determine the effects of a novel free radical scavenger, edaravone, 3-methyl-1-phenyl-2-pyrazoline-5-one (ED), against endothelial barrier dysfunction induced by acute oxidative stress in cultured human umbilical vein endothelial cells (HUVECs). METHODS: To estimate the integrity of the HUVEC monolayer, transendothelial electrical resistance (TEER) was measured for 3 hours. We investigated the permeability change of the monolayer by measuring the concentration of fluorescence in isothiocyanate-labeled dextran (FITC-Dx), and estimated the degree of oxidative stress in terms of hydrogen peroxide (H2O2) in the apical chambers. RESULTS: The TEER changes in both xanthine oxidase (XO)+xanthine (X) and X/XO+ED group were significantly lower than the control group (p < 0.001). The amount of 4 FITC-Dx in the XO+X group was significantly higher than the control group at 3 hours (p < 0.001). In the XO+X group, the concentration of H2O2 was significantly higher than control and the X/XO+ED group (p < 0.001 each). Thus, edaravone improved the disturbed endothelial barrier function induced by acute oxidative stress. CONCLUSIONS: The permeability increase induced by acute oxidative stress was prevented by free radical scavenger edaravone significantly in vitro. This radical scavenger may have clinical applications to protect against endothelial barrier dysfunction.     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

Summary.  Bovine spermatozoa from frozenthawed semen are sensitive to lipid peroxidation. Vitamin E protects sperm membrane against oxidative damage. Sperm capacitation produces structural changes on the plasma membrane. Reactive oxygen species could be involved in the capacitation process. The aim of this work was to study the influence of natural antioxidants on the plasma membrane and the influence of reactive oxygen species during bovine sperm capacitation. Sperm samples were frozen in a standard diluent, with and without vitamin E (1 mg ml^-1). Heparin (60 μg ml^-1) was used as a sperm capacitation inductor. Sperm capacitation was evaluated by chlorotetracycline assay. Lipid peroxidation was determined by the 2-thiobarbituric acid assay. A diminution of thiobarbituric acid reactive substances was observed in sperm samples frozen with vitamin E ( P < 0.05). The addition of vitamin E to the freezing diluent had no effect on the capacitated pattern ( P > 0.05).
When vitamin E and vitamin E + vitamin C were added to the capacitation medium, a significant decrease in the percentage of capacitated spermatozoa ( P < 0.05) was observed in both cases. The addition of superoxide dismutase (0.1 mg ml^-1) or H₂O₂ (50 μM) in the incubation medium, decreased the percentage of capacitated spermatozoa ( P < 0.05). Vitamin E protects the plasma membrane against lipid peroxidation during sperm capacitation, and the presence of superoxide anion would be necessary for frozen-thawed bull sperm capacitation.     

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa

Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable-capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live-capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat-cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten's medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short-term incubation.     

An in vitro promoting role for hydrogen peroxide in human sperm capacitation

A complex process of maturation called capacitation is an essential step for spermatozoa to fertilize oocytes. Recent studies have shown that reactive oxygen species (ROS) can enhance the capacitation of human spermatozoa and sperm-zona interaction. We have investigated whether hydrogen peroxide (H₂O₂) could trigger capacitation of human spermatozoa and the acrosome reaction. The addition of catalase, a specific H₂0₂ scavenger, at the beginning of the capacitation process decreased the levels of both hyperactivation and induced-acrosome reaction whereas catalase added 15 min before the induction of the acrosome reaction by the calcium ionophore had no effect. Supplementation of the medium with H₂O₂ resulted in increased levels of hyperactivation and the acrosome reaction, whereas H₂O₂ added 15 min before induction of the acrosome reaction did not have any stimulatory effect. These results suggest that H₂O₂ may be involved in the capacitation process of human spermatozoa but not in the acrosome reaction.     

Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes.

Mammalian spermatozoa are sensitive to oxygen-induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll-separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS-immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that .O2- and .OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.     

Evaluation of the protective effect of quercetin against cisplatin‐induced renal and testis tissue damage and sperm parameters in rats

The protective effect of quercetin on cisplatin‐induced renal and testicular tissue damage was investigated using biochemical, histopathological and histological approaches. A total of 40 male rats were divided into 5 groups as follows: control; cisplatin alone; quercetin alone; cisplatin + quercetin; and quercetin + cisplatin. Cisplatin was administered to rats at a single dose of 7 mg kg^?1 intraperitoneal. Quercetin was administered by gavage daily for 10 days at dosage 50 mg kg^?1. At the end of the study serum, total antioxidant capacity (TAC) levels and total oxidant status (TOS) were determined. Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and xanthine oxidase (XO) were studied separately in serum, renal tissue and testicular tissue. Renal and testicular morphological alterations were assessed, histopathologically. Epididymal sperm concentration, motility and morphology were investigated. Testicular and renal TAC and TOS values did not alter significantly. Renal CAT levels were increased by cisplatin and cisplatin plus quercetin groups that is reversed by administration of quercetin before cisplatin. MDA, CAT, SOD ve XO levels of testicular tissue did not differ significantly. Cisplatin and cisplatin plus quercetin groups had decreased sperm motility ratio and increased abnormal spermatozoa. Quercetin partially reverses some of the cisplatin‐related pathological effects on kidney and testis.     

Lactate dehydrogenase-C4 is involved in heparin- and NADH-dependent bovine sperm capacitation

Lactate dehydrogenase C4 isoenzyme (LDH-C4) is involved in the energy metabolism of spermatozoa. Sperm capacitation is considered part of an oxidative process; an NADH oxidase of plasma membrane could be responsible for superoxide anion generation which is required for capacitation. The role of LDH-C4 and the requirements of NADH in cryopreserved bovine sperm capacitation were studied. LDH-C4 activity was 5.52 +/- 3.41, 15.72 +/- 6.04 and 15.22 +/- 1.92 Units 1010 spermatozoa-1 in plasma membrane, sperm suspension and cytosol fraction, respectively; these activities were inhibited by sodium oxamate. To study the influence of oxidative substrates in capacitation, three different TALP (T) media were used: TP (pyruvate); TL (lactate) and TC (citrate); heparin or NADH was then added. There were no significant differences in the percentage of capacitation induced by heparin or NADH in TALP medium; similar levels of capacitation were achieved with TL alone or TL +heparin and TP +NADH; capacitation was inhibited with sodium oxamate in all treatments used. Cytosolic NADH may be required as a substrate for sperm oxidase. Lactate influx through plasma membrane may be utilized by cytosolic LDH-C4, increasing reduced coenzymes required for capacitation. Plasma membrane LDH-C4 may participate in the production of lactate to obtain intracellular reducing equivalents to be used by sperm oxidase for in vitro sperm capacitation.     

Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa

We demonstrated previously that pentoxifylline in millimolar concentrations can inhibit superoxide anion production by human spermatozoa. In the present study we have examined the effects of the same concentrations of pentoxifjrlline on experimentally induced lipid peroxidation, as measured by malondialdehyde formation in the thiobarbituric (TBA) assay. Under the experimental conditions used, preincubation of spermatozoa with pentoxifjdline led to a significant dose-dependent stimulation (p<0.005) of malondialdehyde production amounting to 10.77 ± 2.35%, 13.45 ±2.99% and 17.4 ± 1.99% (mean ± SEM) for 1.9, 3.7 and 11.2 mmol/l pentoxifylline, respectively. In the presence of 11.2 mmol/l pentoxifylline, an increase in iron-catalysed lipid peroxidation potential was detected in samples of spermatozoa from 29 infertile men, regardless of their initial levels of malondialdehyde. The results of this study indicate that pentoxifylline might further augment the ferrous ion-stimulated decomposition of pre-accumulation lipid hydroperoxides in the sperm plasma membrane and thus promote malondialdehyde generation in the TBA assay.
It is concluded that the stimulatory effect of pentoxifjdline on iron-induced lipid peroxidation may have an adverse effect on the quality of sperm suspensions prepared for in vitro fertilization, a possibility which should be investigated further.     

Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process.

Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentrations in seminal vesicle secretions. It is degraded by the prostate-specific antigen (PSA) to generate peptides of various biological activities that were found on and inside spermatozoa. Our aim was to determine the effect of Sg on capacitation, which is the series of transformations that spermatozoa must undergo to become fertile. At concentrations of 0.1 to 1.0 mg/mL (600- to 20-fold lower than those of semen), Sg did not affect sperm motility (%) but completely prevented capacitation induced by fetal cord serum ultrafiltrate; a partial inhibition of capacitation was noted with 0.03 mg Sg/mL. There was also a dose-dependent decrease in the tyrosine phosphorylation of fibrous sheath proteins and in the O2-.-related chemiluminescence. Ribonuclease (RNase), which has as high an isoelectric point (pI = 9.7) as Sg (pI = 9.5), also prevented sperm capacitation and O2-.-related chemiluminescence but to a lower extent, suggesting that one mechanism of Sg action on spermatozoa could be related to its positive charge at physiological pH. Sg at 1, but not 0.3 or 0.1 mg/mL, scavenged the O2-. generated by the mix of xanthine + xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects. Therefore, Sg is a potential scavenger for O2-. but probably also affects the sperm oxidase. Spermatozoa rapidly processed Sg; a high proportion of Sg was degraded after 15 minutes of incubation. The resulting polypeptide patterns were reminiscent of those obtained with PSA as a proteolytic enzyme. These data suggest that Sg, its degradation products, or both may be natural regulators of sperm capacitation and could prevent this process from occurring prematurely. One mechanism by which Sg acts could involve an interference with the O2-. that is normally generated during this process.     

Influence of postischemic administration of oxyradical antagonists on ischemic injury to rabbit skeletal muscle

The aim of this study was to determine whether the administration of free radical antagonists, immediately before and during the early minutes of reperfusion, improves muscle survival 24 hr after a period of ischemia. Rabbit rectus femoris muscles were isolated, made ischemic for 3½ hr and treated with either desferrioxamine (DFX), an Fe³⁺ chelator, superoxide dismutase and catalase (SOD & CAT), which quench superoxide and hydrogen peroxide, or allopurinol, an inhibitor of xanthine oxidase (XO). After 24 hr reperfusion, muscle viability (±s.e.m.), measured by the nitro blue tetrazolium (NBT) vital staining technique, was 41.6 ± 11.3% for saline-treated ischemic controls, 30.6 ± 7.6% for DFX-treated, 46.7 ± 10.3% for SOD & CAT-treated, and 43.3 ± 9.5% for allopurinol-treated muscles. None of the treated groups differed significantly from the ischemic control group. Tissue myeloperoxidase, ATP and reduced glutathione levels, and plasma lactate dehydrogenase (LDH) and aspartate transaminase (AST) levels were increased by ischemia and reperfusion in all groups, but the changes did not differ between the treatment groups. Levels of XO in the rabbit muscle were determined and found to be very low in both normal and postischemic muscle. As XO is the target enzyme of allopurinol, its absence provides a basis for the lack of effect of this agent. However, it is not clear why DFX and SOD & CAT had no protective effect © 1997 Wiley-Liss, Inc MICROSURGERY 17:517–523 1996     

Participation of superoxide anion in the capacitation of cryopreserved bovine sperm

Mammalian spermatozoa must undergo a preparation period known as capacitation to become capable of fertilizing oocytes. Controlled amounts of reactive oxygen species (ROS), such as superoxide anion (O2.-) and hydrogen peroxide (H2O2) have been shown essential for capacitation and acrosome reaction. The presence of an oxidase in the sperm plasma membrane has been suggested. The objective of the present study was to provide evidence for the production of O2.- by capacitating cryopreserved bovine spermatozoa. Percentages of capacitation and acrosome reaction were determined by the chlortetracycline assay. The effect of several nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors on capacitation was also studied. H2O2 production was determined by the fluorometric assay using the p-hydroxyphenylacetic acid-horseradish peroxidase system. Superoxide dismutase (SOD) activity was determined spectrophotometrically at 480 nm. Heparin-dependent capacitation was inhibited by all NADPH oxidase inhibitors tested (p < 0.05). Significant levels of H2O2 were produced during capacitation with heparin; such production was inhibited by diphenyleneiodonium, one of the NADPH oxidase inhibitors. The addition of catalase to the incubation medium failed to modify the capacitation rate; inhibition was only observed when SOD was present (p < 0.05). Endogenous SOD activity was diminished during heparin-dependent capacitation (p < 0.05). Similar levels of acrosome reaction induced by lysophosphatidylcholine were obtained in both heparin and O2.--dependent capacitation. Overall results suggest the participation of a sperm oxidase in bovine sperm capacitation. H2O2, generated by O2.- dismutation, failed to participate in capacitation, although this ROS may have been able to decrease endogenous SOD activity. Exogenous O2.- promotes physiological capacitation in cryopreserved bovine sperm, thus allowing the acquisition of fertilizing capacity.     

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

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.     

Capacitation-associated changes in protein tyrosine phosphorylation, hyperactivation and acrosome reaction in hamster spermatozoa

The aim of this study was to evaluate the effects of Ca2+, BSA, NaHCO3 and PVA on the capacitation-associated time-dependent increase in protein tyrosine phosphorylation, hyperactivation, and acrosome reaction in hamster spermatozoa. Hamster spermatozoa when incubated in TALP, a medium that assists capacitation, showed a time-dependent increase in protein tyrosine phosphorylation that correlated with the capacitated state of the spermatozoa. An absence of Ca2+ or NaHCO3 in the capacitation medium delayed the phosphorylation of the proteins, but without both there was a significant decrease in the phosphorylation of the proteins throughout the period of capacitation. An absence of bovine serum albumin also caused a decrease in the phosphorylation of the proteins but this did not occur if polyvinyl alcohol was substituted for it in the medium. The percentage hyperactivation was not affected in the absence of bovine serum albumin if the medium contained polyvinyl alcohol. However, it was delayed in the absence of NaHCO3 and inhibited in the absence of Ca2+. The absence of NaHCO3 or bovine serum albumin had no effect on the acrosome reaction. These results show that hamster spermatozoa undergo capacitation-associated protein tyrosine phosphorylation similar to that of the spermatozoa of other mammals. However, hamster spermatozoa are unique in that the capacitation-associated protein tyrosine phosphorylation is not absolutely dependent on the presence of Ca2+ and NaHCO3. As far as we know, this study is the first to provide evidence that capacitation-associated protein tyrosine phosphorylation is linked to hyperactivation in hamster spermatozoa.     

Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.     

大鼠门静脉高压症及梗阻性黄疸时细菌移位与黄嘌呤脱氢酶和黄嘌呤氧化酶的关系

目的探讨大鼠门静脉高压症（porta; ju[ertemsopm,PH）及梗阻性黄疸（obstructive jaundioe,OJ）时,细菌移位（bacterial translocation,BT）与黄嘌呤氧化酶（xanthine oxidase,XO）、黄嘌呤脱氢酶（xanthine dehydrogenase,XD）之间的关系。方法将雄性SD大鼠60只随机分为对照组（A组）,胆总管结扎组（B组）和门静脉缩窄组（C组）,每组20只。术后第3周取肠系膜淋巴结、脾、肝组织及门静脉、腔静脉血细菌培养,测定门静脉压力（free portal pressure,FPP）,及肠XO,XD活性水平。结果B组及C组细菌移位率明显高于对照组（P〈0．01）,对照组为12％,B组和C组分别为28％和54％;B组和C组空肠XO水平活性明显高于对照组（P〈0．01）,B组和C组门静脉压力也较对照组升高。细菌移位率与XO活性成正相关（r=0．603）。XD活性水平无显著差异。结论门静脉高压症及梗阻性黄疸时可发生细菌移位,可能与肠黏膜屏障被破坏通透性增强有关,肠壁XO水平活性增强引起肠黏膜屏障通透性增高有助于细菌移位发生。     

Speciesspezifische Motilitätsmuster hyperaktivierter Säugetierspermatozoen und die quantitative Auswertung der Hyperaktivierung von Bullenspermatozoen/Species Specific Motility Patterns of Hyperactivated Mammalian Spermatozoa and Quantitative Analysis of the Hyperactivation of Bull Spermatozoa

Movement tracks of spermatozoa of human, lion, tiger, cow, pig and sheep are recorded by dark field photography (fluid layer thickness 16.7 microns, exposure time 1 s). Comparison before and after capacitation by an incubation of 2 h in modified tissue culture medium TCM 199 with 10% fetal calf serum resulted in two quite different patterns of hyperactivated spermatozoa: 1) Tracks are broadened due to enlarged lateral head displacement or radius of rotating head movements respectively (tiger, lion) and show beside that a markedly increase in erratic motility (human). 2) In the studied species of artiodactyla, cow, pig and sheep, a qualitative new, panicle-like pattern arised as a result of superposition of spermatozoa head pendular movements around the axis of forward motility and the other one around the head axis. This new type of tracks allows a simple quantitative analysis of hyperactivation of bull spermatozoa, first described in this report. The method is applied to investigations on efficacy of capacitation media and provides evidence for high individual differences of semen donors in capacitation success.     

Effects of quercetin and fish n‐3 fatty acids on testicular injury induced by ethanol in rats

The aim of this study was to investigate the effects of quercetin and fish n‐3 fatty acids on the changes in testis induced by ethanol. Forty‐five rats divided into five groups, control, ethanol, ethanol+quercetin, ethanol+fish n‐3 fatty acids and ethanol+quercetin+fish n‐3 fatty acids. At the end of 8 weeks, all the rats were sacrificed. Degenerative changes in histopathological analyses, the decreased body weight gain and seminiferous tubule diameters in ethanol group have been observed. TUNEL assay also showed an increase in apoptotic cell number. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px), xanthine oxidase (XO) and testosterone levels were decreased as well as the levels of malondialdehyde (MDA) and nitric oxide (NO) were increased in ethanol group. Histopathological changes caused by ethanol have been improved by quercetin and fish n‐3 fatty acids. It was also found that protection was provided by increasing SOD, CAT and GSH‐Px activities in groups administered quercetin, fish n‐3 fatty acids and quercetin+fish n‐3 fatty acids, and by decreasing the levels of MDA and NO in groups administered both quercetin and fish n‐3 fatty acids together. These results suggest that quercetin and fish n‐3 fatty acids are beneficial agents to reduce testicular injury induced by ethanol except for testosterone levels.