The effect of sulfhydryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro

Immotile spermatozoa from the caput epididymidis become progressively motile when incubated in medium containing theophylline, seminal plasma, and albumin. We previously reported that under these incubation conditions the spermatozoa induced to acquire motility exhibited a marked flagellar angularity, with the sperm head or midpiece bent 90-180 degrees towards the tail. In addition, we demonstrated that sperm flagellar bending did not occur when the sulfhydryl oxidant diamide was added to the motility induction medium. In the present study, we examined further the effect of sulfhydryl oxidation on the morphology and sulfhydryl content of immature caput spermatozoa induced to acquire motility in vitro. We found that flagellar bending was prevented and sperm flagellar straightness was maintained in a dose-dependent manner by diamide. Moreover, flow cytometric analysis of caput sperm sulfhydryls using the sulfhydryl reagent monobromobimane (mBBr) revealed that 1) diamide oxidizes caput sperm sulfhydryls, and 2) less than 15% of the total reactive sperm sulfhydryls were oxidized at diamide concentrations capable of preventing sperm angulation. Sodium tetrathionate (NaTT), another sulfhydryl oxidant, and hamster cauda epididymal fluid (CEF) containing sulfhydryl oxidase enzyme activity also maintained flagellar straightness in induced caput spermatozoa and oxidized sperm sulfhydryls. The flagellar straightness in caput spermatozoa treated with sulfhydryl oxidants, however, was temporary; with extended incubation, diamide- or CEF-treated spermatozoa exhibited flagellar bending. Additional studies showed that the flagellar straightness observed in sulfhydryl-oxidized spermatozoa was sustained when nitrofurantoin, an inhibitor of glutathione reductase, was included in the induction medium. Flow cytometric analysis of nitrofurantoin-treated spermatozoa showed that nitrofurantoin maintained the sperm disulfides formed by diamide and prevented the reduction of sperm disulfides back to sulfhydryls. Taken together, these studies demonstrate the significance of sulfhydryl oxidation in maintaining the morphology of immature caput epididymal spermatozoa induced to acquire motility in vitro and suggest that sulfhydryl oxidation may be important in the development of motility during sperm epididymal maturation in vivo.     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

Coupling of a proton pump with superoxide radical-superoxide dismutase system in maturing mammalian spermatozoa and its association with sperm motility

Mammalian spermatozoa possess a membrane-located 'proton pump' which appears to regulate the acquisition and maintenance of their motility. The 'proton pump' seems to be coupled with the superoxide radical-superoxide dismutase system which, when uncoupled, results in total loss of sperm motility. Addition of sulfhydryl-compounds restarts the proton pump and reactivates sperm motility. Cobaltous ions block the membrane sulfhydryl groups and it is proposed that the sulfhydryl groups may be involved in regulating proton secretion and sperm motility.     

Induction and enhancement of progressive motility in hamster caput epididymal spermatozoa

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

The motility of ram and bull spermatozoa in dilute suspension

1. Ram and bull spermatozoa suspended in a glucose-sodium chloride solution rapidly lose motility at relatively high dilutions. The substitution of chloride-free diluents does not alter the phenomenon. 2. The rapid immobilization of ram and bull spermatozoa due to high dilution may be partially prevented by the addition of supernatants of either ram or bull semen, although motility is not maintained at the same level as in a more concentrated specimen. Various other substances which also partially protect spermatozoa are egg albumin, plasma albumin, plasma gamma globulin, starch, and glycogen. 3. Washing ram spermatozoa six times greatly reduces motility. This is not restored by the addition of ram seminal plasma which, however, reverses the concurrent head agglutination. 4. Washing ram and bull spermatozoa four times results in considerable loss of motility and head agglutination both of which may be reversed by the addition of seminal plasma. 5. Potassium chloride at 0.005 M concentration partially restores the motility of four times washed ram spermatozoa at 24 degrees C. or 37 degrees C. but not that of similarly treated bull spermatozoa.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Cysteine and glutathione in orange juice

The major part of the sulfhydryl compounds of both Valencia and Navel orange juice was found to exist as cysteine and glutathione. These two compounds were isolated and analyzed as their crystalline S-benzyl derivatives from concentrates prepared by preferential mercuric ion precipitation. Cysteine analysis before and after hydrolysis showed that all of the sulfhydryl of the concentrates was cysteine and glutathione. The glutathione analysis was confirmed by Woodward's glyoxalase method.     

Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Effects of transglutaminase substrates and inhibitors on the motility of demembranated reactivated spermatozoa

The effects of transglutaminase (TGase) substrates putrescine, dansylcadaverine, spermine, etc., and the TGase inhibitor cystamine were tested on the motility of demembranated mammalian spermatozoa. These products blocked within a few seconds the motility of demembranated reactivated spermatozoa at concentrations ranging from 0.25 to 5 mM. These minimal inhibitory concentrations could be decreased 5–150-fold when TGase substrates and inhibitor were incubated with demembranated spermatozoa for 15 min prior to the addition of Mg·ATP. The inhibition was reversed by higher concentrations of Mg·ATP but none of these TGase substrates or inhibitor could inhibit bull sperm dynein ATPase. TGase activities, as measured by the incorporation of ³H-putrescine into TCA-precipitable proteins, were present in both sperm Triton-soluble and -insoluble fractions. On the other hand, amine acceptor protein substrates for the TGase-catalyzed reaction were present only in the insoluble fraction. The Triton-soluble TGase was similar to the known “tissue” TGases; the Triton-insoluble TGase activity was calcium independent. The same TGase substrates and inhibitor that blocked the motility of reactivated spermatozoa also blocked TGase activities. Linear relationships were observed between the concentrations of these substances required to block sperm motility and those to block TGase activities. These data suggest the involvement of a TGase activity in sperm motility.     

Partially irreversible cold-induced lipid phase transitions in mammalian sperm plasma membrane domains: freeze-fracture study

In an effort to investigate the nature of the cellular injury caused when mammalian spermatozoa are cooled prior to cryopreservation, the occurrence of thermal phase transitions amongs the lipid components of the sperm plasma membrane was investigated by the use of freeze-fracture electron microscopy. The mechanisms by which glycerol and egg yolk exert protective effects during semen cooling and freezing were also examined. Ram and blackbuck spermatozoa, maintained at 30 degrees C prior to fixation at this temperature, exhibited randomly distributed intramembranous particles over the acrosomal, postacrosomal, and flagellar regions of the plasma membrane. In contrast, spermatozoa fixed at 5 degrees C after slow cooling to this temperature exhibited particle clustering over the postacrosomal region of the head as well as over the tail. These effects were not influenced by the presence of egg yolk or glycerol during the cooling procedure, although these substances protected the spermatozoa against loss of motility. Particle clustering over the sperm tail, induced by the slow cooling process, was found to be only partially reversible. The extensive areas of particle-free lipid, noted to result from the cooling procedure, were absent if the spermatozoa were rewarmed to 30 degrees C; however, the original distribution of particles was not restored and numerous small particle-free domains persisted. It is proposed that this type of irreversible change within the sperm plasma membrane may contribute to the loss of motility and fertility suffered by spermatozoa after cooling and freezing. Furthermore, it is suggested that protective substances such as egg yolk may exert their effects by countering these deleterious changes, rather than by preventing their occurrence.     

Antifertility factors of mammalian seminal fluid

The fertilizing ability of spermatozoa is inhibited by certain substances present in the seminal fluid. Most of these antifertility factors are proteinaceous in nature and differ in their physical characteristics. They inhibit fertilization by inhibiting either motility, capacitation, acrosome reaction or penetration of the ovum investments by the spermatozoa. This review describes and discusses the properties of these factors and their possible role, individually and collectively, in the regulation of fertility.     

Age-related differences of semen quality,seminal plasma,and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.     

Sperm preparation for ART

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll^® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep^®, Nycodenz, SilSelect^®, PureSperm^® or Isolate^® were developed in order to replace Percoll^®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.     

Motility of fish spermatozoa: from external signaling to flagella response

For successful fertilization, spermatozoa must access, bind, and penetrate an egg, processes for which activation of spermatozoa motility is a prerequisite. Fish spermatozoa are stored in seminal plasma where they are immotile during transit through the genital tract of most externally fertilizing teleosts and chondrosteans. Under natural conditions, motility is induced immediately following release of spermatozoa from the male genital tract into the aqueous environment. The nature of an external trigger for the initiation of motility is highly dependent on the aquatic environment (fresh or salt water) and the species’ reproductive behavior. Triggering signals include osmotic pressure, ionic and gaseous components of external media and, in some cases, egg-derived substances. Extensive study of environmental factors influencing fish spermatozoa motility has led to the proposal of several mechanisms of activation in freshwater and marine fish. However, the signal transduction pathways initiated by these mechanisms remain clear. This review presents the current knowledge with respect to (1) membrane reception of the activation signal and its transduction through the spermatozoa plasma membrane via the external membrane components, ion channels, and aquaporins; (2) cytoplasmic trafficking of the activation signal; (3) final steps of the signaling, including signal transduction to the axonemal machinery, and activation of axonemal dyneins and regulation of their activity; and (4) pathways supplying energy for flagellar motility.     

The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures

Cysteine availability is normally the rate-limiting factor in glutathione synthesis. How neurons obtain cysteine from extracellular space is not well established. Here we used mouse cortical neuron cultures to examine the role of the excitatory amino acid transporters (EAATs) in neuronal cysteine uptake. The cultured neurons expressed both EAAT2 and EAAT3. Cysteine uptake was predominantly (> 85%) Na+-dependent, with an apparent Km of 37 microm. Cysteine uptake was reduced by the EAAT substrates l-glutamate and l-aspartate and by synthetic EAAT inhibitors. The non-selective EAAT inhibitor threo-beta-hydroxyaspartate had a significantly greater maximal inhibitory effect than did the EAAT2-selective inhibitor, dihydrokainate, indicating uptake by both EAAT2 and EAAT3. Serine, a substrate of ASC uptake system, had negligible effects on cysteine uptake at 10-fold excess concentrations. To assess the functional importance of EAAT-mediated cysteine uptake in neuronal glutathione synthesis, cultures were treated with diethylmaleate to deplete glutathione, then incubated with cysteine in the presence or absence of EAAT inhibitors. Threo-beta-benzyloxyaspartate and the non-transportable inhibitor threo-beta-hydroxyaspartate both inhibited the cysteine-dependent glutathione synthesis. The findings suggest that neuronal EAAT activity can be a rate-limiting step for neuronal glutathione synthesis and that the primary function of EAATs expressed by neurons in vivo may be to transport cysteine.     

The mechanisms of reversible immobilization of fowl spermatozoa at body temperature

Intact fowl spermatozoa became almost immotile at 40 degrees C, but motility increased significantly at 30 degrees C. The oxygen consumption at both temperatures was 8-11 microliters O2/10(10) spermatozoa.min-1. The ATP concentration at 40 degrees C was higher than that at 30 degrees C but ADP concentration at 30 degrees C was higher than that at 40 degrees C. Consequently, the ATP/ADP ratio at 30 degrees C (1.9-2.2) increased to 3.5-3.7 at 40 degrees C. The motility of intact spermatozoa at 40 degrees C was effectively restored by 2 mM-Ca2+, 10% seminal plasma and 10% peritoneal fluid taken at the time of ovulation. In contrast, these effectors did not restore the motility of demembranated spermatozoa at 40 degrees C. Motility of demembranated spermatozoa was restored at 30 degrees C. These results suggest that the immobilization of fowl spermatozoa at 40 degrees C occurs due to a decrease in flagellar dynein ATPase activity. Furthermore, the action of effectors for motility such as Ca2+ may not be directly on the axoneme, but mediated by solubilized substances which have been removed by demembranation of the spermatozoa.     

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

肌肽棉酚对绵羊精子运动及超微结构的影响

肌肽促进绵羊精子运动增强,直线运动精子数目增多;棉酚则强烈抑制精子运动并使精子的运动方式发生改变;肌肽与棉酚混合处理精子,肌肽可不同程度抵消棉酚对精子产生的有害作用。但当棉酚完全抑制精子运动后,再加入肌肽则不能使精子恢复运动。电镜观察,肌肽对绵羊精子超微结构无任何不良影响;棉酚同造成精子严重损伤,导致生物膜系统损害、轴丝崩解、线粒体螺旋鞘紊乱等。本文通过肌肽,棉酚加入的时序不同,探讨两者对绵羊精子