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BOR - Papers in Press, published online ahead of print June 9, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028407
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BIOLOGY OF REPRODUCTION 71, 1309–1316 (2004)
DOI: 10.1095/biolreprod.104.028407
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Functional Significance of the Pentose Phosphate Pathway and Glutathione Reductase in the Antioxidant Defenses of Human Sperm1

A.C. Williams, and W.C.L. Ford2

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Bristol BS2 8EG, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, H2O2, 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 µg/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% O2. 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.

gamete biology, sperm, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The harmful effects of reactive oxygen species (ROS) on human sperm have been known for a long time. The toxicity of hydrogen peroxide toward sperm was first demonstrated in 1943 [1]. Subsequently, it has become clear that ROS can damage sperm in vitro [2–4] and that production of ROS by sperm suspensions is associated with poor sperm function and subfertility [5–8]. The extent of damage depends on the level of oxidative stress, which in turn depends on the balance between pro- and antioxidant processes, and the latter has been suggested as being a good predictor of fertility [9].

Seminal plasma is rich in antioxidants, including uric acid, {alpha}-tocopherol (vitamin E), and ascorbic acid (vitamin C) [10, 11], and it contains high activities of superoxide dismutase (SOD) and catalase. By contrast, the sparse cytoplasmic volume of the sperm cell limits its antioxidant capacity, and it generally is believed that human spermatozoa become more vulnerable to oxidative stress once separated from seminal plasma [12]. However, spermatozoa do retain some antioxidant capacity. They contain {alpha}-tocopherol [13], but their principal antioxidant defense rests on the enzymes SOD and glutathione peroxidase [14]. The continued activity of glutathione peroxidase depends on the regeneration of reduced glutathione by glutathione reductase, which in turn relies on NADPH, the principal source of which in spermatozoa is the pentose phosphate shunt. The activity of glucose 6-phosphate dehydrogenase, which is the first enzyme in the pentose phosphate pathway, may limit the rate of NADPH production and, hence, the ability of the glutathione peroxidase system to detoxify peroxides [15].

Spermatozoa are unusual in that the predominant form of glutathione peroxidase for both hydrogen peroxide and lipid peroxide reduction is phospholipid hydroperoxide glutathione peroxidase (sometimes termed glutathione peroxidase 4). This enzyme has a number of roles: An oxidatively cross-linked, enzymatically inactive form accounts for 50% of the structure of the mitochondrial capsule of rat epididymal sperm, and it regulates apoptosis during spermatogenesis, cross-linking of protamines during epididymal maturation, and metabolizing peroxides [16]. Dramatic decreases in glutathione peroxidase 4 expression have been observed in spermatozoa from some infertile men [17]. In human spermatozoa, inhibition of glutathione peroxidase with mercaptosuccinate led to a greater than 10-fold increase in the rate of spontaneous lipid peroxidation [18], confirming that glutathione peroxidase has an important antioxidant role.

Here, we investigated whether flux through the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system in human spermatozoa increases in response to oxidative stress and, if so, whether this increased flux is effective in protecting the sperm against lipid peroxidation and loss of function induced by ROS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals were purchased from Sigma Chemical Co. (Poole, Dorset, U.K.) or from BDH Laboratory Supplies (Poole, U.K.) unless otherwise stated.

Spermatozoa

Ejaculates were collected from donors whose semen exceeded World Health Organization [19] criteria for normal semen variables. All the donors provided signed consent for their semen to be used for research and were recruited in accordance with local ethical guidelines. Semen was collected into sterile plastic pots and allowed to liquefy at 37°C for up to 1 h before being layered above 40%/80% discontinuous Percoll density gradients [20]. Gradients were centrifuged at 350 x g for 25 min, and the resultant sperm pellet was washed twice (350 x g, 10 min) in Biggers Whitten and Whittingham buffer (BWW) containing 3 mg/ml of BSA [21]. For measurements of glucose metabolism, sperm were suspended at a concentration of between 20 and 40 million sperm/ml in BWW that contained 0.1 mM glucose. For other experiments, the sperm were suspended at a concentration of 5 million sperm/ml in standard BWW (5.5 mM glucose). In some experiments, leukocytes were removed from the sperm suspension as described previously [22], except that Dynabeads coated with mouse anti-CD45 (Dynal Ltd., U.K.; Wirral, U.K.) were used so that separation could be achieved in one step.

Measurement of Flux Through the Glycolytic and Pentose Phosphate Pathways

Preparation of [1-14C]D-glucose and [3-3H]D-glucose solution The [1-14C]D-glucose and [3-3H]D-glucose were purchased from either Amersham International (Bucks, U.K.) or from Sigma. To remove all traces of tritiated water from the [3-3H]D-glucose, it was reconstituted with 50 µl of 0.6 mM unlabeled glucose (prepared in ultrapure water) and subjected to three cycles of freeze-drying. The labeled glucose was reconstituted using 750 µl of unlabeled, 0.133 mM glucose prepared in BWW. To this solution, 250 µl of [1-14C]D-glucose was added. The resultant solution contained 0.13 mM glucose with specific activities of 500 µCi/µmol of 14C and 2.5 mCi/µmol of 3H. This stock solution was stored at –20°C until required.

Measurement of glucose metabolism Incubations were done in 3.5-ml, cylindrical plastic vials into which a nylon side well had been fixed to the wall. The side-well contained a rolled-up piece of Whatman no. 1 filter paper cut to the appropriate size [23]. On each occasion, incubations were set up in duplicate or triplicate, and the mean was used as the replicate value for statistical analysis. A 100-µl volume of sperm suspension was added to the main compartment, and the total volume was adjusted to 190 µl with BWW containing 0.1 mM glucose together with the experimental reagents described below. The tubes were gassed with 95% air/ 5% CO2 and sealed with gas-tight silicone rubber caps (Suba-Seal; Gallenkamp, Loughborough, U.K.). The tubes were incubated for 1 h at 37°C, and 10 µl of [1-14C]D-glucose and [3-3H]D-glucose solution were carefully added to the main compartment of the tubes. The tubes were then regassed, recapped, and returned to the incubator for a further hour. At the end of this period, the sperm suspension was acidified by the injection of 100 µl of 1 M perchloric acid through the silicone cap (ensuring the retention of any CO2 formed). Zero-time values were obtained by injecting additional flasks with perchloric acid immediately after adding the radiolabeled glucose. At the same time, 50 µl of 1 M KOH were injected into the filter paper wells. The tubes were then transferred into a shaking water bath at 37°C for 30 min. Subsequently, the filter papers were carefully transferred into 3.5 ml of liquid scintillation cocktail (Ultima Gold MV; Packard Instrument Company, Meriden, CT) in mini-scintillation vials (Pony Vials; Packard). The side wells were flushed three times with distilled water (100 µl per flush), and the flushings were added to the appropriate vials. Following a 30-sec vortexing, the scintillation vials were allowed to stand in the dark for 30 min before being counted using a liquid scintillation analyzer-Tri-Carb 1900CA equipped with SecuRia 2200 software (Canberra Packard, Pangbourne, Berks, U.K.).

After flushing, the side wells were carefully removed from the tubes, leaving the acidified sperm suspension in the main compartment. The tubes were then capped and vortexed, and 50-µl samples of the suspension were transferred to the side wells of unused incubation tubes that contained 500 µl of distilled water in the main compartment. The tubes were capped and incubated overnight at 37°C to allow complete equilibration of the tritiated water between the two compartments. The 500 µl of water in the main compartment, containing 500/550 of the tritiated water in the sample [23], were then transferred into 3.5 ml of scintillant and counted as described above.

Effects of oxidative stress on glucose metabolism Cumene hydroperoxide was dissolved in distilled water and stored as a 25.6 mM stock solution at 4°C in the dark. Mercaptosuccinate and diethyl maleate were dissolved in distilled water and prepared daily. Xanthine, xanthine oxidase (XO), FeSO4, ascorbic acid, SOD, and catalase were prepared fresh each day in BWW medium containing 0.1 mM glucose. 1,3-Bis-(2-chloroethyl) 1-nitrosourea (BiCNU; Bristol-Myers Pharmaceuticals Ltd., Hounslow, U.K.) was dissolved in ethanol in a glass vial at a concentration of 33.3 mg/ml and diluted with BWW to provide a working stock solution at 5 mg/ml. According to the manufacturer's information sheet, this solution should remain stable for 48 h when stored at 4°C. The reagents were added to the incubations to give the final concentrations shown in the results, and sperm were incubated with them for 1 h at 37°C before radiolabeled glucose was added and incubation continued for a further hour.

Assessment of Lipid Peroxidation using the Thiobarbituric Acid Assay

Lipid peroxidation was measured using a modified thiobarbituric acid (TBA) assay for malondialdehyde (MDA) following promotion with Fe2+/ ascorbate [24]. Spermatozoa were incubated at 37°C in BWW buffer with xanthine plus XO, H2O2, or cumene hydroperoxide for 1 h or with BiCNU for 2 h at the concentrations indicated in Results. At the end of the incubation, the spermatozoa were centrifuged (350 x g, 10 min) and then resuspended at their original concentration in Hanks Balanced Salts Solution lacking Ca2+ and Mg2+ (HBSS; Gibco BRL Life Technologies, Paisley, U.K.). After addition of 40 µM FeSO4 and 200 µM sodium L-ascorbate, the tubes were incubated for 2 h at 37°C to promote the conversion of lipid peroxides to MDA. At the end of the incubation, 1.4 ml of TBA reagent were added to 800 µl of sperm in plastic, 10-ml centrifuge tubes. The TBA reagent was prepared by the addition of 400 µl of 7% (w/v) sodium dodecyl sulfate, 600 µl of 10% (w/v) phosphotungstic acid, 2 ml of 2% (w/v) 2-TBA acid, and 200 µl of 0.2 mM butylated hydroxytoluene to 4 ml of 0.1 M HCl. The tubes were loosely capped and incubated in a boiling water bath for 45 min. Following cooling, 2 ml of n-butanol was added to each tube to extract the TBA-MDA adduct, and the tubes were vortexed before being left to stand for 10 min to ensure complete butanol separation. Approximately 1.8 ml of the 2-ml butanol fraction were transferred into 4.5 ml of ultraviolet-grade, four-clear-sided plastic cuvettes (Kartell Plastics U.K. Ltd., Cottenham, Cambridge, U.K.), and the MDA concentration was determined fluorimetrically using an LS 50B spectrofluorimeter (Perkin-Elmer, Beaconsfield, Bucks, U.K.) with an excitation wavelength of 510 nm and an emission wavelength of 553 nm.

Effects of BiCNU on Sperm Motility

Spermatozoa were incubated with 0–500 µg/ml of BiCNU under either 95% oxygen/5% CO2 or 95% nitrogen/5% CO2 at 37°C for up to 4 h. A separate tube was prepared for each measurement (six tubes per experimental condition) to avoid the need to open tubes and admit air until the required time had elapsed. At hourly intervals, tubes were removed, and sperm motility was assessed by computer-assisted semen analysis (CASA) using a Hamilton Thorn sperm motility analyzer, version 7, and "Microcells" (depth, 20 µm; Conception Technologies, San Diego, CA) at 37°C [25]. We endeavored to analyze at least 200 motile sperm per sample. This was impossible in some samples exposed to high BiCNU concentrations, however, because too few motile sperm were present.

Statistics

Data were first tested for normality of distribution with the Kolmogorov-Smirnov test and for homogeneity of variance with the Levene test. Normally distributed data with equal variance between groups were analyzed by one- or two-way ANOVA as appropriate. The Scheffe test was used for post-hoc testing where it was appropriate to analyze differences between all groups, and the Dunnett test was used when comparisons were made with a control. Nonnormally distributed data were tested with the nonparametric Kruskal-Wallis test. All calculations were done with the Microsoft Windows-based Statistical Package for Social Science (SPSS; version 8).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cumene hydroperoxide is an excellent substrate for glutathione peroxidase and a weak oxidizing agent. We predicted that low to moderate concentrations would increase flux through glutathione peroxidase, with a consequent increase in flux through the pentose phosphate pathway. Toxic effects, anticipated at high concentrations, would be indicated by a decrease in the rate of glycolysis. The addition of 5 or 10 µM cumene hydroperoxide increased the basal flux through the pentose phosphate pathway by greater than 50% but had no significant effect on the rate of glycolysis. As the cumene hydroperoxide concentration increased further, flux through the pentose phosphate pathway decreased, becoming significantly less at 160 µM compared with its peak at 10 µM and significantly less at 640 µM compared with basal values (Fig. 1). Glycolytic flux was more resistant to cumene hydroperoxide, and the first significant decrease was seen at 320 µM cumene hydroperoxide (Fig. 1). Under these conditions, pentose phosphate pathway flux in the absence of cumene hydroperoxide represented a little less than 1% of the total amount of glucose metabolized through glycolysis. These results were not affected by any low-level leukocyte contamination in the sperm suspensions, because for experiments in which sperm suspensions were divided in two and one part was denuded of leukocytes by treatment with anti-CD45-coated Dynabeads, the results from the intact and the leukocyte-depleted suspensions were identical (data not shown).



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  		FIG. 1. Effect of increasing concentrations of cumene hydroperoxide on the rate of metabolism through (a) the pentose phosphate and (b) the glycolytic pathways in human sperm. Sperm were incubated for 1 h at 37°C in the presence of cumene hydroperoxide and 0.1 mM [1-14C]Dglucose and [3-3H]D-glucose. The rates of pentose phosphate and glycolytic activity were assessed by the release of 14CO2 and 3H2O, respectively. Data are presented as the mean ± SD (n = 6). Different superscripts indicate significant differences (P ≤ 0.05) between cumene hydroperoxide concentrations (one-way ANOVA plus Scheffe post-hoc test)

Addition of 100 µM H2O2 increased flux through the pentose phosphate pathway by greater than 100% but had no effect on glycolytic flux. Addition of 50 µM H2O2 increased pentose phosphate pathway activity to a smaller and statistically insignificant extent, whereas 500 µM significantly inhibited glycolysis and reduced pentose phosphate pathway levels, although not significantly compared to control values (Fig. 2).



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  		FIG. 2. Effect of increasing concentrations of H2O2 on the pentose phosphate pathway and glycolytic flux in human sperm. Sperm were incubated in the presence of H2O2 for 1 h at 37°C, and metabolic rates were measured as described for Figure 1. Data are presented as the mean + SD (n = 3). Different superscripts indicate significant differences (P ≤ 0.05) between H2O2 concentrations (one-way ANOVA plus Scheffe post-hoc test)

In a similar way, pentose phosphate pathway activity was increased by the addition of 1 mM xanthine and increasing amounts of XO (Fig. 3a). This resulted from the effects of H2O2, because the effect was blocked by catalase (Fig. 3b) but not by SOD (Fig. 3c). Xanthine increased pentose phosphate pathway activity above the basal rate even in the absence of added XO, implying that sperm have endogenous XO activity (Fig. 3a). This increase was also attributed to H2O2, because it was also blocked by catalase (Fig. 3b). The 400 U/ml of catalase also significantly reduced the basal rate of pentose phosphate pathway flux from 0.27 ± 0.02 nmol per 108 sperm per hour (mean ± SD, n = 5) in these replicates to 0.16 ± 0.02 nmol per 108 sperm per hour (P < 0.001, paired t-test).



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  		FIG. 3. Stimulation of (a) the pentose phosphate pathway flux in human sperm by the superoxide-generating system, xanthine plus xanthine oxidase, and the effects of (b) catalase and (c) SOD. Sperm were incubated for 1 h at 37°C in the presence of 1 mM xanthine and increasing concentrations of xanthine oxidase together with catalase or SOD as appropriate. Flux through the pentose phosphate pathway was measured as described for Figure 1. Data are presented as the mean ± SD (n = 6). Different superscripts indicate significant differences (P < 0.05) between xanthine oxidase concentrations (one-way ANOVA plus Scheffe's post-hoc test)

To determine whether the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system would respond to lipid peroxidation, sperm were incubated with 200 µM ascorbate with or without 40 µM FeSO4. Ascorbate alone increased pentose phosphate pathway flux from 0.32 ± 0.016 to 0.47 ± 0.11 nmol per 108 sperm per hour (n = 5, P < 0.01, one-way ANOVA plus Scheffe test). The addition of FeSO4 caused only a small and statistically insignificant increase above that produced by ascorbate alone, to 0.50 ± .0.04 nmol per 108 sperm per hour. The effect of ascorbate did not depend on the presence of endogenous iron, because it was unaffected by the addition of either 0.4 mM EDTA or 80 µM desferroxamine (data not shown).

Mercaptosuccinate is an inhibitor of glutathione peroxidase that has been used previously to inhibit this enzyme in sperm. To our surprise, 40–320 µM mercaptosuccinate had no effect on either the basal rate of pentose phosphate pathway flux or the flux in the presence of 10 µM cumene hydroperoxide (Fig. 4a). Diethyl maleate, which we anticipated would complex glutathione, had no effect on the basal rate of pentose phosphate pathway flux but blocked (partially at 40 µM and completely at 80 µM) the increase produced by 10 µM cumene hydroperoxide (Fig. 4b).



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  		FIG. 4. Effect of (a) mercaptosuccinate and (b) diethyl maleate on pentose phosphate pathway flux of human sperm in the presence or absence of 10 µM cumene hydroperoxide. Sperm were incubated for 1 h at 37°C with varying concentrations of mercaptosuccinate or diethyl maleate before the addition of 0.1 mM [1-14C]D-glucose and 10 µM cumene hydroperoxide, after which incubation continued for a further hour. Flux through the pentose phosphate pathway was measured as described for Figure 1. Data are presented as the mean ± SD (n = 7 [a] and 9 [b]). Significant effect of cumene hydroperoxide (paired t-test) is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001. Different superscripts represent significant effects (P < 0.05) of diethyl maleate within cumene hydroperoxide-treated group (one-way ANOVA plus Scheffe post-hoc test)

Treatment of sperm with 10 µg/ml of the glutathione reductase-inhibitor BiCNU consistently increased flux through the pentose phosphate pathway from approximately 0.2 to 0.3 nmol per 108 sperm per hour, whereas 50–1000 µg/ml of BiCNU decreased pentose phosphate pathway activity to approximately 0.1 nmol per 108 sperm per hour (Fig. 5). As before, 10 µM cumene hydroperoxide increased pentose phosphate pathway flux by greater than 50%. In the presence of 10 µM cumene hydroperoxide, 10 µg/ml of BiCNU did not induce a further increase in pentose phosphate pathway activity, but 50–1000 µg/ml of BiCNU inhibited it significantly to the same value as observed in the absence of cumene hydroperoxide (Fig. 5).



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  		FIG. 5. Inhibition of basal and cumene hydroperoxide-stimulated pentose phosphate pathway flux in human sperm by BiCNU. Sperm were incubated for 1 h at 37°C with varying concentrations of BiCNU, followed by the addition of 0.1 mM [1-14C]D-glucose and 10 µM cumene hydroperoxide as appropriate. The pentose phosphate pathway flux was measured during the subsequent hour by the release of 14CO2 as described for Figure 1. Data are presented as the mean ± SD (n = 5). Significant effect of cumene hydroperoxide (paired t-test) is indicated as follows **P < 0.01. Different superscripts indicate significant differences (P ≤ 0.05) between varying BiCNU concentrations within cumene hydroperoxide treatments (one-way ANOVA plus Scheffe post-hoc test)

In experiments with BiCNU, the control rate of flux through the glycolytic pathway (nmoles [3-3H]D-glucose converted to 3H2O per 108 sperm per hour; n = 5) was 25 ± 3.4. This was unaffected by BiCNU at concentrations of 500 µg/ml or less, but it decreased to 12 ± 1.9 with 1 mg/ ml of BiCNU (P < 0.05, one-way ANOVA plus Dunnett test). Sperm viability measured by the hypo-osmotic swelling test (HOST) test behaved in a similar way, with 500 µg/ml or less of BiCNU having no effect but 1 mg/ml decreasing the proportion of viable sperm by 10%.

Lipid peroxidation increased when spermatozoa were subjected to oxidative stress by incubation with 1 mM xanthine plus 10 mU of XO or with 100 µM H2O2, but not with 10 µM cumene hydroperoxide (Fig. 6). Lipid peroxidation was also increased following glutathione reductase inhibition by BiCNU (Fig. 7).



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  		FIG. 6. Influence of oxidative stress on lipid peroxidation in human sperm. Sperm were incubated for 1 h at 37°C in BWW medium with either 1 mM xanthine plus 10 mU xanthine oxidase, 100 µM H2O2, 10 µM cumene hydroperoxide, or an equivalent volume of BWW medium (control). Sperm were then resuspended in HBSS and incubated for 2 h at 37°C with 40 µM FeSO4 and 200 µM ascorbate, after which malondialdehyde release was measured using a modified thiobarbituric acid assay. Data are presented as ratios to control (mean + SD, n = 4). Significant differences (P ≤ 0.05) from control are indicated by different superscripts (one-way ANOVA plus Dunnett post-hoc test)



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  		FIG. 7. Increased lipid peroxidation in human sperm incubated with increasing concentrations of BiCNU. Sperm were incubated in BWW medium with various concentrations of BiCNU for 2 h at 37°C. Sperm were then resuspended in HBSS, and lipid peroxidation was measured as described for Figure 7. Data are presented as the mean ± SD (n = 4)

The importance of glutathione reductase in protecting sperm function was studied by measuring the effect of BiCNU on sperm motility under 95% oxygen and under 95% nitrogen. In the presence of 0–10 µg/ml of BiCNU, both the percentage of sperm that were motile and their progressive velocity (VSL) declined only slightly during the 4-h incubation whatever the gas phase. In the presence of 50 µg/ml of BiCNU, both the percentage of motile sperm and the VSL declined between 3 and 4 h, and as the BiCNU concentration increased to 100 or 500 µg/ml, the decline in motility began earlier and became more severe. With the higher BiCNU concentrations (100 and 500 µg/ ml), the decline was noticeably greater in the incubations under 95% oxygen than in those under 95% nitrogen (Fig. 8, a–d). The effect of oxygen could be seen more clearly by examining the ratio between motility under oxygen and motility under nitrogen. Both the percentage of motile sperm and the VSL clearly declined significantly more rapidly and severely under 95% oxygen in the presence of 100 and 500 µg/ml of BiCNU, and for the percentage of motile sperm, this effect could also be seen with 50 µg/ml (Fig. 8, e and f). Broadly similar changes were seen for the percentage of rapidly motile (average path velocity [VAP], >25 µm/sec) sperm, curvilinear velocity, and VAP (data not shown).



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  		FIG. 8. The effect of 0–500 µg/ml of BiCNU on (a and d) the percentage of progressively motile, (b and e) their straight line velocity (VSL), and (c and f) their lateral head displacement (ALH) when incubated under (a–c) 95% N2/5% CO2 or (d–f) 95% O2/5% CO2 and the ratios of (g) the percentage motile sperm, (h) VSL, and (f) ALH under 95%O2 over the corresponding value under 95% N2. Sperm were suspended in BWW medium and incubated at 37°C in sealed tubes under the appropriate gas phase. Separate tubes were prepared for each time point. At the allotted time, tubes were opened, and a small volume of sperm was transferred to a "Microcell" (depth, 20 µm) for motility assessment by computer-assisted semen analysis. A significantly greater effect of BiCNU under O2 than under N2 is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001 (repeated-measures ANOVA)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These observations clearly demonstrate that flux through the pentose phosphate pathway in human sperm increases in response to moderate oxidative stress. Such levels of stress have no effect on glycolytic flux, supporting the view that the stimulation is specific and that antioxidant defenses in human sperm are capable of protecting sensitive sulfhydryl enzymes, notably glyceraldehyde 3-phosphate dehydrogenase, against oxidative stress [26]. Were the pentose phosphate pathway responding to a stimulatory effect of moderate oxidative stress, we would expect a parallel increase in glycolysis to meet the increased energy demand [23]. The capacity of the antioxidant pathway is limited, and defenses are overwhelmed by high levels of oxidative stress that decrease flux through both the pentose phosphate pathway and glycolysis and through reduced sperm motility. Evidence from other systems suggests that the addition of cumene hydroperoxide is an effective way to increase flux through glutathione peroxidase and to study the subsequent response of the pentose phosphate pathway [27, 28]. The glutathione peroxidase-glutathione reductase-pentose phosphate pathway axis is important in limiting the oxidative damage suffered by human sperm, because blocking glutathione reductase with BiCNU made the pentose phosphate pathway unable to respond to increased demand from glutathione peroxidase and made the cells more vulnerable to lipid peroxidation and loss of motility under aerobic conditions. Our data do not allow the capacity of the pathway to be estimated, because it was necessary to use only 0.1 mM glucose to maintain a high specific activity of [1-14C]glucose 6-phosphate so that 14CO2 release by glucose 6-phosphate dehydrogenase could be measured. Estimates of the maximum activity of glucose 6-phosphate dehydrogenase activity in human sperm range from 3.3 nmol per 108 sperm per minute in sperm permeabilized with Triton X-100 [29] to 11.7 nmol per 108 sperm per minute in sperm permeabilized with digitonin [30], and these values probably provide approximate limits for the maximum activity of the pathway as a whole.

A major anomaly in our results is the failure of mercaptosuccinate to block the increase in pentose phosphate pathway flux induced by cumene hydroperoxide. Mercaptosuccinate has been shown to inhibit glutathione peroxidase activity in human sperm with a concomitant increase in sensitivity to lipid peroxidation [14, 18, 31]. It is possible that an alternative route of glutathione or NADPH oxidation is linked to the presence of cumene hydroperoxide. It is unlikely that this would be glutathione peroxidase 5 derived from the epididymis, however, because this enzyme is present only as an inactive splice variant in humans [32]. Moreover, some nonselenium glutathione peroxidases are sensitive to mercaptosuccinate [33]. Another possibility is that the residual glutathione peroxidase activity is sufficient to account for the rather modest rate of pentose phosphate pathway activity that was achieved in the presence of a low glucose concentration. In the experiments with mercaptosuccinate, pentose phosphate pathway flux in the presence of 10 µM cumene hydroperoxide was approximately 0.31 nmol per 108 sperm per hour (Fig. 4a), whereas glutathione peroxidase activity in sperm treated with 200 µM mercaptosuccinate was 126 nmol per 108 sperm per hour [18], a 400-fold excess. In our hands, however, mercaptosuccinate had no effect on sperm viability or motility [34]. All our other observations are consistent with the metabolism of peroxides by glutathione peroxidase leading to the formation of oxidized glutathione that is regenerated by glutathione reductase leading to a demand for NADPH production through the pentose phosphate pathway.

An important concern in all experiments about ROS metabolism by human sperm is to what extent the results reflect the presence of contaminating leukocytes. Here, removal of leukocytes with anti-CD45-coated Dynabeads had no effect on pentose phosphate pathway activity, so we can be confident that the data represent the metabolic capacity of sperm cells. With this in mind, it is interesting that addition of catalase decreased the basal flux through the pentose phosphate pathway, implying that even in sperm suspensions with a very low level of leukocyte contamination, the sperm need to detoxify hydrogen peroxide. Recent reports suggest that human sperm do not contain measurable NADPH oxidase activity [30, 35], although multiple plasma membrane redox systems are present [36]. The hydrogen peroxide likely is derived from the mitochondria, from peroxidase enzymes, or from one or more of the plasma membrane redox systems. By contrast, although diethyl maleate, which is presumed to complex cell glutathione, inhibited the increase in pentose phosphate pathway flux produced by cumene hydroperoxide, it had no effect on the basal rate, suggesting that the latter rate is independent of glutathione and, by inference, glutathione peroxidase. The basal rate varied between sperm from different donors and between different ejaculates from the same donor. This probably reflects differences in oxidative stress and activity of other redox systems [36], but it will also be influenced by other demands for NADPH, the existence of which is suggested by residual pentose phosphate pathway activity in the presence of high concentrations of BiCNU (Fig. 5).

We were anxious to establish if the pathway would respond to lipid peroxidation, and we tested the effect of exposing the sperm to ascorbate plus ferrous iron. Ascorbate increased pentose phosphate pathway flux. However, no significant additional effect of iron was found even though iron greatly increased malondialdehyde release when added to the HBSS in the subsequent lipid peroxidation assay [34]. These observations are consistent with ascorbate acting as a pro-oxidant and promoting lipid peroxidation de novo, whereas the ferrous iron promoter initiates free radical formation from stabilized lipid peroxides in the cell membrane, leading to malondialdehyde production proportionate to preexisting but stabilized membrane peroxidation [24]. On this premise, the pentose phosphate pathway in human sperm is able to respond to lipid peroxidation as well as to H2O2 and synthetic organic peroxides. The ability of H2O2 to induce lipid peroxidation as well as acting as a glutathione peroxidase substrate may explain why it can increase pentose phosphate pathway flux to a greater extent than cumene hydroperoxide can.

The dose response of the effect of BiCNU on pentose phosphate pathway flux was interesting, because 10 µg/ml of BiCNU consistently produced an increase under control conditions even though it had no effect in the presence of 10 µM cumene hydroperoxide. A possible explanation is that partial inhibition of glutathione reductase leads to an increase in the proportion of oxidized glutathione. This might stimulate the activity of glucose 6-phosphate dehydrogenase, whereas the increased substrate concentration maintains flux through glutathione reductase. Alternatively, it is conceivable that low concentrations of BiCNU might stimulate one of the plasma membrane redox systems [36]. A concentration of 50 µg/ml of BiCNU markedly inhibited flux through the pentose phosphate pathway, and inhibition was stronger still in the presence of 100 µg/ml or more of BiCNU (Fig. 5). This was largely reflected in its effect on lipid peroxidation (Fig. 7). The somewhat lesser effect of 50 µg/ml of BiCNU on lipid peroxidation than on pentose phosphate pathway flux might have resulted from the former being measured in standard BWW that contained 5.5 mM glucose and the pentose phosphate pathway being measured in BWW that contained only 0.1 mM glucose to increase the specific activity of [1-14C]D-glucose. These data support the view that motility is a relatively insensitive indicator of oxidative damage to human sperm [22], because 50 µg/ml of BiCNU had a pronounced effect on lipid peroxidation but only a slight effect on motility (Fig. 8). The ratios between motility under oxygen and under nitrogen (Fig. 8, g–i) make it clear that oxygen increases the toxicity of BiCNU, as its presumed site of action in blocking the glutathione peroxidase/reductase couple would predict. However, BiCNU had a significant inhibitory effect on motility even under nitrogen. We are unable to say if this reflects other toxic pathways or the presence of trace amounts of oxygen during the incubations, but the absence of effects on glycolysis or viability by 500 µg/ml or less of BiCNU favors the latter explanation.

To conclude, we believe our data demonstrate that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system in human sperm can respond dynamically to oxidative stress and is capable of protecting the cells against oxidative damage.


   ACKNOWLEDGMENTS
 
We are grateful for help and support from the late Prof. M.G.R. Hull and other staff of the University of Bristol, Division of Obstetrics & Gynaecology.


   FOOTNOTES
 
1 Supported by a grant from the Wellcome Trust. Back

2 Correspondence: W.C.L. Ford, University Division of Obstetrics & Gynaecology, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, U.K. FAX: 44 117 9285290; chris.ford{at}bristol.ac.uk Back

Received: 12 February 2004.

First decision: 9 March 2004.

Accepted: 3 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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