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Male Reproductive Tract |
Departments of Pharmacology and Therapeutics and of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3G 1Y6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aging, epididymis, seminal vesicles, sperm, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is therefore crucial for the male reproductive system to be well guarded against oxidative injury. As a means of protection from oxidative stress, spermatozoa contain antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, glutathione reductase, and catalase [6] as well as glutathione (GSH) [7] (Fig. 1). The epididymis also plays a major role in shielding spermatozoa from oxidative stress by removing ROS and by secreting antioxidants into the epididymal lumen. The exact nature of the epididymal secretions that modulate ROS is still unresolved, but the role of secreted glutathione peroxidase [8, 9], GSH [10], catalase, and superoxide dismutase [11] seem apparent. The seminal vesicles provide yet another line of defense against oxidative stress; they have been shown to secrete superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, and GSH into the seminal fluid [6], thus offering spermatozoa additional antioxidative support in the highly oxidizing environment of the female reproductive tract [12].
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It is well documented that with advancing age an organism is under greater oxidative stress as the result of impairment of the function of the mitochondrial respiratory chain [13]. This leads to an accumulation of DNA, RNA, and protein free radical damage [14] and causes alterations in antioxidant enzyme levels [15]. However, few studies have addressed the effect that aging has on the antioxidative abilities of the male reproductive system. Knowing the vital role that oxidative stress plays in spermatozoal quality, investigation of the relationships between aging and spermatozoa and epididymal and seminal vesicle antioxidant status becomes important.
In addition to increased overall oxidative damage, aging causes an alteration in antioxidant response to stress [16]. As a result, any type of oxidative stress, such as poor diet, infection, or exposure to chemicals, can render the aging organism more vulnerable to free radical damage [17]. Therefore, it is important to determine how the response of the epididymis, seminal vesicles, and spermatozoa to oxidative challenge is altered by age. Oxidative challenge can be induced experimentally by the administration of L-buthionine-S,R-sulfoximine (BSO), an inhibitor of 
-glutamylcysteine synthetase, resulting in a depletion of GSH [18].
The goals of this study were to characterize the relative levels of GSH, glutathione disulfide (GSSG), and enzyme activities involved in the biosynthesis and breakdown of ROS (Fig. 1) in male reproductive tissues, to investigate the changes that occur with age, to examine how these changes are altered after administration of BSO, and to determine the effects that these age-related changes have on the characteristics of spermatozoal motility. By administering BSO for 7 days we achieved two objectives: 1) over this timeframe GSH became significantly depleted, and 2) spermatozoa were targeted for the duration of their transport along the epididymis, which in rats takes approximately 7 days. In addition to the epididymis and seminal vesicles, we examined the effect of oxidative stress on the liver, a tissue that is well studied and can serve as both a control and reference point for our study. The Brown Norway (BN) rat is a well-established model of male reproductive aging because it is long-lived and rarely develops age-related pathology [19]. Furthermore, these rats undergo similar male reproductive aging as humans, experiencing declines in serum testosterone and impairment in spermatogenesis [19–22].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male BN rats, aged 4 and 21 mo, were obtained through the National Institutes on Aging (Bethesda, MD) from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed on a 14L:10D cycle. Food and water were provided ad libitum. All animal studies were conducted in accordance with the principles and procedures outlined in A Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care (McGill protocol 206).
BSO Treatment
Twenty-four animals were randomly divided into four groups of six animals each: young control (YC), young treated (YT), old control (OC), and old treated (OT). Treatment consisted of subcutaneous injections of a glutathione synthesis inhibitor, BSO, at a dose of 2 mmol/kg at 12-h intervals for 7 days. Control groups received saline in place of BSO. The rats were sacrificed by CO2 asphyxiation 2 h after the last injection. Liver, seminal vesicles, and caput, corpus, and cauda epididymides were collected and flash-frozen in liquid nitrogen. Testes of old rats were examined for regression, and only epididymides from animals with nonregressed testes were used.
Glutathione/Glutathione Disulfide
GSH and GSSG concentrations were determined according to the method of Anderson [23]. Briefly, tissue samples were homogenized in 5% 5-sulfosalicylic acid and centrifuged at 18 000 x g for 5 min. For GSH measurements, the supernatant was added to a reaction cocktail that was equilibrated at 25°C and consisted of stock buffer (143 mM sodium phosphate, 6.3 mM Na4-EDTA, pH 7.5); NADPH (0.247 mg/ml); and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (6 mM). GSSG reductase (266 U/ml) was added to initiate the assay, and the change in absorbance was read at 412 nm. GSSG was determined by the same reaction, except that GSH was blocked from reacting with DTNB by a 1-h derivatization at 25°C with 2-vinylpyridine. It was determined that GSH assay recording remained linear down to 0.005 nmol/mg tissue and GSSG to 0.001 nmol/ mg tissue.
Antioxidant Enzyme Assays
Tissues were prepared for antioxidant enzyme assays according to the method of Ho et al. [24] with some modification. Specifically, tissues were homogenized in ice-cold homogenization buffer (50 mM potassium phosphate, 0.1% Triton X-100) with a Brinkman Polytron homogenizer (Rexdale, ON) for 10 sec, followed by sonication for 5 sec with a Vibra Cell sonicator (Sonics & Materials Inc., Danbury, CT). Protein content was determined using the Bio-Rad protein assay [25]. All enzyme activities were measured from the linear portion of the absorbance/time curve.
Glutathione peroxidase Glutathione peroxidase activity was assayed as indicated in Vernet et al. [26] with the following modifications: tissue homogenate was added to the reaction buffer (100 mM Tris-HCl, pH 7.2, 1 mM GSH, 0.2 mM NADPH, warmed to 37°C), and the reaction was catalyzed by addition of 1 U/ml glutathione reductase and 25 µM of H2O2. The rate of NADPH utilization was followed at 340 nm, and enzyme activity was expressed as nanomoles of NADPH consumed per minute per milligram protein (nmol NADPH min–1 mg–1). The NADPH mM extinction coefficient is 6.22.
Glutathione reductase Glutathione reductase was assayed according to the method of Carlberg and Mannervik [27]. Briefly, activity was assayed at 340 nm in 0.2 M potassium phosphate buffer, pH 7.0, 2 mM EDTA, 2 mM NADPH, 20 mM GSSG, equilibrated at 25°C. Glutathione reductase activity is defined as nanomoles of NADPH consumed per minute per milligram tissue protein (nmol NADPH min–1 mg–1).
Catalase Catalase activity was measured at 240 nm in 50 mM phosphate buffer, pH 7.0, containing 10 mM H2O2 and equilibrated at 25°C [28]. The extinction coefficient for catalase is 0.0394 mM, and activity is expressed in micromoles H2O2 consumed per minute per milligram tissue protein (µmol H2O2 consumed min–1 mg–1). Enzyme activity was measured from the linear portion of the absorbance curve.
Superoxide dismutases The total activity of superoxide dismutase was determined at 550 nm by measuring the inhibition of xanthine/xanthine oxidase-mediated reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; 0.5 mM xanthine in 50 mM potassium phosphate, pH 7.8, 0.1 mM EDTA, xanthine oxidase sufficient to produce a slope of 0.25, 50 µM XTT, 25°C) [29]. One unit of superoxide dismutase activity is defined as the enzyme activity needed to inhibit 50% of XTT reduction. Manganese superoxide dismutase activity was assessed by repeating the above experiment in the presence of 50 mM NaCN to inhibit Cu-Zn superoxide dismutase. Copper-zinc superoxide dismutase activity was then determined by extrapolation.
Spermatozoal Motility
Spermatozoal motility was measured using the method of Slott et al. [30]. Briefly, spermatozoa from the cauda epididymidis were collected into 5 ml of motility buffer (Hanks Balanced Salt Solution, 0.35 mg/ml sodium bicarbonate, 4.2 mg/ml Hepes, 0.9 mg/ml D-glucose, 0.1 mg/ml sodium pyruvate, 0.025 mg/ml soybean trypsin inhibitor, 2 mg/ml BSA, pH 7.4, pre-warmed to 37°C) and were allowed to disperse for 5 min. A volume of 20 µl of the suspension was loaded onto 2x Cel Sperm Analysis Chambers (Hamilton-Thorne Research, Beverly, MA) prewarmed to 37°C. Movement characteristics were assessed using computer-aided sperm analysis (CASA) with a Hamilton-Thorne IVOS Motility Analyzer, version 12 (Hamilton-Thorne Research, Beverly, MA) and included the following primary parameters: average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), as well as two derived parameters, amplitude of lateral head displacement (ALH) and linearity (LIN). Analysis was done using the following settings: stage temperature, 37°C; frames acquired, 30; frame rate, 60 Hz; minimum contrast, 80; minimum cell size, 4 pixels; minimum static contrast, 15; cell intensity, 80; magnification, 0.82; static size limits, 0.29–8.82; and static intensity, 0.18–1.8. The chamber depth was 80 µm, where 20 µl were loaded into each chamber.
Each rat was sampled five times, where a minimum of 100 sperm per sample were analyzed and the mean of the 5 measurements were calculated for each rat.
Cytoplasmic Droplet
The presence of cytoplasmic droplets was determined using the method established by Syntin et al. [31]. Motility images that had been recorded with CASA were still-frozen and spermatozoa were assessed for the presence or absence of cytoplasmic droplet. A minimum of 100 spermatozoa/ animal were evaluated.
Chemicals
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) except for Hanks Balanced Salt Solution (Gibco Invitrogen Co., Grand Island, NY); H2O2 (Fisher Scientific International Inc., New Jersey, NY); and Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA).
Statistical Analyses
All measurements were made on tissues and/or cells obtained from six different animals (n = 6). Data are expressed as means ± SEM. Comparisons between groups were made using t-tests with Bonferroni correction (SigmaPlot, v. 7.0, Aspire Software International, Leesburg, VA). Differences were considered as significant at P < 0.05.
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RESULTS |
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GSH concentrations in the liver were approximately fivefold greater than in seminal vesicles and approximately eightfold greater than in epididymal tissues (Fig. 2A). Since GSH is expressed per milligram tissue, it should be noted that although body weights of old animals were approximately double those of young, there were no significant differences in wet weight between epididymal, seminal vesicle, or liver weights between the two age groups either before or after BSO treatment (data not shown). GSH concentrations were not significantly different in the young and old rats, although there was a trend toward an increase in the seminal vesicles and caput and corpus epididymides. GSSG levels in the liver were similar to those of the seminal vesicles but were about 10-fold higher than those in the epididymis (Fig. 2B). GSSG concentrations increased significantly in the seminal vesicles of aged rats but were not changed with age in the liver or epididymis.
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BSO administration depleted GSH dramatically. Although there was no significant difference between the levels of depletion in young and old animals, there were variations in the extent of depletion in different tissues. In the liver, GSH decreased by 74% and 76% in young and old, respectively, and in the seminal vesicles by 71% and 68%, respectively. In epididymal tissues, the depletion was more pronounced: GSH concentrations in young and old decreased 81% and 83% in the caput, 87% and 91% in the corpus, and 92% and 89% in the cauda epididymidis, respectively (Fig. 2A).
Since GSSG decreased below detection, the GSH/GSSG ratios after BSO administration could not be determined in the epididymal segments, but in the liver and seminal vesicles this ratio was not significantly altered.
Glutathione Peroxidase
Glutathione peroxidase activity was highest in the liver and lowest in the seminal vesicles (Fig. 3A). With age, the activity of glutathione peroxidase increased significantly in the liver and decreased in the cauda epididymidis. After GSH depletion, glutathione peroxidase activity remained unchanged or decreased; this decrease was significant in the seminal vesicles of young rats.
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Glutathione Reductase
In young rats, basal glutathione reductase activity was comparable in the liver, seminal vesicles, and epididymis (Fig. 3B). With aging, enzyme activity increased significantly in the liver and cauda epididymidis. In response to GSH depletion, glutathione reductase activity increased in the caput and corpus epididymidis of young rats; glutathione reductase activity almost doubled after BSO treatment in the corpus epididymidis. GSH depletion resulted in increased glutathione reductase activity in the liver, caput, corpus, and cauda epididymides of old rats.
Catalase
In the young animals, catalase activity was about 40-fold greater in the liver than in the seminal vesicles or the epididymis (Fig. 3C). Catalase activity changed little with age, where a significant increase was observed only in the liver. GSH depletion resulted in an increase in catalase activity in the caput and corpus epididymides of young rats. An increase in catalase activity was found in these tissues and in the cauda epididymidis and liver of aged rats. In contrast, the activity in the seminal vesicles in old animals was significantly decreased with BSO treatment. Similar to the pattern seen with glutathione reductase activity, glutathione depletion affected catalase activity most dramatically in the corpus epididymidis.
Superoxide Dismutase
Total superoxide dismutase activity levels were of a comparable scale in all tissues studied (Fig. 4A). Age resulted in a decrease in activity of the enzyme only in the corpus epididymidis. GSH depletion caused a change in activity only in liver, where it significantly decreased after treatment in the young. Manganese superoxide dismutase activity decreased dramatically with age in all tissues, except for the corpus epididymidis, where activity increased (Fig. 4B). With BSO treatment, Mn superoxide dismutase activity decreased significantly in the liver and seminal vesicles of young rats, but was unaffected in the epididymis. Copper-zinc superoxide dismutase activity was determined by subtracting that of Mn superoxide dismutase from total superoxide dismutase activity. Copper-zinc superoxide dismutase was affected by age only in the corpus epididymidis, where enzyme activity decreased. GSH depletion produced little change (Fig. 4C).
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Spermatozoal Motility and Cytoplasmic Droplets
One parameter of sperm motility, VAP, increased significantly with aging (Fig. 5). In young rats, GSH depletion resulted in a significant decrease in VAP. The effects of GSH depletion were more pronounced in old rats than in young rats. In the aged rats, VAP, VCL, and ALH were decreased significantly after GSH depletion. Although not all changes in motility parameters were statistically significant, they all consistently showed greater sensitivity to BSO treatment with age, where VSL decreased by 6% and 8% in young and old rats, respectively; VAP decreased by 6% and 13%; VCL decreased by 8% and 14%; ALH decreased by 10% and 17%; and LIN increased by 4% and 9%, respectively (Fig. 5). The percent of sperm retaining their cytoplasmic droplet was 25% in young rats and 18% in aged rats; these values were not affected by GSH depletion (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aging is associated with an overall increase in oxidative stress; this is due in large part to changes in the mitochondria, where respiratory chain function becomes impaired with age, resulting in decreased ATP and increased ROS production [13]. Additionally, aging is associated with changes in the levels of antioxidants and antioxidant enzymes. The activity of Mn superoxide dismutase, also known as the mitochondrial superoxide dismutase, has for the most part been reported to increase with age [32]. However, studies using the liver have shown both an increase [33] and a decrease [15, 34]. In our study, liver Mn superoxide dismutase was not changed significantly in aged animals, whereas in the male reproductive tissues the results were both tissue- and segment-specific. The difference in the effect of age on Mn superoxide dismutase in epididymal segments may be influenced by the presence of a recently identified secreted form of Cu-Zn superoxide dismutase [35] that was shown to be expressed in particularly high levels in the cauda epididymidis. To the best of our knowledge, its expression in the seminal vesicles has not yet been assessed.
The effect of age on the concentrations of GSH and GSSG in the liver is controversial; some groups report an increase with advancing age [36], whereas others report a decrease [15]. Our study showed neither GSH nor GSSG to be altered by age. In contrast, glutathione peroxidase and glutathione reductase activities increased, which is consistent with previous studies using the liver [15, 36, 37]. These changes in enzyme activities suggest a general enhancement of the GSH oxidizing-reducing cycle and an overall adaptive response to counterbalance the increased oxidative stress that is seen with aging.
The redox system in the caput and corpus epididymides, as well as the seminal vesicles, was not greatly affected by age. In contrast, a very interesting pattern of changes was observed with age in the cauda epididymidis. Although GSH levels remained the same, glutathione reductase activity increased and glutathione peroxidase activity decreased, suggesting that the system has become more sparing in its GSH utilization; the redox system of the cauda epididymidis may be particularly vulnerable to aging. Previous studies have reported that the expression of glutathione-S-transferases, the enzymes that conjugate electrophiles to GSH, decreased with age in principal cells of the proximal cauda epididymidis, but not in the other epididymal segments [38].
In most studies, the effect of age on human spermatozoa is associated with a change in spermatozoal motility parameters [39]. Our study on the BN rat also reveals some effects of age on spermatozoal motility, consistent with previous reports from our laboratory [31]. Spermatozoa in older animals had a more curved trajectory, reminiscent of the irregular spermatozoal movement in the caput epididymidis, suggesting that spermatozoa in old rats are not maturing as efficiently during their course along the epididymis.
In conclusion, age had a differential effect on redox systems in the tissues examined; the most dramatic changes were found to occur in the liver and cauda epididymidis. Spermatozoal motility characteristics were also affected by age. Since spermatozoa are collected and stored in the cauda epididymidis, it is likely that age-related changes in this tissue play a role in the observed motility change.
Effect of Glutathione Depletion
Although every tissue examined responded to GSH depletion in a unique manner, it is striking that the responses of glutathione reductase and catalase are very similar in all of the tissues evaluated. These enzymes may change in the same manner since they act cooperatively to avert oxidative stress, the function of catalase being to remove H2O2 and that of glutathione reductase to replenish GSH, which in turn also acts to remove H2O2. By comparing the changes in activities of these two enzymes we can gain valuable information about the response of the tissues to stress.
In the liver, catalase and glutathione reductase respond more dramatically with age, suggesting that GSH depletion has less effect in younger animals than in old rats. Glutathione peroxidase was not altered by BSO treatment, but total superoxide dismutase activity decreased in the young animals. A possible explanation for the change in superoxide dismutase activity might be that with less GSH available to conjugate H2O2, the H2O2 becomes elevated to the point that it provides negative feedback on superoxide dismutase [40].
In the caput epididymidis, both catalase and glutathione reductase activities change moderately regardless of age, suggesting that age does not greatly affect the tissue's sensitivity to GSH depletion. The corpus epididymidis is most responsive to GSH depletion, as both catalase and glutathione reductase activities increase dramatically. This leads to the speculation that the strong response of the antioxidant enzymes in the corpus epididymidis is a means of protecting spermatozoa from oxidative stress at a point when it can be particularly disruptive to their integrity. This is consistent with the observation that the basal region of epididymal principal cells is rich in lipid droplets [41], which if oxidized could propagate a lipid peroxidation reaction in the spermatozoal membranes. In the cauda epididymidis, glutathione reductase activity appears to be moderately influenced by BSO treatment while there is a greater influence on catalase activity. The response of antioxidant enzymes to GSH depletion in this epididymal segment was clearly age-dependent.
Redox regulation in the seminal vesicles appears quite different from that of other tissues studied. This is particularly apparent when comparing changes in catalase activity after BSO administration. Although in the liver and caput, corpus, and cauda epididymides, catalase activity increased, in the seminal vesicles it decreased, particularly so with age. This suggests that the seminal vesicles have unique antioxidant enzyme regulation, one that is not seen in the liver or the epididymis. Additionally, after BSO treatment glutathione reductase activity is found to significantly increase in the liver and caput, and corpus epididymides, and to have an increasing trend in the cauda epididymidis. However, not only do the seminal vesicles not present any increase in glutathione reductase activity after BSO treatment, but in aged animals there is even a slight decrease. Therefore, the response of these two enzymes in the seminal vesicles is contrary to what we observed in all of the other tissues, which is truly remarkable.
A distinguishing feature of the seminal vesicles is their capacity for secreting particularly large quantities of prostaglandins [42]. Although prostaglandins are not directly related to oxidative stress, they are formed from the same precursor and have similar structures and properties as isoprostanes, which are the products of lipid peroxidation and well-established markers of oxidative stress [43]. Therefore, the production of large quantities of prostaglandins may play a role in the distinctive response to oxidative stress that is displayed by the seminal vesicles. After BSO treatment the seminal vesicles had the greatest decrease in glutathione peroxidase activity among the tissues studied, indicating an overall decrease in GSH utilization. This is once again surprising, as we noted above that catalase had also decreased, and if both glutathione peroxidase and catalase decreased, the question arises as to whether there might be another antioxidative system important to the seminal vesicles or if they are simply left exceptionally vulnerable to oxidative stress after GSH depletion. These are important issues to explore in order to better assess the origin of oxidative damage in ejaculated sperm.
Spermatozoal motility was affected by both aging and GSH depletion. The profile of changes after BSO treatment of old rats suggests sluggishness in movement. Previous reports have suggested that lipid peroxidation of spermatozoa is strongly negatively correlated with VAP and VCL [44]. Changes in motility parameters in our study may also be due to lipid peroxidation, as it is well established that peroxidation of spermatozoa membranes is a function of antioxidant enzyme activity [45]. A high level of ROS induces changes in spermatozoal movement through several mechanisms; these include an effect on Ca++ channels due to lipid peroxidation of the outer membrane [46, 47] and depletion of spermatozoa ATP stores [48, 49].
In summary, we have shown that the redox system in the liver, seminal vesicles, and cauda epididymidis, as well as spermatozoal motility, are affected by aging. BSO mediated a dramatic depletion of GSH in all organs studied, and this depletion was accompanied by changes in antioxidative enzyme activities. After induction of oxidative challenge, the effects of age on the redox system in the liver and cauda epididymidis were more apparent. As the age-dependent alteration in cauda epididymal redox response was coupled with a decline in spermatozoal motility parameters, we speculate that the increased vulnerability of the cauda epididymidis to GSH depletion affects its capacity to protect spermatozoa from oxidant exposure.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Bernard Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, PQ, Canada H3G 1Y6. FAX: 514 398 7120; bernard.robaire{at}mcgill.ca
Received: 11 February 2004.
First decision: 5 March 2004.
Accepted: 4 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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