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From the Departments of * Pharmacology and
Therapeutics and Obstetrics and Gynecology,
McGill University, Montréal, Québec, Canada.
| Correspondence to: Bernard Robaire, Department of Pharmacology and Therapeutics, 3655 Promenade Sir William Osler, room 104, Montréal, QC, Canada, H3G1Y6 (e-mail: bernard.robaire{at}mcgill.ca). |
| Received for publication August 3, 2006; accepted for publication September 25, 2006. |
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Abstract |
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Key words: Oxidative stress, epididymis, glutathione peroxidase, superoxide dismutase, reproduction, sperm
Spermatozoa are highly susceptible to ROS-induced damage due to the high percentage of polyunsaturated fatty acids (PUFA) in the membrane (Mack et al, 1986; Alvarez and Storey, 1995). Oxidation of these PUFA can lead to decreased fluidity and flexibility of the sperm membrane and can therefore compromise the fertilization potential of the sperm (de Lamirande and Gagnon, 1992, 1995, 1995). In addition to lipid membrane damage, increased ROS in aged spermatozoa has detrimental effects on chromatin quality (Zubkova et al, 2005), as well as on motility (Kidd et al, 2001; Jung et al, 2002) and morphology (Plas et al, 2000) parameters.
In spite of the major consequences oxidative stress may have on male fertility, very few studies have investigated how sperm quality is affected by age. Notably, with the mean age of fathers significantly increasing in recent years, there have been increased concerns about decreased sperm quality with advancing age (Kidd et al, 2001). There is now a significant proportion of men who father children while in their 50s (Plas et al, 2000); men in this age group have a higher incidence of subfertility, and the time to pregnancy is increased (Rolf et al, 1996).
Spermatozoa have long been known as efficient producers of ROS (Tosic and Walton, 1950; Holland and Storey, 1981; Aitken and Clarkson, 1987). ROS such as superoxide anion (O2•–), hydrogen peroxide (H2O2), and the hydroxyl (HO•) radical are formed as byproducts of aerobic cellular processes. While under normal conditions ROS are essential for various sperm-specific physiological processes (de Lamirande et al, 1997), when produced in larger than normal quantities ROS are associated with male infertility (Aitken et al, 1996; Griveau and Le Lannou, 1997). The mechanism for this is likely due to detrimental ROS effects on the motility of spermatozoa, peroxidation of membrane lipids leading to compromised sperm-oocyte fusion, and decreased chromatin quality (Aitken, 1999; Christova et al, 2004; Zubkova and Robaire, 2004).
From the time they are made in the testes through to ejaculation and travel in the female reproductive tract, spermatozoa are constantly exposed to oxidizing environments. Perhaps most critically, the epididymis can be seen as a highly pro-oxidizing environment for the sperm (Vernet et al, 2004). Spermatozoa entering the caput epididymidis are functionally immature; during their maturation they are under constant attack from intracellular and extracellular production of ROS (Vernet et al, 2004).
It is thus crucial that spermatozoa be protected from oxidative damage. To this end, they contain enzymatic antioxidant defense systems available to deal with excess ROS production. Glutathione reductase (GR), glutathione peroxidases (Gpx), and superoxide dismutase (SOD) have been shown to exist in epididymal spermatozoa (Tramer, 1998), while the presence of catalase (CAT) remains controversial (Holland and Storey, 1981; Alvarez et al, 1987; Bilodeau et al, 2000). The glutathione peroxidases are involved in repairing oxidative damage to cellular macromolecules and scavenging H2O2 while utilizing glutathione (GSH) as a reducing equivalent. Their continued activity depends on the ability of GR to recycle oxidized GSH back to the reduced form.
The family of GSH peroxidases currently contains 5 characterized isoforms. Gpx4 is involved in forming a structural part of the midpiece (Ursini et al, 1999), is a protamine thiol peroxidase in the sperm nucleus (Conrad et al, 2005), and is also involved in reduction of oxidized phospholipids in the membrane of the spermatozoa (reviewed in Ursini et al, 1997). The importance of Gpx4 in the maturation process of spermatozoa during their transit through the epididymis has been demonstrated previously (Conrad et al, 2005). In its nuclear form, Gpx4 is responsible for the continual process of protamine thiol oxidation in the sperm nucleus from the testes, to the caput, and the cauda epididymidis (Conrad et al, 2005); this process is critical for proper chromatin condensation and packaging. A schematic representation of spermatozoa, ROS, and the enzymatic antioxidant defense system is illustrated in Figure 1.
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Materials and Methods |
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Animals
Male Brown Norway rats (aged 4 months and 21 months) were obtained from the
National Institute of Aging (Bethesda, Md) and housed on a 14L:10D cycle. Food
and water were provided ad libitum. All animal studies were conducted in
accordance with the guidelines outlined in A Guide to the Care and Use of
Experimental Animals prepared by the Canadian Council on Animal Care
(McGill protocol 206).
Sperm Extraction and Purification
Animals were euthanized by CO2 asphyxiation, and testes were
checked for regression in the 21-month-old animals. Epididymides with
corresponding regressed testes were not used for sperm extraction. In total, 6
young (4 months) and 5 old (21 months) animals were used for the study. Caput
and cauda epididymides were rapidly removed and placed into buffer on ice, and
sperm were collected as previously described
(Seligman et al, 1991).
Spermatozoa were released into either 1x PBS (for lipid peroxidation and
ROS measurements) or the antiperoxidant media BSPDR (for enzymatic analyses,
0.01 mol Tris-Cl (pH 7.5), 0.12 mol NaCl, 1 mmol phenylmethylsulfonyl fluoride
(PMSF), 0.1 mmol deferoxamine mesylate, and 0.1 mmol resveratrol) on ice and
filtered. Purification and fractionation was carried out essentially as
described in Tramer et al
(1998). Final pellets were
suspended in 1 mL of either 1x PBS or BSPDR and were aliquoted, counted,
and stored at –80°C for subsequent analysis.
Enzymatic Analyses
Prior to analysis, purified epididymal sperm (3 x 106/mL)
were sonicated (Vibra-Cell; Sonica and Materials Inc, Danbury, Conn) on ice 3
x 20 seconds and then incubated with 0.2% peroxide-free Triton-X at room
temperature for 5 minutes. Activities were measured on a Beckman DU-7
spectrophotometer (Beckman Coulter, Fullerton, Calif) using the kinetic
function. Final reaction volumes in all enzymatic assays were 1 mL. All enzyme
activities were measured within the linear portion of the absorbance/time
curve. Assays were run in triplicate.
Gpx1 activity was determined using t-butyl hydrogen peroxide as the substrate by following the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm in the presence of an excess of GR (EC 1.6.4.2; Sigma), as previously described (Flohe and Gunzler, 1984). Kinetic analysis was done over 10 minutes recording the rate of decrease in absorption at 340 nm. Final results were expressed in nmol NADPH oxidized/min/106 cells.
GR activity was determined by following the oxidation of NADPH at 340 nm via addition of oxidized GSH (GSSG), as previously described (Goldberg and Spooner 1983). Final results were expressed in nmol NADPH oxidized/min/106 cells.
Total SOD activity 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-tertrazolium-5-carboxanilide (XTT), as previously described (Zubkova and Robaire, 2004). One unit of superoxide dismutase activity is defined as the amount needed to inhibit 50% of XTT reduction. The assay was run simultaneously with varying known concentrations of SOD to generate the standard curve. Results are expressed in Units of SOD activity/106 cells.
Gpx4 activity was determined by following the oxidation of NADPH at 340 nm with the addition of an excess of GR. Activity was calculated in assay conditions exactly as for Gpx1, except that phosphatidylcholine hydroperoxide (PCOOH) was used as the substrate (Maiorino et al, 1990). PCOOH was isolated via the reaction of phosphatidylcholine (Type IV/S; Sigma) with soybean lipoxygenase (EC 1.13.11.12, Type V; Sigma) in a C-18 Sep Pak cartridge (Waters Corp, Milford, Mass), as previously described (Maiorino et al, 1990). PCOOH was extracted from the solid phase using methanol. The final substrate concentration was quantified spectrophotometrically at 234 nm. Results were expressed in nmol NADPH oxidized/min/106 cells.
Gpx4 Immunofluorescence and Quantification
For the determination of Gpx4 immunofluorescence, epididymal spermatozoa
were treated with either 40 mmol GSH or 40 mmol dithiothreitol (DTT) for 40
minutes at room temperature before being spun onto glass slides using the
StatSpin Cytofuge (Iris Sample Processing, Westwood, Mass). Treatment with GSH
facilitates head staining, while DTT treatment is specific for midpiece Gpx4
staining (Haraguchi et al,
2003). Cells were fixed in –20°C methanol for 10
minutes, blocked, and incubated with a polyclonal anti-Gpx4 primary antibody
(1:50). Secondary antibody was FITC-conjugated (1:200); imaging was done using
standard fluorescence microscopy.
Quantification of immunofluorescence was done with the microcomputer imaging software (MCID, version 7.0; Imaging Research, St Catherine's, Canada), using the relative optical density (ROD). GSH-treated spermatozoa were used to assess relative head fluorescence intensity, and DTT-treated sperm were used to assess relative midpiece fluorescence. ROD values were converted to gray levels by the formula [(1/10ROD) x 256]. A minimum of 100 sperm were counted per group (n = 3).
ROS Measurements
H2O2 Steady State Measurements—
Determination of H2O2 steady state concentration in
epididymal sperm suspensions after 10 minutes incubation was done by
fluorometric assay using the horseradish peroxidase
(HRP)–p-hydroxyphenylacetic acid (pHPA) system, as previously described
(Panus et al, 1993).
Spermatozoa were suspended in 1x PBS, 0.05 mg/mL A23187 in 1x PBS,
or 200 U/mL CAT in PBS and incubated for 10 minutes at 37°C. Suspensions
were then diluted (1:2.5) in prewarmed (37°C) 100-mmol phosphate buffer
(pH 7.4) containing 2.8 U/mL HRP and 40 µmol p-HPA. Final reaction volume
was 1.25 mL. Fluorescence of the samples was measured at 37°C on a
SpectraMax Gemini fluorescence spectrophotometer (Molecular Devices,
Sunnyvale, Calif) at 317 nm excitation-414 nm emission wavelengths.
H2O2 concentration was determined by subtracting the
value of the sample treated with 200 U/mL CAT from the untreated samples
(buffer alone or buffer + A23187). A standard curve was run in parallel with
known concentrations of H2O2. H2O2
concentration after 10-minute incubation was then calculated using the
following formula: [H2O2] = fluorescence intensity
x Vfinal /slope x Valiquote.
Superoxide Production— Superoxide production was determined by chemiluminescence using a computer-driven BMG LUMIStar Galaxy chemiluminometer (BMG Labtech Inc, Durham, NC), as previously described (de Lamirande and Gagnon, 1995, de Lamirande and Gagnon, 1995) at 5-minute intervals for 70 minutes, in the integration mode (output summed for 10 seconds). Spermatozoa were suspended in either 1x PBS, 0.05 mg/mL A23187 in 1x PBS, or 200 U/mL SOD in PBS (negative control). Epididymal spermatozoa, at a final concentration of 1 x 106/mL, were diluted 1:2 in 100 mmol phosphate buffer (pH 7.8) and incubated at 37°C. Chemiluminescent measurements were started immediately after addition of the probe, 2-methyl-6-(p-methoxyphenyl)-3, 7-dihidroimidazo [1,2-a] pyrazin-3-one (MCLA), at a concentration of 20 µmol, from a prepared 10-mmol stock in DMSO stored in the dark at –80°C. Due to the potential for autoxidation of MCLA in the assay, every sample was run in parallel with the same sample supplemented with SOD. The SOD signal was then subtracted from the sample data to determine the SOD-inhibitable signal. Because the medium itself can generate superoxide, it was also tested by itself, with or without SOD.
Lipid Peroxidation
Lipids were extracted and lipid peroxide products quantified directly using
the LPO kit from Cayman Chemical Co. Briefly, lipids were extracted from
epididymal spermatozoa into deoxygenated chloroform. Lipid peroxide products
were then quantified directly using a SpectraMax Plus (Molecular Devices)
spectrophotometer via the chromogenic detection of the reaction of ferrous
ions with lipid peroxides (Milhaljevic et al, 1996). Known concentrations of
lipid peroxide were run simultaneously to generate the standard curve, and
appropriate blanks were run. The chromogen-peroxide complex was measured at
500 nm. Results were expressed in µmol lipid peroxides/107
cells.
Statistical Analysis
Data were analyzed by SigmaStat 2.03 software (SPSS Inc, Chicago, Ill),
using 2-way ANOVA and the Holm-Sidak post hoc test, unless otherwise
indicated. Error bars represent SEM. Differences were considered significant
at P less than .05.
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Results |
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Gpx4 Immunofluorescence
Spermatozoa were treated in either 40 mmol GSH
(Figure 3a) or 40 mmol DTT
(Figure 3b) in order to allow
the Gpx4 antibody to gain access to the head or midpiece, respectively. DAPI
counterstaining was done for comparison purposes.
Figure 3a shows that in both
GSH-treated caput and cauda epididymal spermatozoa, aging significantly
decreased the intensity of fluorescence for Gpx4 in the head of spermatozoa;
these levels are quantified in Figure
3c. In both caput and cauda epididymal sperm, Gpx4 staining was
twice as intense in the young compared to the old. GSH treatment also appeared
to facilitate midpiece staining in the young animals
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Discussion |
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The extent to which the decrease in the thiol-dependent antioxidant enzymatic activity could potentially be explained by a decrease in the availability of substrates has not been fully explored. The degree to which Gpx1, GR, and Gpx4 (all thiol-dependent enzymes) utilize GSH as a substrate is unclear due to the very low levels of GSH in rat epididymal spermatozoa (Tramer, 1998). If GSH does exist at appreciable levels, there is evidence to suggest that these levels decrease in aging (Maher, 2005); in particular, GSH concentrations have been shown to decrease in the testis in the mouse model (Rebrin et al, 2003). In light of these observations, it is possible that the sustained levels of GR activity in aged rat spermatozoa observed in the current study represent a cellular compensatory mechanism for maintaining the levels of GSH. Another candidate as a substrate for the thiol-dependent antioxidant enzymes is cysteine, which has also been shown to decline in aging (Droge, 2005). In fact, under conditions in which the substrate concentrations are low, these enzymes are more prone to oxidative damage and degradation (Miller, 1987).
Of particular interest is the effect of age on Gpx4 activity. Gpx4 expression is drastically decreased in infertile men (Imai et al, 2001); combined with other lines of evidence (Garrido et al, 2004), these data point to a direct correlation between decreased Gpx4 activity and abnormal sperm morphology. Here we show that Gpx4 activity is significantly lower in aging rat spermatozoa and that Gpx4 expression is significantly lower in the head and midpiece of spermatozoa from aged animals. Previous data from our lab have demonstrated that aging decreases the number of disulfide bonds that exist between the protamines (as assessed using the mBBr technique, there were 25% fewer disulfide bonds in aged cauda spermatozoa) (Zubkova et al, 2005), a phenomenon directly associated with chromatin condensation (Conrad et al, 2005). Fewer disulfide bonds indicate a more open structure, and hence a DNA more vulnerable to oxidative damage. In light of the proven role of Gpx4 and protamine thiol oxidation in the sperm nucleus, the current study provides a novel potential mechanism for this decrease in disulfide bond formation. Further, the decreased expression of Gpx4 in the midpiece may indicate a compromised structure, potentially impacting motility. Previous data from our lab in the same model have shown that aging does affect certain sperm motility parameters in the rat (Zubkova and Robaire, 2004).
We have demonstrated that spermatozoa from the caput epididymidis have significantly higher antioxidant enzymatic activity then spermatozoa from the cauda region. This is in agreement with a previous report by Tramer et al (1998), although this study noted no difference in GR activity between segments. An explanation for the phenomenon of higher antioxidant enzymatic activity in the immature caput spermatozoa may reflect the greater need for control of ROS-related maturational events at this stage of spermatozoa maturation. That is, the sperm in the cauda epididymidis are already mature and capable of carrying out their biological function. Hence this loss in antioxidant activity could simply be interpreted as a diminished need for the mediation of ROS-controlled sperm maturational events.
In order to gain a more comprehensive insight into the extent of oxidative stress in aged spermatozoa, we examined H2O2 and O2•– production. Following previously noted age-associated increases in oxidant generation in other mammalian tissues such as heart (Sohal et al, 1994), brain (Gabbita et al, 1997), and testis (Chen et al, 2001), we noted that spermatozoa from the aged rat showed significant increases in H2O2 and O2•– production. Interestingly, the age-related increase in H2O2 production in cauda epididymal spermatozoa was only significant in the sperm incubated under conditions that induce the acrosome reaction, that is, with A23187. Functionally, this may mean that aged spermatozoa under capacitating conditions exhibit bursts of H2O2 that are much higher than required for capacitation and most probably are deleterious to the cell. Further, in both young and old animals, H2O2 production was twofold higher in caput epididymal spermatozoa than in spermatozoa from the cauda region. This trend was similar in the O2•– production of the 4-month-old animals, albeit at a lower level of significance. These results support previous findings that rat caput epididymal spermatozoa produce more ROS then spermatozoa from the cauda epididymidis (Vernet et al, 2001).
A23187 did not significantly induce either H2O2 or O2•– production in the spermatozoa from the caput epididymidis that were analyzed. This is expected, given that spermatozoa from the caput are less capable of undergoing the Ca2+-mediated processes of the acrosome reaction and capacitation, and hence would be less affected by an initiator of this process. In fact, it has been shown previously that induction of these Ca2+-initiated events is dependent on epididymal maturation (Sirivaidyapong et al, 2001), and that Ca2+ accumulation in sperm increases as the sperm transit through the epididymis (Okamura et al, 1992). Further, it has been demonstrated that there is a difference in the type of activated Ca2+ channels in spermatogenic cells compared to mature spermatozoa (Serrano et al, 1999). These findings suggest a difference in the response to Ca2+ between immature (caput) and mature (cauda) spermatozoa. Here we support these findings, as only spermatozoa from the cauda epididymidis were affected by A23187 with respect to ROS production.
Using a more direct method of assessing lipid peroxidation, we demonstrated
that age had a highly significant effect on sperm membrane peroxidation in
spermatozoa from both the caput and cauda epididymal regions. Peroxidation
levels were drastically increased (
twofold) in aged spermatozoa. The
increased extent of lipid peroxidation in sperm from aged animals indicates
serious deficiencies in sperm membrane quality, potentially interfering with
lipid membrane fluidity and flexibility, leading to compromised fusogenic
potential (Aitken and Sawyer,
2003). Because of the demonstrated importance of various lipids in
promoting sperm-egg fusion (Riffo et al, 1997;
Weerachatyanukul et al, 2001),
this increase in peroxidation of spermatozoa from old rats also provides a
plausible explanation for the increase in infertility generally associated
with aged men (Mathieu et al,
1995; Jung et al,
2002).
Taken together, these data suggest an overall decreased capacity for aged spermatozoa to manage excess oxidative challenge. A decreased antioxidant capacity coupled with an increased ROS production is a hallmark of aging and, as demonstrated with the lipid peroxidation end point, can have significant effects on macromolecular integrity. In light of the aforementioned susceptibility of spermatozoa to ROS damage, it is likely that these processes contribute significantly to the decline in sperm quality parameters that have been observed in aging males.
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Footnotes |
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