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From the * Department of Biochemistry and
Molecular and Cell Biology, School of Veterinary Medicine, University of
Zaragoza; and the Department of Animal
Production, Centro de Investigación y Tecnología Agroalimentaria
(CITA), Zaragoza, Spain.
| Correspondence to: Dr J. A. Cebrián-Pérez, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, C/ Miguel Servet, 177. 50013 Zaragoza, Spain (e-mail: pcebrian{at}unizar.es). |
| Received for publication June 5, 2007; accepted for publication December 11, 2007. |
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Abstract |
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Key words: Reactive oxygen species, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase
Reactive oxygen species (ROS) are generated by sperm metabolism. On the one hand, ROS are required for maturation, capacitation, and acrosome reaction—all processes needed for oocyte fecundation—and on the other, they are able to modify many of peroxidable cellular compounds (Jones and Mann, 1977). Thus, DNA structural damage (Reiss and Tappel, 1973), loss of membrane integrity, and enzyme inactivation (Roubal and Tappel, 1966; Wills, 1971) can originate serious injuries to cellular structures, altering their stability and, therefore, their functioning (Jones and Mann, 1977). Mammalian spermatozoa can thereby lack all the necessary resources to protect themselves from oxygen injuries (Abu-Erreish et al, 1978). The enzyme system comprising superoxide dismutase (SOD; Nissen and Kreysel, 1983; Alvarez et al, 1987), glutathione peroxidase/glutathione reductase (GPx/GR; Li, 1975; Alvarez and Storey, 1989), and catalase (CAT; Jeulin et al, 1989) has been described as functioning as a defense against lipid peroxidation in mammalian sperm. A defect of these enzyme activities could produce a loss of cell function.
The relationship found between "in vitro" capacitation and superoxide anion production (de Lamirande and Gagnon, 1995; de Lamirande et al, 1998) suggests that free radicals could play a role in the cryopreservation-induced damage. Some authors have, therefore, proposed that "cryocapacitation" is at least partially induced by ROS originated during semen processing (Bailey et al, 2000; Allamaneni et al, 2005). Furthermore, several authors have described the production of ROS during cryopreservation (Álvarez and Storey, 1992; O'Flaherty et al, 1997) and the higher sensitivity of frozen sperm to lipid peroxidation (Trinchero et al, 1990; Salamon and Maxwell, 1995).
Antioxidants exert a protective effect on the plasma membrane of frozen bovine sperm, preserving both metabolic activity and cellular viability (Beconi et al, 1993). Although a significant negative correlation between the ROS levels and the in vitro fertilization rate has been found (Agarwal et al, 2005), controlled quantities of ROS have shown to be essential for the development of capacitation and hyperactivation (de Lamirande and Gagnon, 1993), 2 physiologic processes of the spermatozoon that are necessary to ensure fertilization. The maintenance of a suitable ROS level is, therefore, essential for adequate sperm functionality.
The presence of antioxidant enzymes, SOD, GPx, and CAT, in human (Mann and Lutwak-Mann, 1981), bull (Beconi et al, 1993), and ram (Abu-Erreish et al, 1978; Marti et al, 2003; Marti et al, 2007) semen, and the effect of semen dilution in reducing their protective capacity (Maxwell and Stojanov, 1996) have been shown. Therefore, to further the understanding of the antioxidant defense system in ram semen, in this study we determined the activity of certain enzymes involved in ROS elimination and their changes during the cryopreservation process in order to investigate the hypothesis that the antioxidant defense system is involved in the maintenance of frozen sperm quality. The effect was also evaluated of different cryoprotectants on the activity and distribution of the most representative antioxidant enzymes in ram spermatozoa throughout the freezing/thawing process.
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Materials and Methods |
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The sires were kept apart, and semen was collected every 2 days, in 2 successive matings each day (0830–0930 hours). Under these conditions, and using second ejaculates, individual differences are very low, as we have already reported (Ollero et al, 1996), and pooled ejaculates provide a uniform, good-quality sperm sample suitable for representative studies of ram semen.
For each experiment, semen was pooled from 4 rams. The number of assays repeated on different days with different batches of pooled semen is indicated in each case.
Sperm Extenders and Cryopreservation
Sperm concentration of ejaculates was assessed by loading 5 mL diluted
semen (1:2000) into a Neubauer chamber (Marienfeld, Germany) and counting in
duplicate using a phase-contrast microscope at x100.
A milk-yolk medium (MY) was used as an extender up to a sperm concentration of 100 million sperm per straw. This extender was added in 2 fractions based on the diluent described by Soderquist et al (1997). Fraction 1 (F1) was performed by adding 5% egg yolk (v/v) and antibiotics (45 000 IU penicillin and 0.4 g streptomycin/100 mL) to skimmed milk. Fraction 2 (F2) consisted of the same quantity egg yolk and antibiotics, 14% glycerol (v/v) and 224 mM galactose. An initial extension of the semen was carried out with F1 at 37°C prior to a slow cooling rate of 0.4°C/min to 5°C. Dilution with a half-volume of F2 (of the F1) was used at 5°C and at 90 minutes before the freezing process. This mode of addition allows time for equilibration of glycerol with the cytosol. Samples were packed in 0.25-mL plastic straws and sealed with polyvinyl alcohol powder before cooling to –70°C for 10 minutes. They were then stored at –170°C in a liquid nitrogen container until use. Frozen straws were thawed in a bath at 37°C for 30 seconds.
The following additives were added directly to F1: ram seminal plasma proteins (SPPs; 4 mg/100 million sperm) that were proven able to prevent (Pérez-Pé et al, 2001) and repair (Barrios et al, 2000) cold shock sperm membrane damage, as well as increase frozen/thawed ram semen quality (Ollero et al, 1998); alfa-lactalbumin (11 µM, shown to increase sperm quality parameters in frozen ram semen; Ollero et al, 1998); beta-lactoglobulin (11 µM, as milk extender is widely used for the cryopreservation of ram semen); vitamin E acetate (2 mM); and linoleic/oleic acid–bovine serum albumin (BSA; 25 µM), as we have demonstrated that impaired function of cold shocked ram spermatozoa can be prevented by their addition to the medium (Pérez-Pé et al, 2001). No additive was added to control samples.
Assessment of Semen Parameters
Progressive individual motility was subjectively assessed under a coverslip
on a warm stage (37°C) by phase-contrast microscopy (x10 objective)
after dilution in the 2-phase system medium. Two magnifications were used
(x100 and x400), and the percentage of progressively motile
spermatozoa was estimated. The same person throughout the study performed
semen motility evaluations.
Cell viability is defined here as both intact plasma and acrosomal membranes. It was assessed by fluorescent staining with 6-carboxifluorescein diacetate and propidium iodide (Sigma Chemical Co, Madrid, Spain; Harrison and Vickers, 1990). The cells were then examined under a Nikon Labophot-2 fluorescence microscope with a B-2A filter (Nikon, Tokyo, Japan) at x400 magnification. The numbers of fluorescein-positive (plasma membrane–intact) and propidium iodide– positive (plasma membrane–damaged) spermatozoa per 100 cells were estimated and recorded. At least 200 cells were counted in duplicate for each sample.
The hypoosmotic swelling test (HOS test) was determined after dilution in a hypotonic solution and further incubation at 37°C for 45 minutes (Jeyendran et al, 1992).
These tests were undertaken immediately after semen collection, after cooling to 5°C, and after freezing/thawing.
SPP Preparation
Seminal plasma was obtained by spinning 1 mL semen at 12 000 x
g for 5 minutes in microfuge at 4°C. The supernatant was
centrifuged again, and 400 µL undiluted seminal plasma was removed and,
after filtration through a 0.22-µm Millipore membrane (Millipore
Ibérica, Madrid, Spain) and addition of 10% of a protease and
phosphatase inhibitor cocktail (Sigma Chemical Co, St Louis, Missouri), was
kept at –20°C.
SPPs were obtained by filtering the whole seminal plasma through Microsep microconcentrators of 3-kd molecular weight cutoff (Filtron Technology, Northborough, Massachusetts), spinning for 6 hours at 3000 x g at 4°C. The obtained sample concentrate was diluted with 5 volumes of a medium containing 0.25 M sucrose, 0.1 mM ethylene glycol tetraacetic acid, 4 mM sodium phosphate pH 7.5, 10% (v/v) of 10x buffer stock Hepes (50 mM glucose, 100 mM Hepes, and 20 mM KOH), was centrifuged again, and the SPPs were then recovered and stored at –20°C.
Protein concentration was assessed using the Bradford method (Bradford, 1976).
Enzymatic Activity Determination
Aliquots of 800 µL of each sample (fresh, cooled at 5°C, and
frozen/thawed, diluted in MY, with or without cryoprotectants) were diluted
1:1 with a sucrose medium (Marti et al,
2003) based on the formulation of Quinn et al
(1985) and centrifuged at room
temperature at 9600 x g for 6 minutes. The supernatant was
removed, and the pellet was incubated with 3% Triton X-100 for 30 minutes at
room temperature. The obtained supernatant was then collected by
centrifugation at 600 x g for 6 minutes, mixed with the one
previously obtained, and kept in ice until enzyme determination.
The determination procedures for SOD, GPx, and GR activities have been described by Marti et al (2003).
Enzyme Immunocytochemistry
Aliquots of 0.5 mL fresh and of 1 mL cooled or frozen/thawed samples with
4% paraformaldehyde were incubated at room temperature for 15 minutes. The
fixed cells were recovered by washing through a sucrose gradient
(Harrison et al, 1982) by
centrifuging at 200 x g for 5 minutes and at 750 x
g for a further 10 minutes. A total of 8 x 106 cells
were resuspended with 300 mL phosphate-buffered saline (PBS) and centrifuged
at 600 x g for 5 minutes to eliminate the sucrose buffer. The
sperm were incubated with 300 mL PBS/5% BSA for 40 minutes at 37°C. The
medium was then removed by adding 300 mL PBS and centrifugation (600 x
g for 5 minutes). The sperm were then incubated for 90 minutes with
0.1 mL of a primary antibody (anti-GR, anti-GPx, or anti-SOD), diluted 1:50
for GR and GPx antibodies and 1:250 for SOD antibody, in PBS/1% BSA, at
37°C. After a 5-minute wash in PBS, the cells were incubated for 90
minutes with 0.1 mL fluorescein-labeled immunoglobulin G (IgG) as a secondary
antibody, diluted 1:600 in PBS/1% BSA. After washing in PBS for 5 minutes, the
cells were resuspended in 0.25 mL PBS. The samples were assessed on a Nikon
Eclipse E-400 microscope under epifluorescence illumination using a B-2A
filter. At least 200 sperm per slide were scored.
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Results |
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SPP added either alone or with other compounds showed a protective effect and accounted for significantly higher values of motility, viability, and HOS test response in cooled and frozen/thawed samples (Table). The 3 enzyme activities were also increased in fresh, cooled, and frozen/thawed samples. As seminal plasma contains SOD, GPx, and GR (Marti et al, 2007), we determined the activity of each enzyme in the SPP sample so as to subtract the corresponding activities in the 40 mg of SPP added to samples (0.0911, 0.3501, and 32.93 µmol/min/mL of SOD, GPx, and GR, respectively). Therefore, the obtained data correspond to the real enzyme activities in the samples, excluding those of the SPP added. Figure 2 shows the percentage of increase of each enzyme activity in fresh, cooled, and frozen/thawed samples related to controls (MY alone). The inclusion of either SPP with oleic/linoleic acids or SPP with vitamin E accounted for the highest SOD activity value (39% and 28.3% increases, respectively; Figure 2A). Likewise, the additives produced a notable increment in GPx activity (Figure 2B). Furthermore, the mixture of SPP and oleic/linoleic acids produced increases in GPx activity with relation to control samples of 240% and 30% in cooled and frozen/thawed samples, respectively. Similarly, GR activity was strongly preserved using a mixture of SPP, oleic/linoleic acids, and vitamin E, resulting in a significant rise (P < .001) of 760% in cooled samples and an increase of about 260% in frozen/thawed samples (Figure 2C). A significantly higher enzyme activity value was also found in frozen/thawed compared with fresh samples (150% ± 30% increase in fresh vs 260% ± 40% in frozen/thawed samples; P < .05). The addition of vitamin E together with oleic/linoleic acids accounted for a higher value of GR activity than SPP alone.
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The cryopreservation process induced significant changes in the enzyme distribution, which depended on the presence of cryoprotectants (Figure 6A). Control samples (frozen with MY) underwent considerable changes in the proportion of each sperm type. The signaling in the acrosome and postacrosome (C and D types; Figure 3) was lowered, and the proportion of type A was increased (entire surface reactivity; Figure 3A), and this is, therefore, the most representative (49%). It is worth pointing out that when using a mixture of seminal plasma proteins, oleic and linoleic acids, and vitamin E as additives, the SOD distribution after freezing/thawing was very similar to that in fresh samples (Figure 6A), although a significant increase was found in the acrosomal labeling.
GPx distribution on the sperm surface showed different patterns (Figure 4), the main types being A, with labeling on the entire surface (Figure 4A); type B, on the tail (Figure 4B); type C, on the acrosome, postacrosome, and tail (Figure 4C); type D, with only postacrosome staining (Figure 4D); and type E, on the apical head, postacrosome region, and tail (Figure 4E). The freezing/thawing process brought about big changes in the GPx distribution (Figure 6B). Thus, in frozen control samples (MY), we found only 2 subpopulations: A and E. The addition of SPPs, oleic/linoleic acids, and vitamin E slightly restored the original distribution of fresh samples (Figure 6B) and accounted for a significant increase in the type A subpopulation, which was the most abundant (76%; Figure 6B).
GR distribution in fresh sperm was very different from the other 2 enzymes studied. The main subpopulation showed GR labeling only on tail (75%, type B; Figures 5B and 6C), and about 23.5% showed slight reactivity on the entire surface (type A; Figures 5A and 6C); some cells also showed a bright band on the equatorial segment (type D; Figures 5D and 6C). Again, cryopreservation produced considerable changes. Thus, in control samples (MY), we found a new GR location, type C, which showed reactivity on postacrosome and tail (Figures 5C and 6C). The proportion of type B was still the most abundant, although the amount of types A and D increased slightly (Figure 6C). The use of cryoprotectants restored the proportion of type B, whereas type A decreased and type D was significantly increased.
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Discussion |
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Our results show that 3 relevant antioxidant enzymes are present in ram sperm and that the cryopreservation process modifies their activities and distribution. The obtained values for the enzyme activities in control samples indicate that SOD is the enzyme most affected by cryoinjury, with a decrease of 65% after freezing/thawing (Figure 1). These results agree with those of Lasso et al (1994), who found that the SOD activity of a given human sperm sample was always lower after cryopreservation than in a fresh ejaculate. Likewise, we have proven that SOD activity diminishes in the 20°C to 0°C range, particularly the Manganese isoform of the enzyme (data not shown), which might be due to a partial inactivation of the enzyme. Although an increment in SOD activity in frozen boar sperm has been described (Cerolini et al, 2001), it may be due to the different efficiency in obtaining the enzymatic extract from frozen or fresh cells. The lower SOD activity found in this study after the freezing/thawing process could, therefore, be a result of the loss of the enzyme and/or a partial enzyme inactivation due to low temperature. GPx and GR activities were less affected by cryopreservation, which is in agreement with the results of a previous report in bull (Bilodeau et al, 2000).
The addition of SPP, particularly a mixture of SPP with oleic/linoleic acid and vitamin E, accounted for an increase in the enzyme activity levels, not only in the fresh sample but also after cooling and freezing/thawing (Figure 2). Likewise, as the intrinsic enzyme activity of the SPP added was discounted, there was a real increase in the activities related to the control samples. These findings suggest that this pool of proteins would provide a protector role of the sperm enzymes, and it may well be relevant if we consider that peroxidative damage is considerable in the majority of assisted reproduction techniques, which involve sperm washing methods and, consequently, long incubation periods without seminal plasma (Yeung et al, 1998).
The obtained results suggested studying the distribution of these enzymes on the sperm surface in control samples, processed only with a general diluent (MY), compared with other samples using a mixture of SPP, oleic/linoleic acids, and vitamin E as additives. We found that the antioxidant studied enzymes are located over several sperm regions and, therefore, we can define several subpopulations taking each enzyme into account (Figures 3, 4, 5). Furthermore, the distribution of each enzyme on the sperm surface corresponded to different membrane domains. Thus, the most representative SOD distribution is on the acrosome, postacrosome, and tail, whereas GPx is localized on the postacrosome and apical head, and GR distributes on the tail (Figure 6A through C). A similar pattern of SOD has been reported in human sperm by Lasso et al (1994), who also described that about 10% to 30% of intact cells showed immunofluorescence over the mid-piece and neck.
The cryopreservation process clearly affected the antioxidant enzyme distribution on ram sperm. Thus, the number and percentage of the fresh defined subpopulations changed considerably toward an entire surface distribution (Figure 6A through C). The mixture of SPP, oleic/linoleic acids, and vitamin E showed a tendency to maintain and recover the fresh distribution, particularly of SOD, although this was not completely achieved. These additives also proved useful for preventing the redistribution of GR, although the activity was hardly affected by the freezing/thawing process. These results are in agreement with the obtained enzyme activities that were better maintained throughout the cryopreservation process when these additives were used, which clearly verifies the protective effect of these compounds.
The results of this study show that the resistance to cryodamage and the defense against peroxidation in any given sperm sample is not uniform. Although the use of pooled ejaculates provides a uniform semen sample suitable for representative studies of ram semen, differences between treatments might have been overestimated by underestimating the variation between individual rams. However, the possibility of using the specific distribution of these enzymes as indicators of semen quality should not be ruled out.
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Acknowledgments |
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Footnotes |
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These authors share senior coauthorship. 
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