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From the Department of Biochemistry and Molecular and Cell Biology, School of Veterinary Medicine, University of Zaragoza, 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 March 13, 2006; accepted for publication June 18, 2006. |
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key words: Swim-up, caspase activity, annexin V
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although apoptosis is a phenomenon which has been known and studied in somatic cells for a long time, it is still a subject of controversy in ejaculated sperm (Gadella and Harrison, 2002; Oehninger et al, 2003; Martin et al, 2004; Said et al, 2006). Thus, germ cell apoptosis plays an important role in sperm production during normal spermatogenesis (Sakkas et al, 1999). Apoptosis in sperm would then be activated as a mechanism of elimination of abnormal or senescent spermatozoa or in response to environmental stress or injures. Ejaculated spermatozoa of humans (Gorczyca et al, 1993) and bulls (Anzar et al, 2002) have been shown to exhibit certain characteristics of apoptotic somatic cells.
Apoptosis can be initiated by many stimuli from outside (extrinsic apoptosis pathways) or inside the cell (intrinsic apoptosis pathways), including ligation of cell surface receptors, treatment with cytotoxic agents or irradiation, DNA damage, or oxidative stress. A central component of the apoptotic machinery involves, in most cases, a family of aspartic acid–directed cysteine proteases called caspases (cysteinyl aspartate-specific proteases). They are expressed as catalytically inactive proenzymes that are activated by proteolytic cleavage. Caspases involved in mammalian apoptosis are divided into 2 groups: initiator caspases, such as caspase-2, -8, -9, and -10, and effector or executioner caspases, such as caspase-3, -6, and -7. All initiator caspases are activators of downstream caspases, which execute the disassembly of the cell by cleaving a variety of cell structure proteins and generation of DNA strand breaks. Translocation of the phospholipid phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is an early feature of the terminal phase of apoptosis (Koopman et al, 1994), thus conveying cells to phagocytosis.
In the present study, we investigated the effects of 4 sperm preparation techniques (a dextran/swim-up procedure, discontinuous Percoll density gradient centrifugation, sucrose washing, and filtration) on ram sperm quality parameters. Besides evaluation of the viability and capacitation state, we also analyzed the apoptotic status of the sperm samples by assessing the PS translocation and caspase-3 and -7 activities to determine which method is the least harmful for spermatozoa.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dextran Swim-Up Procedure
The swim-up procedure was performed following the method we developed
previously for ram semen
(García-López et al,
1996). A 4-step procedure was carried out. Briefly, aliquots of
0.5 mL of unprocessed semen were pipetted into round-bottomed, 15-mm diameter
tubes under 0.5 mL of dextran solution (30 mg/mL) and then overlaid with 1.5
mL of SM medium (200 mM sucrose, 50 mM NaCl, 18.6 mM sodium lactate, 21 mM
HEPES, 10 mM KCl, 2.8 mM glucose, 0.4 mM MgSO4, 0.3 mM sodium
pyruvate, 0.3 mM K2HPO4, 1.5 UI/mL penicillin, and 1.5
µg/mL streptomycin (pH 6.5, 323 mOsm/kg) containing 5 mg/mL bovine serum
albumin. The tubes were kept at 37°C in a vertical position for 15
minutes. The top 0.75 mL of the layer containing the spermatozoa was removed
and replaced by careful addition of 0.75 mL of fresh medium. The incubation
sequence was then repeated 3 more times so that 4 supernatants were obtained.
The first supernatant was rejected because it contained lower-quality
spermatozoa and plasma component contamination
(García-López et al,
1996). The 3 top layers obtained from the final 3 consecutive
swim-ups were then combined to give 2.25 mL of sperm suspension.
Discontinuous Percoll Density Gradient Centrifugation
The 70% and 35% working density solutions were prepared by diluting
commercial Percoll with a saline buffer (v/v)
(Vincent and Nadeau, 1984)
composed of 135 mM NaCl, 10 mM glucose, and 20 mM HEPES (pH 7.6). A
discontinuous 2-layer Percoll density gradient was created by carefully
pipetting 4 mL of 35% solution over 2 mL of 70% Percoll solution into a
round-bottomed, 15-mm diameter tube. An aliquot of 0.3 mL of raw semen was
layered onto the top of the Percoll discontinuous gradient, and the tube was
centrifuged for 5 minutes at 200 x g followed by a second centrifugation
at 1200 x g for 15 minutes at 20°C. The pellet was washed twice by
resuspension in a dilution buffer (0.25 M sucrose, 0.1 mM EGTA, 4 mM sodium
phosphate [pH 7.5], and 10% [v/v] of 10x buffer stock HEPES [composed of
50 mM glucose, 100 mM HEPES, and 20 mM KOH]; the osmotic pressure was 330
mOsm/kg) and centrifugation for 5 minutes at 700 x g.
Sucrose Washing
Raw semen was diluted in 4 volumes of SM medium, and 1 mL of the diluted
sample was carefully pipetted over 7.5 mL of sucrose buffer (232 mM sucrose,
2.5 mM KOH, 20 mM HEPES, 10 mM glucose, 0.5 mg/mL polyvinyl alcohol, and 0.5
mg/mL polyvinylpyrrolidone (PVP); the osmotic pressure was 305 mOsm/kg;
Harrison et al, 1982) into a
conical-bottomed tube. The sample was centrifuged for 5 minutes at 200 x
g followed by a second centrifugation at 750 x g for 10 minutes. The
supernatant was removed and the pellet was collected in a conical tube.
Filtration
The filtration process was carried out by diluting the sample twice with 20
volumes of a dilution buffer (0.25 M sucrose, 0.1 mM EGTA, 4 mM sodium
phosphate [pH 7.5], and 10% [v/v] of 10x buffer stock HEPES) and
filtering through a 5-µm pore size Millipore disk (Millipore
Ibérica, Madrid, Spain). Sperm were recovered by sweeping the last mL
of the sample across the surface of the filter
(Pérez-Pé et al,
2001).
Viability Staining
Cell viability was defined as intact plasma and acrosomal membranes. It was
assessed by fluorescent staining with 6-carboxyfluorescein diacetate (6-CFDA)
and propidium iodide (Sigma Chemical Co, Madrid, Spain)
(Harrison and Vickers, 1990).
Sperm samples were diluted to 5 x 106 sperm/mL.
The cells were then examined under a Nikon Labophot-2 fluorescence microscope with a B-2A filter at 400x magnification. The number of fluorescein-positive (plasma membrane–intact) and propidium iodide–positive (plasma membrane–damaged) spermatozoa per 100 cells was estimated and recorded. At least 200 cells were counted in duplicate for each sample.
Assessment of Capacitation Status
Sperm capacitation was evaluated using the chlortetracycline (CTC) assay as
previously reported
(Pérez-Pé et al,
2002) using the technique described by Ward and Storey
(Ward and Storey, 1984). For
the evaluation of CTC patterns, the samples (5 x 106
sperm/mL) were observed through a Nikon Eclipse E-400 microscope under
epifluorescent illumination using a V-2A filter at 1000x magnification.
At least 200 cells were counted in duplicate for each sample.
Annexin V Staining
Annexin V is a calcium-dependent phospholipid-binding protein with high
affinity for PS. The translocation of PS residues to the outer layer of the
plasma membrane was detected with the Annexin V-Cy3.18 Apoptosis Detection Kit
(Sigma). To differentiate between live cells (with or without PS
translocation) and dead cells, we used 6-CFDA along with Ann V-Cy3.18. The
nonfluorescent 6-CFDA enters the cell and is converted to the green
fluorescent compound 6-carboxyfluorescein. This conversion is a function of
the esterases that are present only in living cells. Thus, no green
fluorescence can be observed in dead cells.
Sperm samples were diluted with 1x binding buffer (commercial kit) up to 500 µL (5 x 106 sperm/mL) and stained with 5 µL 6-CFDA (1 mM in DMSO) and 2 µL Ann V-Cy3.18 (commercial antibody provided in the kit).
Each sample was placed on a slide and analyzed at 1000x magnification by epifluorescence microscopy. Live sperm (6-CFDA+) were visualized in green with a standard fluorescein (Nikon B-2A) filter, and apoptotic sperm (Ann V-Cy3.18+) in red with an ultraviolet (Nikon G-2A) filter. Three patterns of fluorescence were observed: 1) CFDA+/AnnV–: Live cells without PS exposure—this subpopulation was labeled "intact cells"; 2) CFDA+/AnnV+: Live cells with PS exposure—this subpopulation was labeled "apoptotic cells"; and 3) CFDA–/AnnV+: "Dead cells"—this subpopulation showed annexin labeling in the entire cell. These dead cells could have died either by an apoptotic or necrotic process. A total of 400 spermatozoa were counted per slide.
Protein Extraction and Caspase Activity Measurement
Each sample (2 x 107 cells) was washed with
phosphate-buffered saline and centrifuged at 30 000 x g for 15 minutes
at 4°C. Supernatants were discarded, and pellets were stored frozen at
–80°C until analysis. Each pellet was then resuspended in the lysis
buffer provided by the manufacturer and electropermeabilized in predetermined
conditions (1 pulse at 2 Kv, 200 
, and 50 µF). Lysed cells were
centrifuged at 11 000 x g for 5 minutes at 4°C, and supernatants
were collected and stored at –80°C until analysis. Protein
concentration in supernatants was determined by the Bradford assay
(Bradford, 1976).
We used a commercial kit (EnzChek Caspase-3 Assay Kit #2; Molecular Probes Inc, Eugene, Ore) that allows the detection of apoptosis by assaying the increase in caspase-3 and other DEVD-specific protease activities. As caspase-7 recognizes the same amino acid sequence, the result of the measurement with this kit represents the total activity of both caspases together.
The substrate used in this test is rhodamine 110-bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide), which is a derivative of rhodamine 110 containing DEVD peptides covalently linked to each of R110's amino groups, thereby suppressing the dye's visible absorption and its fluorescence. Upon enzymatic cleavage, the nonfluorescent bisamide substrate is converted into fluorescent R110. The substrate is a synthetic product, and the enzyme recognizes a synthetic sequence. Therefore, it is not a species-specific recognition. We confirmed that the observed fluorescence signal was specifically due to the activity of caspase-3–like proteases using an inhibitor provided with the kit (Ac-DEVD-CHO inhibitor) (data not shown). The assay was used according to the manufacturer's instructions, and the activity was monitored using a fluorometer (Tecan Spectrafluor Plus; Tecan Ibérica, Barcelona, Spain). Fluorescence units were converted to nM R110/min·mg protein.
Statistical Analysis
Results were expressed as the mean ± standard error of the mean
(SEM) of the number of samples. Means were compared by analyses of variance
(ANOVA) tests to determine whether there were any significant differences
between samples using INSTAT for Windows (version 3.01). P less than
.05 was considered to be statistically significant. If ANOVA was appropriated
for data type and distribution was investigated by the Kolmogorov-Smirnov
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Capacitation State
Evaluation of the capacitation state by CTC staining confirmed the higher
quality of the swim-up sample (Table
2). The highest percentage of noncapacitated spermatozoa was
recovered by the swim-up procedure (62.2 ± 2.3%), thus increasing the
initial proportion present in fresh semen (55.5 ± 2.1%), although
differences between both were not significant. Consequently, swim-up samples
contained a lower percentage of capacitated and acrosome-reacted spermatozoa
than fresh semen. With the other 3 washing procedures, the percentage of
noncapacitated spermatozoa was significantly lower (P < .05) than
that in fresh semen and the swim-up sample (32.5%, 35.5%, and 27.0% in Percoll
density gradient, sucrose washing, and filtration samples, respectively).
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Phosphatidylserine Translocation
Changes in sperm membrane involving PS translocation after the washing
processes were analysed using Annexin V/6-CFDA staining. As shown in
Table 3, the population of
apoptotic cells decreased in all washed samples compared with fresh semen. It
is worth pointing out that only the swim-up procedure accounted for a
significant effect, with a decrease (P < .05) in the proportion of
apoptotic cells of 19.1 ± 5.1%. The decreases after Percoll density
gradient and sucrose washing (13.0 and 2.8%, respectively) were not
significant. Moreover, filtration hardly reduced the presence of apoptotic
sperm (0.6 ± 1.0%).
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Caspase Activity
To analyze whether other apoptotic markers were modified after these
preparative techniques, caspase activity was determined. The mixed activity of
caspase-3 and -7 was comparatively studied in cellular extracts of fresh and
treated samples (Table 1).
Decreases in caspase activity were found in the samples obtained by swim-up
(23.9 ± 4.1 nM substrate/min·mg protein), sucrose washing (16.1
± 2.7 nM substrate/min·mg protein), and filtration (22.1
± 2.5 nM substrate/min·mg protein) procedures compared with
fresh semen (31.1 ± 6.1 nM substrate/min·mg protein), although
the differences were not significant. Surprisingly, caspase activity in the
Percoll density gradient sample was hardly detected (0.0 ± 1.3 nM
substrate/min·mg protein). To clarify this result, an additional
experiment was carried out to test the effect of Percoll on caspase activity.
Increasing amounts of Percoll were mixed with a solution of a commercial
caspase-3 (0.025 µg/mL of recombinant human caspase-3; Sigma), and enzyme
activity was determined. The concentration of caspase used in this assay was
chosen on the basis of its activity to obtain a value similar to that in our
samples. As shown in the Figure, the higher the Percoll concentration, the
lower the enzyme activity was found. With a Percoll concentration similar to
that used in the gradient (70%), caspase activity was almost zero.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sperm preparative techniques used in animal reproduction are usually directed towards maintaining motility and viability. The accurate measurement of the semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. As the low fertility rate obtained with frozen-thawed semen is possibly due to premature capacitation-like changes (Thomas et al, 2006), assessment of the sperm capacitation state as well as other sperm characteristics such as apoptotic markers, could give substantial information about the sperm fertilizing capacity.
The reasons why apoptotic sperm are present in ejaculated semen are not
very clear. Some authors attribute it to the existence of immature sperm
(Paasch et al, 2004), others
to a phenomenon of abortive testicular apoptosis
(Sakkas et al, 2004), and some
to pathologic causes (Oehninger et al,
2003). Whatever the cause, the presence of apoptotic spermatozoa
in seminal doses could also be one of the reasons for poor fertility, as has
been reported in humans (Taylor et al,
2004; Said et al,
2006) and bulls (Anzar et al,
2002). Thus, it appears that the selection of nonapoptotic
spermatozoa is one of the prerequisites for achieving good results after
assisted reproduction (Paasch et al,
2005). Therefore, efforts must be focused on the selection of a
sperm preparative technique able to discard apoptotic
spermatozoa.
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In the present study, we compared 4 preparative methods taking into account not only the percentage of viable recovered sperm but also the capacitation state and apoptotic markers. Apart from the inherent interest of this study, it is essential to mention that, to our knowledge, this is the first report about apoptosis in ram sperm.
Although there are numerous studies comparing swim-up and Percoll gradient methods, the obtained results are contradictory. Some investigators have found no differences between samples obtained by either method (Smith et al, 1995), some found swim-up more advantageous (Palomo et al, 1999), while others have demonstrated that the use of the Percoll gradient resulted in better sperm quality (Ding et al, 2000; Somfai et al, 2002). Both the classical swim-up method (Berger et al, 1985) and the Percoll gradient (Arcidiacono et al, 1983) involve centrifugation steps, which have detrimental effects on ejaculated spermatozoa (Aitken and Clarkson, 1988; Alvarez et al, 1993; Mortimer, 1994). It is worth pointing out that the dextran/swim-up technique that we previously developed (García-López et al, 1996) and used in this study does not include centrifugation, thereby avoiding sperm viability loss.
Our comparison of the dextran/swim-up method, Percoll density gradient centrifugation, sucrose washing, and filtration procedure revealed that the swim-up technique resulted in the best quality spermatozoa. The dextran/swim-up sample showed the highest percentages of viable and noncapacitated spermatozoa, the lowest degree of PS translocation, and a caspase activity value lower than fresh semen. We already described the enrichment in viable sperm in the dextran/swim-up sample (García-López et al, 1996) and the sperm capacitation state (Pérez-Pé et al, 2002); however, the apoptosis status in this selected sample was not evaluated until this current study.
The higher level of PS translocation in Percoll-selected sperm compared with swim-up samples was not correlated with a higher caspase activity, despite the fact that PS translocation has been related to caspase activity (Martin et al, 1996). This suggests that the almost null caspase activity in the Percoll sample is due to an artifact more than a lack of activity. The additional experiment that we carried out with Percoll and a commercial caspase suggests that Percoll interferes with the enzyme determination as has been documented by others (Andersson and Hjorth, 1984; Awasthi and Misra, 2001).
The study of the capacitation state showed that Percoll density gradient yielded a higher percentage of capacitated spermatozoa than the swim-up technique, even greater than the proportion in the fresh sample. This observation is in accordance with that of Moohan and Lindsay (Moohan and Lindsay, 1995), who found that Percoll-selected spermatozoa showed an increased hyperactivation and an ability to undergo capacitation more readily than swim-up samples. This could be due to the fact that the Percoll method selects spermatozoa with different surface membrane properties than those selected by swim-up. Particularly, the Percoll-separated spermatozoa did not possess coating envelopes or a high level of surface fucose residues in comparison with the swim-up spermatozoa, and this may facilitate capacitation (Tanphaichitr et al, 1988). This fact, which could be desirable in sperm preparation techniques for human-assisted reproduction, could be a disadvantage in animal artificial reproduction when timing between ovulation and insemination is not always completely synchronized or the time interval between sperm preparation and insemination could be extended.
The other 2 methods studied, sucrose washing and filtration, yielded a similar sperm viability value, both lower than the fresh sample. Furthermore, the percentage of acrosome-reacted spermatozoa increased after both treatments compared with the control, swim-up, and Percoll samples. In addition, although caspase activity was lower after sucrose washing, it was not accompanied by a significant decrease in spermatozoa with exposed PS.
These results indicate once more that methods involving mechanical stress, such as centrifugation, are harmful to ram spermatozoa. In Percoll density gradient or sucrose washing, 1 or more centrifugation steps are carried out. Although the first one is more effective than the second in selecting nonapoptotic spermatozoa, both exert damage to the cell membranes that results in a decrease in viable and noncapacitated sperm. Furthermore, Percoll, which is a PVP-coated silica, has deleterious effects on sperm membranes (Strehler et al, 1998).
Filtration also exerts mechanical damage on sperm membranes as revealed by the decrease in viability. This could be due to the effect of the vacuum and/or the recurrent pippetting to which sperm are subjected during the process. In addition, the inefficient elimination by filtration of some factors in the seminal plasma with negative effects on spermatozoa cannot be ruled out (Ollero et al, 1997). Also, in the filtration process only seminal plasma is removed, while debris and all dead cells are kept and could affect live spermatozoa probably by production of high levels of reactive oxygen species, which have been reported as inductors of apoptosis (Wang et al, 2003; Grunewald et al, 2005).
In conclusion, results of this study confirm that the dextran/swim-up is a suitable method for selecting high-quality sperm. This is in accordance with our previous observation that ram sperm selection by this simple procedure increases fertilization rates following intrauterine insemination in superovulated ewes (Grasa et al, 2004). The high sperm concentration in ram ejaculates is not a drawback for the yield of the technique. The high percentage of noncapacitated spermatozoa and the low levels of apoptotic markers in the dextran/swim-up sample could, together with the high motility and viability values, contribute to the increased fertility rate of the selected sample compared with the control one. This is the first report on apoptotic markers in ram sperm, and it also confirms the higher capacity of the dextran/swim-up procedure to select nonapoptotic, viable, and noncapacitated sperm, compared with the other 3 sperm preparation methods. This should be considered to enhance the results of AI techniques in ovine reproduction.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this article and share senior
coauthorship. 
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