1 Department of Biochemistry and Molecular Biology, University of Granada, Campus Universitario Fuentenueva, 18701 Granada, 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain and 3 Laboratoire dEylau, 55 rue Saint Didier, 75116 Paris, France
4 To whom correspondence should be addressed at: MAR&Gen, Gracia 36, 18002 Granada, Spain. e-mail: cmendoza{at}ugr.es
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Abstract |
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Key words: apoptosis/cell death/DNA fragmentation/phosphatidylserine externalization/spermatozoa
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Introduction |
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This study was undertaken to examine the relative contribution of apoptosis and necrosis to the demise of ejaculated spermatozoa.
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Materials and methods |
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Each sample was divided into three aliquots. One aliquot was diluted (1:10) in phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 220 g for 10 min. The seminal plasma supernatant was discarded and the resulting pellet was resuspended in 1 ml of Menezo B2 medium (BioMérieux, France). A small sample (100 µl) of this cell suspension was removed immediately after this step for the evaluation of sperm motility, viability, phosphatidylserine externalization and DNA integrity (see below). The rest of this aliquot was divided into equal parts which were incubated in B2 medium equilibrated with 5% CO2 at 37°C for 4 and 24 h, respectively.
The second aliquot was layered on to a two-layer discontinuous (4080%) Percoll isotonic gradient and centrifuged at 200 g for 20 min. The medium used to dilute the Percoll was modified Hams F10 medium (Sigma, St Louis, MO) supplemented with 1% bovine serum albumin and 25 mM HEPES pH 8.0 (both purchased from Sigma). The 40% fraction was discarded, and the higher density layer was resuspended in 10 ml of PBS and centrifuged again at 600 g for 10 min. The resulting pellet was resuspended in 1 ml of B2 medium. As in the former group, an aliquot of 100 µl was removed for the evaluation of sperm motility, viability, phosphatidylserine externalization and DNA integrity (see below), and the rest of the aliquot was divided into two equal parts which were incubated in vitro for 4 and 24 h, respectively.
The third aliquot was prepared using a simple wash in PBS and centrifugation at 220 g for 10 min. After the removal of the seminal plasma, 1 ml of B2 medium was gently layered onto the sperm pellet followed by incubation (vertically) for 1 h at 37°C under 5% CO2 in air. At the end of the incubation, 500 µl of the overlying medium was separated to select the motile sperm fraction. As previously, an aliquot of 100 µl of the resulting cell suspension was removed for sperm analysis, and the resting cell suspension was subjected to in vitro incubation.
Motility analysis
The proportions of sperm that were motile and immotile were evaluated with the use of a Makler chamber. A drop of sperm sample was placed into the chamber and directly observed under a 40x objective. The operation was repeated twice and the mean of the two evaluations was taken into account. Each time, at least 100 spermatozoa were counted.
Supravital eosin staining
To evaluate sperm viability, sperm samples were centrifuged at 220 g for 10 min. The supernatant was discarded, and the pellet resuspended in 100 µl of PBS heated to 37°C. To assay sperm viability, 10 µl of the resulting cell suspension was mixed with 10 µl of the vital stain eosinnigrosin and incubated for 30 s. The specimens were then smeared on a glass slide, air dried and observed with a 100x oil-immersion objective. For each smear, at least 200 spermatozoa were examined and the percentage of unstained spermatozoa was recorded as the percentage of viable spermatozoa.
Evaluation of annexin-V binding and supravital propidium iodide staining
Externalized phosphatidylserine was visualized by incubating sperm samples at 37°C with annexin-Vfluorescein labelling reagent and 20 µl of propidium iodide in 1 ml of HEPES buffer (Boehringer Mannheim, Mannheim, Germany) according to the manufacturers instructions. The sperm cell suspensions were then smeared on glass slides and air dried. Slides were observed under a fluorescent microscope with a 100x oil-immersion objective. For each smear, 500 spermatozoa were examined. Spermatozoa stained either by propidium iodide alone or by both propidium iodide and annexin-V were considered as necrotic, whereas those stained with annexin-V only were considered as apoptotic. Unstained cells were considered alive.
Evaluation of DNA fragmentation
The presence of apoptosis-related DNA strand breaks in spermatozoa was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), using the Apoptosis Detection System, Fluorescein (Boehringer Mannheim, Mannheim, Germany). Washed sperm samples were smeared on microscope slides, fixed with 4% paraformaldehyde in PBS at 4°C for 25 min and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The rest of the procedure was carried out according to the manufacturers instructions. Briefly, the specimens were washed twice in PBS, equilibrated with the equilibration buffer at room temperature for 15 min and incubated at 37°C for 1 h in TUNEL incubation buffer containing nucleotide mix and the enzyme terminal transferase. After stopping the enzyme reaction, the slides were washed three times in PBS for a total time of 2030 min to ensure complete removal of non-specific fluorescence. Slides were observed in a fluorescent microscope with a 100x oil-immersion objective. For each slide, 500 spermatozoa were evaluated. The negative and the positive control were performed, respectively, by omitting the enzyme terminal transferase and by pre-incubating fixed and permeabilized sperm samples with DNase I (1 mg/ml) for 20 min at room temperature.
Statistical analysis
The significance of differences in the percentage of immotile, eosin-positive, propidium-positive, annexin-V-positive and TUNEL-positive spermatozoa in individual sample groups was evaluated by 2 test using the StatView II statistical package (Abacus Concepts, Berkeley, CA).
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Results |
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Sperm samples prepared by discontinuous density gradient centrifugation
In samples prepared by discontinuous density gradient centrifugation (Table II), the percentage of immotile and non-viable (eosin-positive and propidium-positive) spermatozoa was lower than in crude sperm samples prepared by simple washing (Table I). However, similar to the crude sperm samples, the percentage of immotile and non-viable spermatozoa remained unchanged during the first 4 h of in vitro incubation but increased after 24 h of incubation (Table II).
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Sperm samples prepared by washing followed by swim-up
Of the three sperm preparation methods used in this study, sperm washing followed by swim-up selection resulted in the isolation of sperm populations with the lowest percentages of immotile and non-viable (eosin-positive and propidium-positive) spermatozoa (Table III). However, the pattern of change for sperm motility, viability and apoptosis during subsequent in vitro incubation was similar to that of the other sperm preparation methods (Table III).
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Discussion |
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Irrespective of the preparation technique used, the percentage of motile and viable spermatozoa remained unchanged during the first 4 h of in vitro incubation, followed by a significant increase in the percentage of immotile and non-viable spermatozoa observed in all groups after 24 h of incubation. This increase was roughly proportional to the initial incidence of immotile and non-viable spermatozoa in individual groups. However, the overall increase in the percentage of immotile and non-viable spermatozoa was not accompanied by any detectable increase in the population of spermatozoa showing phosphatidylserine externalization and DNA fragmentation, two distinctive signs of an active apoptotic process. Thus, these data suggest that sperm cell death during in vitro incubation at 37°C occurs by necrosis rather than by apoptosis.
Several authors have described the presence of apoptotic markers (phosphatidylserine externalization, DNA fragmentation and Fas expression) in spermatozoa from men with normal and deranged spermatogenesis (Sakkas et al., 1999a; Gandini et al., 2000
; Oosterhuis et al., 2000
; Tesarik et al., 2001
; Ricci et al., 2002
). However, it is not clear whether ejaculated spermatozoa still retain the ability to activate the apoptotic signalling cascade or whether the apoptotic markers detected in ejaculated spermatozoa are merely relics of an abortive apoptotic process started before ejaculation (Sakkas et al., 1999
a). The abortive outcome of the apoptotic process may be related to a premature release of spermatids at early stages of apoptosis from Sertoli cells which normally should actively participate in the elimination of such cells. The present data strongly suggest that the presence of apoptotic markers in human ejaculated spermatozoa is a consequence of processes started before ejaculation and that the death of healthy ejaculated spermatozoa occurs by necrosis rather than by apoptosis.
In fact, if the increase in the percentage of spermatozoa supravitally stained with propidium iodide and eosin, observed after 24 h of in vitro incubation, resulted from an apoptotic process started in healthy ejaculated spermatozoa at the beginning of incubation, an increase in the percentage of spermatozoa with externalized phosphatidylserine would be expected to be detectable at least at the 4 h time point. In agreement with the present findings, the presence of active caspases, which are mediators of the classical apoptotic process in most cell types, was only rarely detected in human post-meiotic germ cells, and the presence of caspase activity did not correlate with DNA fragmentation (Tesarik et al., 2002).
The association of apoptotic markers with the death of human spermatozoa, observed by different authors (Sakkas et al., 1999a,b; Blanc-Layrac et al., 2000
; Ramos and Wetzels, 2001
), may be explained by an increased sensitivity of those spermatozoa whose apoptosis has been started in a late phase of spermatogenesis and interrupted after spermiation to the effects of external damaging agents. This may be due to membrane or mitochondrial damage caused by the incipient apoptotic process. In this study, however, all sperm samples came from sperm donors with normal basic sperm parameters in whom the occurrence of aborted apoptosis in ejaculated spermatozoa can be expected to be low. This may explain why simple necrosis, rather than atypical apoptosis, was responsible for cell death of most of the spermatozoa that did not survive during the 24 h incubation period.
In conclusion, these data strongly suggest that ejaculated spermatozoa lack the capacity of initiating the apoptotic pathway of cell death, and their demise occurs mainly by necrosis. Because sperm cell necrosis is accompanied by the loss of motility, there appears to be no significant increase in the risk of mistaking dying spermatozoa for viable ones after prolonged in vitro incubation periods. Consequently, sperm in vitro incubation for up to 24 h is unlikely to compromise outcomes of assisted reproduction using ICSI, at least when normozoospermic sperm samples are used. The death patterns of ejaculated spermatozoa from men with deranged spermatogenesis remain to be evaluated.
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Submitted on August 26, 2003; resubmitted on October 20, 2003; accepted on November 18, 2003.