Hum. Reprod. Advance Access originally published online on March 25, 2004
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Human Reproduction, Vol. 19, No. 5, 1127-1134,
May 2004
© 2004 European Society of Human Reproduction and Embryology
Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters
1 INSERM U459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex and 2 Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandres et Laboratoire d’Histologie, Faculté de Médecine, F-59037 Lille Cedex, France
5 To whom correspondence should be addressed at: INSERM U 459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France. e-mail: philippe.marchetti{at}lille.inserm.fr
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Abstract |
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BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.
Key words: cell death/flow cytometry/IVF/spermatozoa
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Introduction |
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It has been hypothesized that sperm cell death is associated with male infertility. Indeed, reports indicated that cell death-associated changes, including reduction of the mitochondrial membrane potential (
m) (Donnelly et al., 2000
; Marchetti et al., 2002) and reactive oxygen species production (Whittington et al., 1999) are more frequent in men with poor semen quality. Moreover, semen from infertile men contain a high proportion of spermatozoa with typical signs of apoptosis, including morphological damage (Baccetti et al., 1996; Gandini et al., 2000), nuclear DNA fragmentation (Barroso et al., 2000; Gandini et al., 2000) and loss of asymmetric distribution of phosphatidylserine at the plasma membrane (Barroso et al., 2000; Oosterhuis et al., 2000). However, membrane changes assessed by annexin V staining in ejaculated sperm do not correlate with semen abnormalities (Ricci et al., 2002), questioning the validity of apoptosis detection to evaluate sperm quality. Caspases are cysteine proteases that promote apoptosis in mammals. To date, 14 caspases have been described in human (for review, see Nicholson, 1999). These proteins are initially synthetized as single inactive polypeptides (zymogens) that undergo activation via proteolytic processing into a large (
20 kDa) and a small subunit (10 kDa). Caspases are usually divided on the basis of the substrate specificities and also into functional sub-families. Some apoptotic caspases are involved in a hierarchically ordered proteolytic cascade. Initiating caspases (caspases-8, -6, -9, -10) activate effector caspases (caspases-3, -7, -2) which are in turn responsible for the cleavage after aspartate residues of crucial substrates in the final degradation phase of apoptotic cell death. Caspase-3 activity, responsible for activation of specific DNases inducing for nuclear apoptosis, has been strongly implicated in human pathologies (Reed, 2001). However, until recently the involvement of caspases in apoptotic ejaculated sperm cells was unclear. In mouse sperm, caspases are absent (Weil et al., 1998) whereas caspase-3 enzymatic activity was detected in a relatively small proportion of human sperm with low motility (Veis et al., 1993; Weng et al., 2002). In agreement with these results, a higher percentage of spermatozoa with activated caspases were found in infertile men (Paasch et al., 2003) confirming the existence of a caspase-dependant apoptotic pathway in ejaculated human sperm.
It was proposed that methods detecting cell death could serve as a test to evaluate sperm quality, thus sperm fertilizing capacity in vitro (Host et al., 2000; Marchetti et al., 2002). Assays based on flow cytometry are particularly well adapted for clinical purposes because they provide a rapid, easy and objective analysis of high numbers of sperm cells. Consequently, cytofluorometric analysis of DNA fragmentation (TUNEL method) (Sun et al., 1997; Oosterhuis et al., 2000), and m (Troiano et al., 1998; Marchetti et al., 2002) were both previously found to correlate with sperm characteristics. As mentionned above, results with annexin V staining are conflicting (Oosterhuis et al., 2000; Ricci et al., 2002) indicating that annexin V binding is not a valuable test to assess sperm quality. Recently, detection of activated caspases in living spermatozoa was performed using a cell-permeable fluorescent derivative of the inhibitor peptide VAD-FMK, expected to detect the overall activation status of caspases (Paasch et al., 2003).
Consequently, in this study, we analysed by flow cytometry sperm samples from patients enrolled in an IVF programme stained with a cell-permeable fluorescent derivative of the inhibitor peptide VAD-FMK (FITC-VAD-fmk).
This approach allowed us to correlate the global caspase-activation status: (1) with other cell death markers (TUNEL method, m determination); and (2) with the parameters of sperm evaluated by conventional light microscopy analysis in semen samples prepared for IVF.
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Materials and methods |
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Materials
Ferticult medium was purchased from Fertipro NV (Beernem, Belgium) and PureSperm gradient from NidaCon International AB (Gothenburg, Sweden). 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] was obtained from Molecular Probes Inc., (Eugene, OR, USA). The CaspACETM FITC-VAD-FMK In Situ Marker obtained from Promega (Madison, WI, USA) was used for analysis of the global caspase-activation status. The caspase-3 intracellular activity assay kit I (Phi-Phi Lux G1D2) was purchased from Calbiochem (EMD Biosciences Inc., Merck KGaA, Darmstadt, Germany). The TUNEL kit was from Promega. Antibodies against caspase-3 (clone 19) were from Becton Dickinson Biosciences (Le Pont de Claix, France) and the secondary antibodies (anti mouse IgG peroxidase) from Bio-Rad, (Marnes la Coquette, France). The ELISA kit Quantikine detecting human active caspase-3 was obtained from R&D Systems Europe Ltd (Abingdon, UK). All other reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA).
Collection of semen samples
We studied male subjects who underwent seminal fluid evaluation at the Laboratory of Reproductive Biology (CHRU, Lille). All subjects were the partners of women who had failed to conceive after 2 years of unprotected intercourse. Patient information remained confidential and within the institution. This study was conducted according to guidelines established for research on human subjects (Ethical committee, CHRU Lille). The samples were collected by masturbation into sterile plastic jars, after 3–5 days of sexual abstinence. Within 1 h after sample collection, a routine semen analysis was performed on samples from 105 subjects using light microscope to determine sperm quality. According to the World Health Organization criteria (1999), normal sperm parameters were defined as ‘a+b’ type motility or progressive motility 
50% and ‘a’ type motility or forward motility 25%; sperm cell concentration 20 x 106 cells/ml; and sperm cells with altered morphology <70%. Based on these criteria, semen profiles were classified into normal (n = 33) or abnormal (n = 72) sperm parameters.
Preparation of semen samples
To isolate spermatozoa, an aliquot of semen was purified using a three-step discontinuous Pure Sperm gradient (90–70–50%) diluted in Ferticult medium. After centrifugation at 400 g for 20 min, purified population of highly motile spermatozoa (from the 90% layer) were recovered, washed in Ferticult medium, and resuspended in 1 ml of the same medium. Prepared spermatozoa were counted and the percentage of forward motile spermatozoa was calculated. Prepared sperm was used for IVF and aliquots taken for experiments. Within 2 h both neat semen samples and purified motile spermatozoa were either subjected to flow cytometry or prepared for caspase-3 determination.
Western blot for caspase-3
Sperm cells (10 x 106) were washed twice in phosphate-buffer saline (PBS) and resuspended in RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 µg/ml aprotinin, leupeptin and 5 mM phenylmethylsulfonyl fluoride (PMSF). Cells were kept at 4°C under constant agitation for 1 h. Lysates were briefly sonicated then centrifugated at 14 000 g and 75 µg of supernatant was loaded on 12.5% polyacrylamide gels, electrophoresed and transfered onto nitrocellulose filter. Caspase-3 was detected using a monoclonal mouse anti-caspase-3 antibody (1:1000) which recognizes both the 32 kDa unprocessed pro-caspase-3 and the cleaved products of the active caspase-3.
Cytofluorometric assessment of activated caspases
FITC-VAD-fmk, a cell-permeant fluorochrome derivative of caspase inhibitor Val-Ala-DL-Asp-fluoromethylketone, was used to detect activated caspases in sperm cells by flow cytometry. Cells were washed in PBS and resuspended in 1 ml of PBS at the concentration of 1 x 10exp6 cells/ml. Then, FITC-VAD-fmk was added at the final concentration of 5 µM and cells were incubated for 20 min at room temperature (RT) in the dark. After washing twice in PBS, cells were then fixed in 0.5% paraformaldehyde for 20 min at RT in the dark. Finally, prior to flow cytometry analysis the cells were washed once in PBS. Fluorescence was measured at 530 nm (excitation 488 nm) in the FL1 channel.
To detect caspase-3 activity we used Phi-Phi Lux G1D2 and followed the original procedure with minor modifications. Briefly, 1 x 106 cells were washed in PBS and 50 µl of 10 µM Phi-Phi Lux G1D2 substrate solution was mixed with the cell pellets. After 1 h of incubation at 37°C in a 5% CO2 incubator, cells were washed once in 1 ml of ice-cold PBS buffer then once in 1 ml of ice-cold flow cytometry dilution buffer provided with the kit. Finally, pellets were resuspended in 1 ml of ice-cold flow cytometry dilution buffer and samples were examined within 60 min after incubation at 37°C. Fluorescence was measured at 530 nm (excitation 488 nm) in the FL1 channel.
Detection of active caspase-3 by ELISA Quantikine kit
Sperm cells (10 x 106) were washed twice in phosphate-buffer saline (PBS) and lysates were prepared to detect active caspase-3 following the original protocol provided by the manufacturer (R&D Systems Europe).
Fluorescence microscopy
Immediatly after FITC-VAD-fmk staining procedure (see above), counterstaining of nuclei was performed with 1 µg/ml Hoescht 33342 for 10 min in the dark. Then, cells were washed once in PBS and resuspended in Vectashield H-100 mounting medium (Vector Laboratories, Burlingame, CA), coverslipped and analysed with a Zeiss Axiophot 2 epifluorescence microscope (Carl Zeiss, Le Pecq, France). Images were captured using Quips Smart Capture software (Vysis, CA, USA).
Analysis of nuclear apoptosis by TUNEL and determination of mitochondrial membrane potential
Nuclear apoptosis was detected with the TUNEL kit (Promega) used according to the manufacturer’s protocol with minor modifications as previously described (C.Marchetti et al., 2002). Mitochondrial membrane potential (m) was measured using 3,3'-dihexyloxacarbocyanine iodie (DiOC6(3)) (Molecular Probes Inc., Eugene, OR) as previously described ((C.Marchetti et al., 2002).
In all cytofluorometric experiments, debris was gated out based on light scatter measurements. For each analysis, 10 000 cells were examined. All experiments were performed on a Coulter XL cytofluorometer (Coulter Corp., Hialeah, FL).
Statistical analysis
Data are presented as mean values ± SEM. Results were analysed using GraphPad Prism version 3.00 (GraphPad Software, San Diego CA, USA). For comparison of percentage of F-VAD positive cells in neat semen and prepared spermatozoa from the same ejaculate, a Wilcoxon matched rank test was employed. For comparison of two groups (normal and abnormal sperm parameters), a two-tailed, Mann–Whitney U-test was performed. The Pearson rank correlation test was used to calculate the correlation coefficient between TUNEL or mitochondrial membrane potential and F-VAD positive cells. The Spearman’s rank correlation test was employed to evaluate the relationship between semen analysis parameters and cytofluorometric results. Statistical significance was set at P < 0.05.
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FITC-VAD-fmk labeling of semen samples
Figure 1A shows fluorescence intensity profile of a semen sample labeled with FITC-VAD-fmk measured by flow cytometry. Sperm cell fluorescence was heterogenous. Whereas the major subpopulation incorporated only insignificant amount of dye with a cytofluorometric profile almost similar to unlabeled cells (Figure 1A, dotted lines), a subpopulation with a distinctly increased fluorescence (called F-VAD+ cells) was present in the semen samples. Incubation of semen sample with FITC-VAD-fmk followed by immediate examination under fluorescence microscopy revealed green staining of spermatozoa (Figure 1B). The F-VAD+ spermatozoa were counterstained with the DNA marker Hoechst 33342. The green FITC-VAD-fmk fluorescence had cytoplasmic localization, and overlapping with the blue nuclear fluorescence was not observed (Figure 1B). Moreover, FITC-VAD-fmk fluorescence was predominant in the midpiece region of morphologically normal sperm cells (Figure 1B, upper panel). An intense green fluorescence was also present in spermatozoa with abnormal morphological midpiece (Figure 1B, lower panel).
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FITC-VAD-fmk staining and caspases determination
The FITC-VAD-fmk is a fluorescent cell-permeable peptide expected to bind irreversibly to the most activated caspases, thus allowing their detection in situ. In order to determine the capacity of FITC-VAD-fmk to stain activated caspases in semen, we performed control experiments comparing the staining with FITC-VAD-fmk with other techniques of detecting active caspases (Figure 2). Semen samples (neat semen or motile spermatozoa purified by gradient) containing a low or high percentage of F-VAD+ cells were then used to prepare lysates for caspase-3 immunoblotting (Figure 2A). The inactive proform of caspase-3 was detected in all sperm lysates as an
32 kDa band, but only samples containing a high percentage of F-VAD + cells exhibited the cleaved products of the activated caspase-3 (17 and 21 kDa) (Figure 2A, arrows).
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Also, we compared the percentage of F-VAD+cells with the proteolytically activated caspase-3 determined by ELISA (Figure 2B). Semen samples (neat semen or motile spermatozoa purified by gradient) were used to prepare lysates for caspase-3 assay and 10 000 cells were analysed by flow cytometry after FITC-VAD-fmk staining. In semen samples, the percentage of F-VAD+ cells determined by flow cytometry correlated significantly with the quantity of active caspase-3 measured in the lysates. Finally, a comparison was done between the percentage of F-VAD+ cells and the percentage of cells with activated caspase-3 determined by flow cytometry using the profluorescent caspase-3 substrate, Phi-Phi lux (Figure 2C). Phi-Phi lux determines caspase-3 activities based on a different basic principle than F-VAD. Whereas F-VAD is a fluorochrome-labeled inhibitor of caspases, Phi-Phi lux contains the caspase-3 peptide substrate covalently coupled to fluorophores. When the peptide is intact (absence of active caspase-3), the fluorophores form a non fluorescent ground-state dimer. Peptide cleavage by caspase-3 abolishes the dye–dye interaction, resulting in an increase in fluorescence. In sperm samples, we found a high significant correlation between the percentage of F-VAD+cells and the frequency of cells with active caspase-3.
Overall, these results suggest that FITC-VAD-fmk detected sperm cells containing activated caspases.
Correlation between F-VAD+ cells and cell death markers in semen
Estimated by cytofluorometric analysis, the percentage of F-VAD+ sperm cells was compared with either the percentage of sperm cells with DNA strand breaks (determined by the TUNEL method) indicative of nuclear apoptosis (Figure 3A) or with the percentage of cells with high m [DiOC6(3) staining], identifying viable cells (Figure 3B). A statistically significant positive correlation was found between the percentage of F-VAD+ cells and the rate of apoptosis as defined by DNA fragmentation (Figure 3A). A negative correlation between the percentage of F-VAD+ cells and the percentage of viable cells with high m (Figure 3B) was also identified. We identified a statistically significant correlation between the TUNEL method and DiOC6(3) staining (correlation between the % of TUNEL positive and the % of DiOC6(3) high cells: r = –0.33 Pearson correlation test; P = 0.01; n = 62)
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These results suggest that FITC-VAD-fmk preferentially labeled sperm cells with reduced
m and which were undergoing nuclear apoptosis.
Correlation between F-VAD+ cells and sperm characteristics in semen before and after gradient separation
In sperm samples defined as abnormal by the World Health Organization (1999) criteria, the percentage of F-VAD+ cells was significantly higher than in normal sperm samples (Figure 4A; mean 46 ± 2.5% versus mean 28 ± 1.9%; *P < 0.0001). Regarding the relationship between the percentage of F-VAD+ cells and standard semen parameters, a significant negative correlation was found with the sperm concentration (Figure 4B) and with the progressive motility (Figure 4C) assessed by light microscopy.
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We compared the percentage of F-VAD+ cells in neat semen with the percentage determined in the motile sperm prepared by PureSperm gradient from the same ejaculates. In prepared sperm, the proportion of F-VAD+ cells was significantly lower than in neat semen (Figure 4D).
We also determined the percentage of F-VAD+ cells in the motile sperm prepared by Pure Sperm gradient from normal and abnormal semen (Figure 4E). The prepared sperm from normal semen had a lower percentage of F-VAD+ cells than the prepared sperm from abnormal semen (Figure 4E; mean 9 ± 1.4% versus mean 21 ± 3.4%; *P = 0.03). Furthermore, the detection of cells stained with FITC-VAD-fmk correlated negatively with the forward motility of prepared sperm (Figure 4F).
Correlation between F-VAD+ cells and fertilization rate
In both neat semen and prepared sperm, the percentage of F-VAD+ cells correlated negatively with the fertilization rate after IVF (Table I).
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Discussion |
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Apoptosis represents the physiological mode of sperm death during spermatogenesis and some forms of male infertility have been associated with an abortive programme of germ cell death (Sakkas et al., 1999
). In most experimental models, caspases are activated in response to death-inducing stimuli and therefore caspase activation serves as a marker of irreversible commitment to cell death. Although the biochemical pathways of apoptosis are well established in somatic cells, little is known about the molecules involved in apoptosis of germinal cells. In mouse sperm, the apoptotic programme seems mainly independent of the family of executioner proteins called caspases (Weil et al., 1998). It is only recently that several groups detected the presence of caspase-3 in human sperm (Bohring et al., 2001; Kim et al., 2001; Yazawa et al., 2001; Weng et al., 2002). Interestingly, caspase-3 activity was also found in ejaculated human sperm suggesting the existence of a caspase-dependent apoptosis in human semen (Weng et al., 2002). To assess caspase activities in intact sperm cells, we utilized flow cytometry analysis of cells stained with the cell-permeable inhibitor peptide FITC-VAD-fluoromethylketone. It is a fluorescein isothyocyanate conjugate of the irreversible pan-caspase inhibitor z-VAD-fluoromethylketone that binds to the active center of most of the caspases. The replacement of the N-benzyloxycarbonyl (z-group) with the FITC label leads to the production of a fluorochrome-labeled inhibitor of caspases allowing in situ labeling of activated caspases in living cells (for review, see Grabarek and Darzynkiewicz, 2002). FITC-VAD-fmk is considered as a specific and convenient marker of apoptosis associated with caspase activation (Pozarowski et al., 2003). FITC-VAD-fmk was previously used to detect apoptosis in various cells including tumor cells (Yang et al., 2002), dendritic cells (Yang et al., 2002), cerebellar granule neurons (Vaudry et al., 2002), pancreatic cells (Embury et al., 2001) and even cells of the tobacco plant (Elbaz et al., 2002). Now, new fluorochrome-labeled inhibitors of caspases with the capacities to detect specific caspases have been developped (Smolewski et al., 2001; Grabarek and Darzynkiewicz, 2002). Here, we provide evidence that FITC-VAD-fmk is also able to stain sperm cells in situ. Thus, we noticed that the subcellular localization of the green FITC-VAD-fmk fluorescence was exclusively cytoplasmic and mainly in the midpiece region. This distribution is compatible with known intracellular localization of active caspases in sperm obtained previously by different methods (Weng et al., 2002; Paasch et al., 2003). We also detected active caspase-3 by western blot only in sperm samples with the highest percentage of F-VAD+ cells. Finally, we found a positive correlation between the percentage of F-VAD+ cells and active caspase-3 determined by ELISA or with cells stained with the profluorescent caspase-3 substrate. These results suggest that the FITC-VAD-fmk inhibitors penetrate into the sperm cells and are expected to stain cells with activated caspases. Because the binding of FITC-VAD-fmk to activated caspases may involve additionnal mechanisms despite the affinity labeling of the active site of the enzymes (Pozarowski et al., 2003), the specificity of FITC-VAD-fmk staining should be interpreted with caution. Thus, we cannot exclude the possibility that the high fluorescence also results from the increased permeability of the plasma membrane associated with cell death. Moreover, we observed a non specific background of fluorescence in all sperm cells if washing steps after staining with FITC-VAD-fmk (data not shown) were ommitted. Nevertheless, the cell-permeable FITC-VAD-fmk appears to be better retained in dead cells than in living sperm cells.
The utilized flow cytometry analysis of the fluorochrome-labeled inhibitor of caspases not only confirms the presence of activated caspases in sperm cells but also has the advantage of being an easily applied test for evaluation of sperm samples from infertile patients. The correlation found between the frequency of F-VAD-positive cells and the dead associated changes (Figure 3) suggests that fluorochrome-labeled inhibitor of caspases may be a good marker of apoptosis in sperm cells. In most of the sperm samples, the percentage of F-VAD+ cells was higher than the frequency of TUNEL positive cells (Figure 3A), indicating that most likely some cells are F-VAD positive but TUNEL negative. This result is compatible with the fact that, in most experimental systems, caspase activation precludes nuclear degradation. We are now developing a flow cytometry analysis of the double-stained sperm cells that would detect and correlate activated caspases and nuclear cell death within the same cell. In this study, we decided to compare the rate of activated caspases with the conventional sperm analysis used during sperm preparation for IVF. The FITC-VAD-fmk staining was able to discriminate between patients with normal and abnormal sperm parameters not only in neat semen (Figure 4A) but also after gradient preparation (Figure 4E). This analysis indicates that there is a significant negative correlation between the proportion of F-VAD+ cells and both concentration (Figure 4B) and progressive motility (Figure 4C) of sperm in the native samples. As expected from sperm prepared by PureSperm gradient (Marchetti et al., 2002), the motile fraction of sperm contained fewer cells stained with FITC-VAD-fmk. In prepared sperm, however, a negative correlation with forward motility still existed. Thus, the cytofluorometric method utilizing fluorochrome-labeled inhibitor of caspase assesses the sperm parameters with high sensitivity. These results are in agreement with western blot testing activated caspase-3 in lysates from infertile patients vs fertile donors (Weng et al., 2002). However, we and others (Paasch et al., 2003) noticed a high proportion of F-VAD+ cells in samples from patients with normal sperm parameters (28% in neat semen and 9% in prepared sperm). Considering the low proportion of cells with active caspase-3 found by others (Weil et al., 1998; Weng et al., 2002), our results are surprising. One explanation could be that the execution phase of sperm apoptosis involved the activation of numerous caspases (other than caspase-3) which are detected by FITC-VAD-fmk staining and not by the antibody specific for the active caspase-3. The possible involvement of caspases other than caspase-3 in human sperm apoptosis requires further analysis. We could, however, speculate that the initiator caspases (as caspases-8 and -10), which are activated by ligated death receptors, are involved in sperm apoptosis since high levels of the death receptor Fas/CD95/APO-1 were found in spermatozoa from infertile men (Sakkas et al., 1999). Importantly, we found that FITC-VAD-fmk staining was able to predict successful IVF providing additional evidence supporting the importance of the evaluation of cell death markers to test male infertility.
In conclusion, we observed that staining of sperm cells with FITC-VAD-fmk, a fluorochrome reacting with various activated caspases, provides a valuable test assessing sperm apoptosis and may be effective in evaluation of sperm prepared for IVF.
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We thank Dr Renata R. Polakowska (INSERM U 459) for the critical reading of the manuscript. We are indebted to Nathalie Jouy (IFR 114) for technical help. This work was supported by grants from CHRU Lille, IFR114-IMPRT, INSERM, Faculté de Médecine, Université de Lille II. M.-A. Gallego received a fellowship from CHRU Lille.
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Submitted on October 21, 2003; accepted on November 22, 2003.
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