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 dHistologie, 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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Key words: cell death/flow cytometry/IVF/spermatozoa
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Introduction |
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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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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 35 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 (907050%) 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 manufacturers 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, MannWhitney 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 Spearmans 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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Results |
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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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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 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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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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Acknowledgments |
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References |
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Submitted on October 21, 2003; accepted on November 22, 2003.
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