1 INSERM U459, Faculté de Médecine 1 and 2 Laboratoire Histologie-Biologie de la Reproduction, Hôpital Jeanne de Flandre, Lille Cedex, France
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Abstract |
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Key words: cell death/DNA fragmentation/infertility/mitochondria/sperm
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Introduction |
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Recently, growing attention has been paid to the studies on determination of cell death pathways. Nuclear morphology and presence of chromosomal DNA strand breaks, easily detectable by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method, are typical nuclear changes occurring during the final degradation phase of cell death. In numerous experimental models, the advanced DNA fragmentation is preceded by severe perturbations in mitochondrial function detected as a decrease in mitochondrial membrane potential (m). This reduction in
m is accompanied by the production of reactive oxygen species (ROS) contributing to cell death (Kroemer et al., 1997
).
The quality of sperm samples is one of the factors determining successful IVF. Currently sperm quality is evaluated by conventional semen analysis, determining sperm concentration, motility and morphology using light microscopy. These parameters comply with the World Health Organization (World Health Organization, 1999) criteria recommended for sperm classification. Although the conventional analysis of semen gives considerable information, new methods are still needed to make the evaluation of sperm fertilizing capacity in vitro easier and more reliable.
Numerous reports indicate that human sperm from infertile patients contain fragmented DNA and are less viable than that of fertile men (Garner et al., 1986; Host et al., 1999
). Mitochondrial dysfunction (Troiano et al., 1998
) and increased ROS production have also been reported (Sharma and Agarwal, 1996
).
In the present paper, we performed concomitantly four well-defined cytofluorometric assays to assess m, ROS generation, DNA fragmentation and sperm cell viability in semen samples from infertile patients enrolled in an IVF programme. The aim of this study was to establish correlation, if any, between these death-associated changes and the quality of sperm evaluated using conventional light microscopy analysis during the preparation of sperm for IVF.
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Materials and methods |
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Collection of semen samples
We studied 111 subjects who underwent seminal fluid evaluation at the Biology of Reproduction Laboratory (CHU, Lille, France). 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, CHU Lille). The samples were collected by masturbation into sterile plastic jars, after 35 days of sexual abstinence. Within 1 h of collection, a routine semen analysis was performed using a light microscope to determine sperm quality. Semen profiles were classified into normal (n = 39) or abnormal sperm parameters (n = 72). According to the World Health Organization criteria (World Health Organization, 1999), normal sperm parameters were defined as (a + b) type motility or progressive motility
50% and `a' type motility (forward motility)
25%; sperm cell concentration
20x106 cells/ml; and sperm cells with altered morphology
70%.
All samples fulfilled the criteria to be selected for IVF rather than ICSI.
Preparation of semen samples
To isolate sperm, an aliquot of semen was purified using a three-step discontinuous PureSperm® gradient (907050%) diluted in Ferticult® medium containing 0.4% HSA. After centrifugation at 400 g for 20 min, purified population of motile sperm (from the 90% layer) were recovered, washed in Ferticult® medium, and resuspended in 1 ml of the same medium. Prepared sperm were counted and the percentage of forward motile sperm (`a' type motility, WHO, 1999) was estimated. Prepared sperm were then used for IVF and an aliquot taken for cytofluormetric analyses. Both neat semen and purified sperm from the same semen samples were subjected to flow cytometry within 2 h.
Assessment of mitochondrial membrane potential
Mitochondrial membrane potentials (m) were measured by means of DiOC6(3) staining as previously described (Marchetti et al., 1996a
,b
). Briefly, 5x105 cells were incubated for 15 min at 37°C in 500 µl of 40 nmol/l DiOC6(3), immediately followed by analysis on a cytofluorometer with excitation and emission settings of 488 nm and 525 nm (FL1 channel) respectively. Cells were kept on ice before analysis. Control experiments were performed in the presence of 50 µmol/l mClCCP (15 min, 37°C), an uncoupling agent that abolishes the
m.
Determination of reactive oxygen species by flow cytometry
It is possible to determine the production of ROS using HE, a substance that is oxidized by superoxide anion to become ethidium bromide, emitting red fluorescence (Rothe and Valet, 1990). Cells were exposed for 15 min at 37°C to 2 µmol/l HE before cytofluorometric analysis (excitation: 488nm; emission: 625nm in the FL3 channel). Control cells were incubated in the presence of 1 mmol/l menadione, a superoxide anion generator, for 1 h at 37°C, washed twice and labelled with HE.
Cytofluorometric analysis of nuclear apoptosis by TUNEL assay and determination of cell viability
Nuclear apoptosis was assessed by TUNEL assay. This assay was performed with the TUNEL kit according to the manufacturer protocol with minor modifications. Briefly, 1x106 washed sperm cells were fixed with 4% paraformaldehyde (Sigma Chemical Co.) for 30 min at room temperature. Cells were washed twice in phosphate buffered saline (PBS) followed by permeabilization with 0.2 % Triton X-100 for 2 min on ice. After washing with equilibration buffer, cells were incubated with 50 µl of TUNEL mix (dUTP-FITC and TdT enzyme in equilibration buffer) for 1 h at 37°C. Positive control was obtained by incubating one sample with 10 mg/ml DNAse I for 10 min at room temperature. To the negative control, TdT enzyme was not added. Cells were washed twice in PBS before analysis.
To determine cell viability, cells were incubated for 10 min in propidium iodide (PI) (10 µg/ml in PBS) at 37°C and analysed by cytofluorometry as described previously (Marchetti et al., 1999a). For all cytofluorometric experiments, forward and side scatters were gated on the major population of normal-size cells and a minimum of 50 000 cells was analysed.
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 positive cells in neat semen and prepared sperm 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 cytofluorometric analysis. The Spearman rank correlation test was employed to evaluate the relationship between semen analysis parameters and cytofluorometric examination. Statistical significance was set at P < 0.05.
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Results |
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DNA strand breaks, indicative of advanced apoptosis, were studied by TUNEL staining which was negative for a major subpopulation of sperm cells (Figure 1C). Positive controls were performed using DNase I.
Finally sperm viability was assessed as cells excluding propidium iodide staining (PI- cells). Results in Figure 1D show that sperm cell population is heterogenous. It contains viable cells (PI negative) and dead cells (PI positive).
We identified significant relationships between all four cytofluorometric methods detecting death-associated changes in semen samples (Table II).
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Discussion |
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The correlation found among the methods assessing sperm cell damages (Table II) indicated that mitochondrial dysfunction, ROS generation, DNA fragmentation and loss of viability are likely to represent the interrelated aspects of the overall dead status of sperm. However, these assays assess the sperm quality with a different sensitivity. Indeed, we showed that determination of the mitochondrial membrane potential represents the most sensitive test by which to evaluate sperm quality. To determine mitochondrial membrane potential (
m), several fluorochromes have been previously used for sperm samples evaluation including Rhodamine 123, J-C 1 or MitoSensor (Troiano et al., 1998
; Donnelly et al., 2000
; Gravance et al., 2000
). All of these fluorochromes stained mitochondria of live cells and validated usefulness of the cytofluorometric analysis. Rhodamine 123 is the most commonly used fluorochrome in sperm sample evaluation, however it appears to assess
m incorrectly (Troiano et al., 1998
). In this study, we used the carbocyanine dye DiOC6(3), which is cell permeable and stains mitochondria at low concentration (Zamzami et al., 2000
). The mitochondrial uptake of DiOC6(3) is dependent on the mitochondrial membrane potential. Compared to Rhodamine 123, the DiOC6(3) offers the important advantage of not causing major quenching effects (Zamzami et al., 2000
). Consequently, DiOC6(3) fluorochrome is one of the most commonly used to monitor mitochondrial changes preceding nuclear apoptosis in other cell types, including various tumour cells, neurones, hepatocytes, thymocytes (Kroemer et al., 1997
) as well as lymphocytes from patients infected by HIV-1 (Macho et al., 1995
). Our results suggest that DiOC6(3) is also a valuable probe to measure
m in sperm samples. The analysis of sperm cells after DiOC6(3) staining indicated that there is a significant correlation between the proportion of
m high cells and the motility of sperm in both a native sample and after PureSperm® gradient separation. These results confirmed those obtained after JC-1 staining of sperm from infertile men (Troiano et al., 1998
; Donnelly et al., 2000
) and rats (Gravance et al., 2001
) and suggested that sperm motility is associated with the functional status of mitochondria. Indeed, mitochondrial alterations can result in the reduction of sperm motility since this motility is an ATP-dependent process, thus dependent on the functional mitochondria producing energy powering the flagellar motion.
The m reduction is a general feature of cell death. Decrease in the
m defines an early stage of apoptosis preceding other manifestations of this process such as DNA fragmentation, ROS production and the late increase in membrane permeability (Kroemer et al., 1997
). The significance of the
m reduction encountered in infertile sperm remains unknown but assumption that it manifests an early stage of cell death could explain why detection of mitochondrial changes represents the most sensitive test in our study. Several arguments indicating that mitochondria may be involved in the cell death pathways in germ cells support our interpretation. For example, it was found that partial oxygen pressure induced mitochondrial permeability transition and apoptosis in human testis, and that mitochondria from apoptotic germ cells were swollen (Erkkila et al., 1999
). This and other findings strongly imply association of male infertility with mitochondrial alterations observed during apoptosis. However, mitochondrial alterations have been associated with male infertility irrespective of apoptosis (Bourgeron, 2000
).
As expected from sperm isolated by classical Percoll gradient (Donnelly et al., 2000), the sperm fraction obtained using PureSperm® gradient contained more sperm characterized by the high
m, less ROS generation, less DNA fragmentation and they were more viable than sperm initially present in the neat semen. PureSperm® gradient is a Percoll-based density gradient in isotonic salt solution used to enhance semen fertilizing ability applied in medically assisted reproductive procedures. This gradient, containing silanized silica particles, efficiently separates sperm subpopulations and allows for a good recovery of sperm with a high motility and quality characteristics (Yamamoto et al., 1997
; Ding et al., 2000
). Another method, a swim-up sperm preparation requiring more repeated manipulations, may be responsible for the high content of ROS (Agarwal et al., 1994
; Aitken, 1994
) seen in deficient sperm not detected in our study. Also, we showed that sperm separated by PureSperm® gradient had only few signs of DNA fragmentation. In contrast, using the swim-up method one could not avoid getting sperm with DNA strand breaks (Host et al., 2000a
). These findings are particularly important for the assisted reproductive technologies such as IVF and ICSI, requiring sperm with enhanced DNA integrity in order to avoid the inadvertent use of DNA-damaged sperm. Since preparation with PureSperm® gradient removed most of the damaged cells, the forward motility of prepared sperm lacked correlation with ROS production, DNA fragmentation or loss of viability (Figure 6
). Importantly, prepared sperm with high
m always correlated with forward motility, thus confirming the strong link between the functional status of mitochondria and sperm cell quality.
Additional evidence supporting the evaluation of cell death markers to test male infertility is provided by the negative correlation between DNA fragmentation in neat semen and IVF fertilization rate (Table III; Host et al., 2000b
). In contrast to others (Sun et al., 1997
) we did not detect any negative correlation between DNA fragmentation and the fertilization rate in prepared sperm, perhaps because they used the swim-up preparation. Our study determined that fertilization rate significantly correlated with the proportion of sperm with a high
m both in neat semen and after separation. Thus, the evaluation of
m appears to be the most efficient test of human sperm assuring successful IVF. This is not surprising, because values of
m link mitochondrial function to motility of sperm. Indeed, concentration of motile sperm was one of the most significant parameters in predicting the chance of natural conception (Larsen et al., 2000
) and was significantly and positively correlated with the fertilizing ability of sperm in vitro (Zollner et al., 1999
).
To conclude, we observed that detection of m changes is the most efficient test to evaluate sperm quality during the preparation steps for IVF. The
m determine functional mitochondria that have been related mainly to sperm motility. Our data indicate the
m value could represent an important determinant of high quality sperm in neat semen and prepared sperm of infertile men, predicting successful IVF.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 19, 2001; resubmitted on October 25, 2001; accepted on December 17, 2001.