1 INSERM U459, 3 Laboratoire d'Histologie, 4 IFR 114-IMPRT, Faculté de Médecine 1, Place Verdun, 59045 Lille Cedex and 2 Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandre, 59037 Lille Cedex, France
5 To whom correspondence should be addressed. Email: philippe.marchetti{at}lille.inserm.fr
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Abstract |
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Key words: flow cytometry/IVF/mitochondria/spermatozoa
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Introduction |
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Analysing mitochondrial function may offer a means of assessing the motility of sperm. This can be achieved by determining the inner mitochondrial membrane potential (m) in sperm cells. The
m is a sensitive indicator for the energetic state of the mitochondria and the cell, and can be used to assess the activity of the mitochondrial respiratory chain, electrogenic transport systems and the activation of the mitochondrial permeability transition (for a review see Ly et al., 2003
). Thus, determination of
m is widely used for characterization of cellular metabolism, viability and apoptosis in various cellular models. In human, a correlation exists between poor sperm mitochondrial function detected by reduction of
m, and diminished motility and reduced fertility (Troiano et al., 1998
; Donnelly et al., 2000
; Marchetti et al., 2002
; Piasecka and Kawiak, 2003
; Wang et al., 2003
).
Several fluorimetric methods using cationic lipophilic dyes have been utilized to measure the m. The cationic lipophilic dyes accumulate in mitochondria depending on
m, and the fluorescence of the accumulated fluorochromes corresponds to this potential. During the past decades, rhodamine 123 (Rh123) has been widely used as a fluorescent probe to assess
m. However, contradictory data indicated that this probe was not fully satisfactory to measure
m because of the existence of several energy-independent Rh123-binding sites (Lopez-Mediavilla et al., 1989
).
To overcome these drawbacks, several potential sensitive dyes were developed including rosamines, rhodamine and carbocyanine derivatives: (i) Chloromethyl-X-rosamine (CMX-Ros) dye, that contains a mildly thiol-reactive chloromethyl moiety, is much more photostable than Rh123 and constitutes a valuable dye to analyse mitochondrial morphology and function (Poot et al., 1996); (ii) The rhodamine derivative tetramethylrhodamine ethyl ester (TMRE) which has reduced hydrophobic character, also exhibits less potential-independent binding to cells than other rhodamines and has been described as one of the best fluorescent dyes for
m measurements in living cells (Loew et al., 1993
). However, like other rhodamines, TMRE used at high concentration induces fluorescence quenching so that an increase in mitochondrial fluorescence corresponds to depolarization (O'Reilly et al., 2003
); (iii) The carbocyanine derivative 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] offers the important advantage of not causing quenching effects (Metivier et al., 1998
) but DiOC6(3) uptake depends on both mitochondrial membrane and plasma membrane potentials (Salvioli et al., 1997
); (iv) The carbocyanine fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) has been proposed to evaluate changes in
m accurately (Salvioli et al., 1997
).
Thus, interpreting and evaluating changes in m may be somewhat confusing because there are substantial variations between these dyes depending on various susceptibilities to the surrounding environment. Therefore, comparing results obtained with different potentiometric dyes is useful to select the most accurate probe for a particular application.
In recent years, many investigators have used lipophilic cationic fluorochromes including Rh123 (Troiano et al., 1998), JC-1 (Donnelly et al,. 2000
; Piasecka and Kawiak, 2003
) and DiOC6(3) (Marchetti et al., 2002
; Wang et al., 2003
) for
m determinations in sperm samples. A comparison of the ability of these potentiometric dyes to evaluate
m in sperm samples has not been made. To our knowledge, fluorescence from CMX-Ros or TMRE has never been analysed in human spermatozoa. Flow cytometry, when used in conjunction with
m-dependent fluorochromes, could be an ideal method to study the mitochondrial potential in sperm samples. Indeed, flow cytometry provides a rapid, accurate and reliable estimation of the
m in a large number of cells and is of considerable relevance for laboratory practice.
Therefore, we have studied sperm samples from infertile patients enrolled in an IVF programme in flow cytometry after concomitantly staining with CMX-Ros, DiOC6(3), TMRE and JC-1.
We have compared the results obtained with these dyes and have established the correlation with the quality of sperm evaluated by conventional light microscopic analysis in spermatozoa prepared for IVF. This approach allowed us to discuss the advantages and limitations of m-dependent cytofluorometric assays for assessment of sperm quality in the reproductive biology laboratory.
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Materials and methods |
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Collection of semen samples
We studied 28 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.
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 300 g for 20 min, purified populations 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 cytofluorometric experiments.
Conservation of spermatozoa before m labelling
Typically, purified spermatozoa were subjected to flow cytometry within 1 h. In some experiments, we set up conditions of spermatozoa conservation that allow for the retention of the m. For this experiment, samples were stored at 4°C, room temperature or 37°C in medium until the times (0, 30, 60 and 120 min) that the staining with the potentiometric dyes were performed (see below).
Cytofluorometric assessment of mitochondrial membrane potential
Stock solutions of JC-1 (2.5 mmol/l), TMRE (1 mmol/l) and CMX-Ros (1 mmol/l) were made in dimethylsulphoxide (DMSO). DiOC6(3) (4 mmol/l) was dissolved in ethanol. Stock solutions were stored in small aliquots at 20°C, and subsequent working solutions [dilutions 1:250 for JC-1, 1:40 for TMRE; 1:200 for CMX-Ros; 1:2000 for DiOC6(3)] were made in experimental medium immediately before use. A total of 5 x 105 spermatozoa were incubated in the Ferticult medium with the fluorochromes at 37°C, followed by analysis on a cytofluorometer. Except when indicated for Figures 1 and 2, we used optimal conditions of staining defined as 50 nmol/l CMX-Ros for 20 min, 20 nmol/l DiOC6(3) for 20 min, 250 nmol/l TMRE for 20 min, and 1 µmol/l JC-1 for 30 min. All flow cytometry experiments were performed on a Coulter XL cytofluorometer (Coulter Corp., Hialeah, FL). Data were acquired using Expo 32 software (Coulter). The analyser threshold was adjusted on the forward scatter channel to exclude subcellular debris. Forward and side scatters were gated on the major population of normal size cells and a minimum of 10 000 cells was analysed. The fluorescent probes DiOC6(3), JC-1, CMX-Ros and TMRE were excited with the 488 nm argon laser. Signals from the DiOC6(3) fluorescence were collected through the FL1 detector (525±5 nm band pass filter), CMX-Ros through the FL3 detector (620±5n m band pass filter) and TMRE through the FL2 channel (575±5 nm band pass filter). The fluorescence signals of JC-1 monomers and aggregates were detected through the FL1 (525±5 nm band pass filter) and FL2 channels (575±5 nm band pass filter), respectively. Control experiments were performed in the presence of carbamoylcyanide m-chlorophenylhydrazone (ClCCP) or the K+ ionophore valinomycin. ClCCP is a protonophore that uncouples oxidation from phosphorylation by dissipating the chemiosmotic gradient and induces dissipation of m. Spermatozoa were incubated with 0.5 µmol/l ClCCP or 100 nmol/l valinomycin for 30 min at 37°C then stained with the potentiometric dyes as described above.
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Spermatozoa were exposed for 20 min at 37°C to CMX-Ros (50 nmol/l) and YOPRO-1 [200 nmol/l in phosphate-buffered saline (PBS)], to DiOC6(3) (20 nmol/l) and PI (5 µg/ml) or to TMRE (250 nmol/l) and YOPRO-1 (200 nmol/l in PBS). Immediately after staining, spermatozoa were analysed by flow cytometry.
Fluorescence microscopy
Immediately after the m staining procedure, counterstaining of nuclei was performed with Hoescht 33342 1 µg/ml 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 Leica DMLR epifluorescence microscope, using a Leica 63x/1.32 HCX PL APO objective (Leica S.A., Rueil Malmaison, France). Images were captured using Leica software.
Statistical analysis
Results were analysed using GraphPad Prism version 3.00 (GraphPad Software, San Diego CA). The Pearson rank correlation test was used to calculate the correlation coefficient between flow cytometric analyses. The Spearman rank correlation test was employed to evaluate the relationship between semen analysis parameters and cytofluorometric results. Statistical significance was set at P<0.05. To test the reproducibility of the assays, 10 replicates from a single sample were processed and acquired by the same operator for each staining, then typical intra-assay precision tests were performed.
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Results |
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To monitor the m, we have also chosen the carbocyanine dye JC-1 which has been shown to be more reliable than other fluorescent dyes for detecting changes in
m due to its dual emission characteristics (Cossarizza et al., 1996
). Mitochondria with
mhigh concentrate JC-1 into aggregates (red-orange fluorescence in the FL2 channel), while in depolarized mitochondria JC-1 forms mainly monomers (green fluorescence in the FL1 channel). A two-parameter fluorescence display of JC-1-stained spermatozoa reveals that most of the cells emitted relatively high levels of both green and orange-red fluorescence, whereas a subpopulation exhibited a reduced JC-1 aggregation and a decrease in the orange-red fluorescence emission, a finding that indicates a drop in
m (Figure 1D). By flow cytometry, a high correlation has been found previously between mitochondrial membrane potential values in isolated mitochondria and fluorescence ratio (mean red-orange fluorescence intensity/mean green fluorescence intensity corresponding to the FL2/FL1 ratio) (Cossarizza et al,. 1996
). Therefore, in order to determine the population of cells with
mhigh in sperm samples after JC-1 staining, we evaluated both the percentage of cells which concentrate JC-1 into aggregates (high fluorescence of JC-1 red-orange in the upper left quadrant) called
mhigh cells and the values of the fluorescence ratio (JC-1 red-orange/JC-1 green or FL2/FL1 ratio) (Figure 1D).
Optimization of conditions for spermatozoa labelling
Incubation of spermatozoa with different concentrations of fluorochromes revealed that the percentage of cells emitting high fluorescence was influenced by the concentration of dye used (Figure 2A). This study indicated that the minimal dose of dye required to achieve an effective spermatozoa loading was 50 nmol/l of CMX-Ros, 20 nmol/l of DiOC6(3), 250 nmol/l of TMRE and 1 µmol/l of JC-1.
Figure 2B represents the time course for the uptake of dyes used at optimal concentrations. Under these conditions, a 20 min period of incubation was enough for DiOC6(3), CMX-Ros and TMRE to equilibrate into the cells. For JC-1 staining, a 30 min period of incubation was needed and allowed a better separation of the high fluorescence peak of JC-1 red-orange (data not shown). These patterns of cell fluorescence were stable and remained unchanged for at least 120 min of incubation in the medium at 37°C, except for the FL2/FL1 ratio.
In the optimal conditions of staining based on the above results, fluorescence microscopy (Figure 3) was used to verify that each dye accurately measured the correct sperm compartment. The spermatozoa stained with the m-sensitive fluorescence probes in conditions defined above were counterstained with the DNA marker Hoechst 33342. In each case, a high level of fluorescence was associated with the sperm midpiece where mitochondria are located. No other portion of spermatozoa displayed fluorescence that could be detected microscopically, indicating a characteristic mitochondrial uptake of all fluorochromes.
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These results indicate that under our experimental conditions, the four potentiometric fluorochromes reveal a m variation in spermatozoa. However, when we displayed the percentage of ionophore-treated spermatozoa which retain a high
m (grey profiles in Figure 4A and B), the remaining fluorescence was much lower in JC-1-stained spermatozoa than in cells labelled with the other fluorochromes.
We also found a highly significant relationship between all four cytofluorometric methods in samples (Table II), confirming that any potential-sensitive fluorochromes detect m changes of spermatozoa.
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Conservation of spermatozoa before m labelling
Results showed that spermatozoa stored at 4°C undergo a decrease in the percentage of mhigh cells irrespective of the
m-dependent dye used to stain spermatozoa (Figure 5). In any case, prepared spermatozoa stored at room temperature or at 37°C maintained a constant proportion of
mhigh cells at least for the first 60 min.
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Discussion |
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Thus, determining advantages and limitations of different m-sensitive fluorescence probes in spermatozoa is an essential step to develop efficient test(s) to evaluate sperm quality.
We first optimized the conditions of dye labelling. To increase the actual contribution of m to cell fluorescence, it has been suggested to reduce dye concentrations, i.e. the dye/cell ratio. Indeed, lowering the dye/cell ratio reduces the toxicity, the quenching effect and the importance of the
p in cell fluorescence (Rottenberg and Wu, 1998
). Thus, we have chosen the lowest concentration of fluorochromes (and the shortest incubation time) that do not reduce the percentage of
mhigh spermatozoa and that allow us to discriminate unambiguously between spermatozoa with high and low
m. However, even in these optimal conditions, we cannot exclude that
m-independent processes could contribute at least in part to the fluorescence observed.
For an ideal m-sensitive fluorescence probe, specific alterations of the
m should result in decreased uptake of the fluorescence probe in the mitochondrial matrix. To test the capacity of the dyes to detect
m variations, we pre-incubated spermatozoa with uncouplers (Figure 4). We used the protonophore ClCCP and the K+ ionophore valinomycin because it has been demonstrated that ClCCP may change the
p in human spermatozoa (Guzman-Grenfell et al., 2000
). Valinomycin was used at concentrations that modulate the
m without collapsing the
p (Rottenberg and Wu, 1998
). In these experimental conditions, we confirmed that the four classical dyes, DiOC6(3), CMX-Ros, TMRE and JC-1, are sensitive enough to detect changes in
m induced by mitochondrial uncouplers. ClCCP and valinomycin caused a complete modification of JC-1 fluorescence, suggesting that JC-1 had an exclusive distribution to mitochondria. This observation is consistent with the fact that JC-1 is considered as a fluorochrome which can measure
m with great accuracy in intact cells (Salvioli et al., 1997
) including cardiomyocytes (Mathur et al., 2000
) and spermatozoa (Troiano et al., 1998
). In contrast, the uncoupler-induced decrease in CMX-Ros, DiOC6(3) and TMRE fluorescence was much smaller as the peak was in an intermediate position between those of spermatozoa with high and low fluorescence (Figure 4), indicating that CMX-Ros, DiOC6(3) and TMRE staining respond not only to
m changes but also to other non-specific (
m-independent) processes. Thus, JC-1 appears to be the best probe to detect specifically
m changes in spermatozoa.
For an ideal probe suitable for explicit determination of m in clinical samples, the resolution between the
mhigh and
mlow fluorescence peaks should be maximal. As shown here, cytofluorometric profiles of spermatozoa stained with either CMX-Ros, DiOC6(3) or TMRE unambiguously revealed two distinct populations with
mhigh and
mlow. In any case, the determination of the percentage of
mhigh spermatozoa was easily done by setting markers on histograms and using statistical functions of the software. JC-1 differs from rhodamines and other carbocyanines because it produces two fluorescence emission peaks that reflect the existence of two forms of the dye. The JC-1 monomers which emit green fluorescence are predominant at low
m, while the JC-1 aggregates (red-orange fluorescence) are predominant at high
m. Typically, it is described that upon lowering the
m, the JC-1 aggregates dissipate into monomers and lead to a shift from red to green fluorescence. In fact, the intensity of the green fluorescence from the JC-1 monomer form seems to be insensitive to
m changes (Cossarizza et al., 1996
) and was instead used to monitor changes in mitochondrial mass (Mancini et al., 1997
). In spermatozoa, we found that disruption of
m does not lead to a significant increase in green fluorescence even after incubation with valinomycin (Figure 4B). Consequently, we have relied on the red-orange fluorescence emission of JC-1 aggregates to monitor changes in
m. Nevertheless, as shown in Figure 1, the discrimination of spermatozoa with reduced and high
m was rather difficult since the resolution between the two fluorescence peaks of JC-1 aggregates was weak in many samples (see Figure 1D). Thus, it is more subjective to set markers on the JC-1 aggregate histograms. In our study, the use of JC-1 would lead to variations in the determination of the percentage of
m and could explain why the correlation factor among
m-dependent fluorochromes is lower with JC-1 (Table II). One other possibility is to employ JC-1 as a ratiometric probe since a strict correlation has been found between the FL2/FL1 ratio and the
m in isolated mitochondria (Cossarizza et al., 1996
). Consistent with previous results (Gravance et al., 2000
; Mathur et al., 2000
), assessment of JC-1 staining by a ratiometric analysis did not provide better results. This is not surprising because variations in the mitochondrial mass, which can influence the FL2/FL1 ratio independently of changes in
m, have been demonstrated in some cases of asthenospermia (Piasecka and Kawiak, 2003
). Thus, the analysis of the percentage of
mhigh stained with JC-1 seems to be more subjective than after staining with other potential-sensitive dyes.
To develop objective measurements of m in semen samples, it is fundamental to achieve standardized protocols. First, we demonstrated acceptable reproducibility. Secondly, it is important to establish what, if any, is the influence on the
m of storage conditions of spermatozoa before flow cytometric analysis. Indeed,
m-dependent fluorochromes are used on living cells and inadequate storage could seriously alter the
m of spermatozoa. The results of our study indicate that a conservation period at 4°C of spermatozoa in culture medium decreases the percentage of
mhigh spermatozoa irrespective of the fluorochromes used. The recommendations from our study are that spermatozoa should be stored in culture medium at room temperature or 37°C for a maximum period of 60120 min before flow cytometric analysis if they cannot be used immediately.
We compared the evaluation of mhigh by four fluorochromes with respect to their ability to correlate with forward motility. Prepared sperm with high
m correlated with forward motility, thus confirming the strong link between the functional status of mitochondria and sperm cell quality (Marchetti et al., 2002
; Wang et al., 2003
). Whereas JC-1 and DiOC6(3) have been used previously for sperm sample evaluation (Donnelly et al., 2000
; Marchetti et al., 2002
; Piasecka and Kawiak, 2003
; Wang et al., 2003
), it is, to our knowledge, the first report describing CMX-Ros and TMRE as valuable probes to measure
m in sperm samples. Importantly, we found that all
m-dependent fluorochromes were able to predict successful IVF, providing additional evidence supporting the importance of a flow cytometric
m-based test in evaluation of spermatozoa for clinical studies.
Spermatozoa need to possess many attributes including a high motility in order to fertilize an oocyte, and sperm may be infertile for numerous reasons. Therefore, measuring multiple sperm parameters simultaneously on individual spermatozoa should provide a better indication of fertilizating capacity than one single parameter. One advantage of the flow cytometry is the possibility of evaluating, in combination, multiple fluorescent markers associated with individual spermatozoa in a population (Graham, 2001). Thus, flow cytometry should be used to analyse multiple sperm parameters (including the determination of
m and cell viability) to enhance the capacity to estimate the fertilizing potential of semen samples. For this reason, it is important to have several reliable
m-dependent probes emitting in different fluorescence channels, which can be used in combination with other probes evaluating different sperm attributes. In order to evaluate
m and cell viability simultaneously, we developed double staining protocols. We used CMX-Ros, DiOC6(3) and TMRE as potential-sensitive dyes because they produce a single fluorescent emission peak allowing the combination with supravital fluorochromes. In contrast, JC-1 emits two fluorescence peaks (green and orange-red) which preclude simultaneous assessment of cell viability by commonly used supravital probes, because of fluorescence overlap. However, it should be noted that JC-1 has been used recently in combination with the impermeant dye TOTO-3 to investigate cell death (Zuliani et al., 2003
). Nevertheless, the major limitation of this method is the need to use a cytofluorometer equipped with multiple lasers to excite both TOTO-3 and JC-1. Thus, in contrast to JC-1, the fluorochromes CMX-Ros, DiOC6(3) or TMRE permit the development of a simple method in combination with other probes for multiparametric evaluation of sperm quality.
In conclusion, our results indicate that four classical m-dependent fluorochromes provide valuable tests assessing changes in mitochondrial membrane potential of human spermatozoa and may be usable for evaluation of sperm sample quality from infertile patients. These
m changes can be easily detected using cytoflurometric analysis of spermatozoa. Whereas JC-1 detects
m changes in spermatozoa more specifically than other dyes tested, CMX-Ros, DiOC6(3) and TMRE fluorescence is easily analysed and these fluorochromes are particularly suitable for multiparametric staining. The choice of the
m-dependent fluorochromes will depend on their fluorescence characteristics in order to use them in combination with other sperm attribute-dependent fluorescent markers.
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Acknowledgements |
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Submitted on February 13, 2004; resubmitted on May 5, 2004; accepted on June 22, 2004.
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