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Regular Article |
a Departments of Equine Sciences and
b Farm Animal Health of the Graduate School of Animal Health,
c Department of Biochemistry and Cell Biology of the Institute of Biomembranes, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands
ABSTRACT
The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca2+-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO2 on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca2+ ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus
3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10 000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca2+ ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.
calcium, cAMP, fertilization, sperm, sperm capacitation/acrosome reaction, sperm motility and transport
INTRODUCTION
"Capacitation" is a collective term for the changes that a spermatozoon undergoes when it comes in contact with the female reproductive tract. These changes include reorganization of membrane proteins, metabolism of membrane phospholipids, a reduction in membrane cholesterol levels, and hyperactivation [1]. These changes, together with the subsequently induced acrosome reaction (AR), an irreversible exocytotic event, are essential if a sperm is to bind to and penetrate the zona pellucida and thereafter fuse with the oocyte plasma membrane [1]. Capacitation is thus a critical event in the process of fertilization. However, differentiating capacitated from noncapacitated spermatozoa remains an inexact science, despite the almost half-century since Chang [2] and Austin [3] first described the phenomenon of capacitation, and it is frustrating that a straightforward, validated, and easy-to-interpret method for assessing capacitation is still not in common use.
On the other hand, chlortetracycline (CTC) staining has been used to assess the capacitation state of spermatozoa [4–7] and it is currently the assay of choice because it distinguishes three different stages of sperm activation; noncapacitated, capacitated acrosome-intact, and capacitated acrosome-reacted. However, a clear understanding of how CTC interacts with the sperm surface at the molecular level is lacking and, unfortunately, the evaluation of CTC staining is performed on fixed sperm cells.
Merocyanine 540 staining is another technique that may be useful for assessing the capacitation status of spermatozoa. Merocyanine 540 is a hydrophobic dye that has been shown to stain cell membranes more intensely if their lipid components are in a higher state of disorder [8, 9], as is the case with capacitated spermatozoa. In this latter respect, merocyanine 540 has recently been used to monitor alterations in the lipid architecture of the boar sperm plasma membrane during capacitation [10], a process that appears to be due to bicarbonate-induced transbilayer scrambling of phospholipids [11]. One major advantage of merocyanine 540 over CTC is its suitability as a probe for assessing capacitation flow in a cytometric fashion, because the latter technique should allow for more objective analysis of larger numbers of unfixed (and therefore relatively undamaged) sperm samples. Furthermore, with use of a flow cytometer, merocyanine 540 staining can be combined with the membrane-impermeable DNA binding probe, Yo-Pro-1, to allow coincident analysis of membrane lipid status and cell viability, while the AR of living stallion spermatozoa similarly can be assessed with flow cytometry using fluorescein isothiocyanate (FITC)-conjugated peanut (Arachis hypogea) agglutinin (PNA) as a label [12, 13].
Capacitation also involves changes in sperm motility, known as hyperactivation [1], which are believed to aid sperm progression up the oviduct, by enabling spermatozoa to move away from the oviductal epithelium [14] and to provide the motile thrust needed for penetration of the zona pellucida [15].
The primary aim of this study was to establish a flow cytometric technique for assessing capacitation of stallion spermatozoa using a reporter probe with known sperm-binding characteristics rather than CTC, which, however, is empirically accepted but is laborious to use and its working mechanism is scientifically unexplained. To this end, we investigated whether CTC identification of capacitation and the AR in fixed sperm preparations correlated with flow cytometric detection of the same processes in unfixed sperm samples, in which capacitation was indicated by changes in membrane lipid fluidity, as demonstrated by merocyanine 540 staining, and the AR was detected by FITC-PNA staining. In both cases capacitation was induced using a modified Tyrodes medium, as described by Harrison et al. [10], in the presence of 15 mM HCO3-/5% CO2 (Tyr+bic) and the results were compared with control samples incubated in HCO3-/CO2-free Tyrodes medium (Tyr). Furthermore, Ca2+ ionophore was added to aliquots of sperm samples incubated by both Tyr+bic and Tyr in an attempt to induce the AR. For all treatments, the alterations in CTC, merocyanine 540, and FITC-PNA labeling patterns were monitored over time and, in addition, temporal changes in sperm motility and hyperactivation were assessed using a computer-assisted sperm analysis (CASA) system.
MATERIALS AND METHODS
Materials
CTC and Ca2+ ionophore A23187 were purchased from Sigma Chemical Co. (St. Louis, MO). Merocyanine 540, ethidium homodimer (EthD-1), Yo-Pro-1, and propidium iodide (PI) were obtained from Molecular Probes Inc. (Eugene, OR). FITC-PNA was purchased from EY Laboratories Inc. (San Mateo, CA).
Media
A modified Tyrode medium was used for incubating sperm in "capacitating" conditions (Tyr+bic). The complete Tyrode medium contained 96 mM NaCl, 3.1 mM KCl, 2 mM CaCl2, 0.4 mM MgSO4, 0.3 mM KH2PO4, 50 µg kanamycin/ml, 20 mM Hepes, 5 mM glucose, 21.7 mM sodium lactate, 1 mM sodium pyruvate, 15 mM NaHCO3, and 7 mg/ml BSA [16]. For incubating sperm in the control, "noncapacitating" conditions, a modified Tyrode medium without bicarbonate (Tyr) was prepared. In both cases, stock solutions minus the CaCl2, BSA, and pyruvate were prepared; filtered through a 0.2-µm filter; and stored at 4°C [11]. The remaining three ingredients were added 20–24 h prior to the experiment and the medium was maintained in equilibrium with 5% CO2 in air at 37°C until the time of the experiment. The pH and osmolality of both media were maintained at 7.4 and 300 mOsm/kg, respectively.
Semen Collection and Preparation
During the breeding season, semen was collected from three adult (4- to 10-yr-old) stallions once a day for three consecutive days (total of three ejaculates per stallion). All nine ejaculates had a gel-free sperm concentration of >150 million cells/ml and a progressive sperm motility of >65%. The semen was collected using an artificial vagina, filtered through gauze to remove the gel and any large particles of debris, and immediately processed for use. Ejaculates were processed separately. Two milliliters of semen was transferred to a prewarmed 15-ml tube, mixed with 6 ml Tyr, and centrifuged at 900 x g for 10 min to allow removal of seminal plasma. After removal of the supernatant the pellet was resuspended with Tyr to a final volume of 2 ml (equal to the volume of semen used) and incubated at 37°C for 30 min for equilibration and subsequently divided into two equal portions, one of which was diluted with Tyr+bic and the other with Tyr, to final sperm concentrations of 25 x 106/ml. These samples were divided further into two portions, one of which was supplemented with Ca2+ ionophore to a final concentration of 1 µM [17]. All four samples were then incubated at 37°C [4] for 5 h. At 0, 0.5, 2, 3.5, and 5 h, aliquots of each sample were recovered and the state of capacitation, AR, or both was assessed using all three staining methods, as described below, and sperm motility was analyzed for the incidence of hyperactivation.
CTC/EthD-1 Staining
CTC staining was made freshly by dissolving CTC and L-cysteine in a chilled, 20 mM Tris buffer supplemented with 130 mM NaCl to produce final concentrations of 0.75 mM CTC and 5 mM L-cysteine, respectively. The pH of the final solution was adjusted to 7.8, and it was kept in the dark at 4°C until it was used. For staining, a 100-µl aliquot of sperm suspension was mixed with 100 µl of a 2 mM solution of EthD-1 (a supravital stain) in PBS, and this mixture was incubated for 5 min before 100 µl of the CTC stain was added. Thereafter, the sample was fixed with 30 µl of 12.5% glutaraldehyde in 1 M Tris (pH 7.0) to produce a final concentration of 1.1% fixative, and a 10-µl drop of the fixed sperm suspension was mixed with 5 µl of antifade on a glass microscope slide. Next, the droplet was covered with a coverslip, and the slide was gently but firmly pressed under two folds of a tissue paper to absorb any excess fluid. The prepared slide was then stored in the dark until it was analyzed, within 1 h of preparation. For analysis of the CTC staining, an epifluorescence microscope (BH-2; Olympus, Tokyo, Japan) equipped with a wavelength band-pass excitation filter of 458 ± 15 nm, a 470-nm dichroic mirror, and a 500-nm long-pass emission filter was used to asses at least 100 spermatozoa at a magnification of 400x. This combination of filters enabled simultaneous identification of dead cells (EthD-1 positive) versus live cells (EthD-1 negative) and CTC fluorescence patterns. For more detailed visualization, CTC-stained sperm were examined with a spectral confocal microscope (Leica TCS SP; Leica GmbH, Germany). The CTC and EthD-1 stains were excited with the 458-nm argon laser line (emission selected at 495–535 nm), and resolution was optimized by using extended focus made up of three optical Z-sections (0.25 µm step size; average of three X-Y scans per section) and recombined into one image.
Merocyanine 540/Yo-Pro-1 Staining
For flow cytometric analysis of capacitation status, sperm cells were incubated in Tyr+bic or Tyr containing 2.7 µM merocyanine 540 (a reporter probe for phospholipid scrambling; [10]), 25 nM Yo-Pro-1 (a membrane-impermeable nucleic acid stain; [10]), 0.5 mg/ml polyvinyl alcohol, and 0.5 mg/ml polyvinylpyrolidone. In vitro capacitation was performed in air-tight, sealed, 5-ml flow cytometer tubes (Becton Dickinson, San Jose, CA) containing 3 ml of medium. The tubes were flushed with air containing 5% CO2 before closing, and incubated for approximately half an hour in a shaking water bath at 37°C before flow cytometric analyses. Sperm cell analysis was performed using a flow cytometer (FACS Vantage SE; Becton Dickinson). The system was triggered by the forward light scatter signal (FSC). The Yo-Pro-1 and merocyanine 540 probes were both excited by an argon ion laser (Coherent Innova, Palo Alto, CA) with 200 mW laser power at a wavelength of 488 nm. Fluorescence of the Yo-Pro-1 probe was then measured using a band-pass filter of 520 ± 15 nm (fluorescence detector equipped with a photomultipler tube), while merocyanine 540 emission was deflected with a 560-nm short-pass dichroic mirror in the emission pathway and measured using a band-pass filter of 575 ±15 nm. Sperm cells were analyzed at a rate of between 8000 and 10 000 per sec using PBS as sheath fluid, and for each sample, 10 000 events were stored in the computer for further analysis with Cell-Quest software (Becton Dickinson). Sideward light scatter and FSC were recorded so that only sperm cell-specific events, which appeared in a typical L-shaped scatter profile, were selected for further analysis. During measurement, the sample input tube in the FACS Vantage SE was kept at 37°C and 5% CO2 to maintain constant incubation conditions during the entire analysis using a controlled temperature bath/circulator.
For visualization of individual sperm cells, samples were placed in a life chamber at 37°C in which the bicarbonate/CO2 equilibrium was maintained by continuous infusion of humidified air containing 5% CO2; thus, they were maintained under physiological conditions. The sperm were examined using an inverted spectral confocal microscope (Leica TCS SP; Leica) fitted with a 488-nm argon laser line for exciting fluorescent probes. Yo-Pro-1 emission was detected using photomultiplier tube 1, which selected emissions in the wavelength range of 500–550 nm, while merocyanine 540 emission was detected using photomultiplier tube 2 (580–620 nm). Single scans were made to record the labeling patterns of motile sperm cells.
FITC-PNA/PI Staining
The acrosomal status of sperm was examined by staining the incubated samples with 5 µg/ml FITC-PNA (as a marker for acrosomal leakage [18]) and 1 µM PI (as marker for cell death [19]), and analyzing the labeled sperm on a flow cytometer (FACScan; Becton Dickinson) as described before [18, 20].
For visualization of individual sperm cells, samples were placed in a life chamber at 37°C in which the bicarbonate/CO2 equilibrium was maintained by continuous infusion of humidified air containing 5% CO2 in a spectral confocal microscope (Leica TCS SP; Leica) and excited with the 488-nm argon laser line. The emission of PNA-FITC was detected using photomultiplier tube 1, focused for emissions in the 500–550 nm wavelength range, and the emission of PI was detected using photomultiplier tube 2 (600–700 nm). Single scans were made to capture the labeling patterns of (hyper) motile sperm cells.
Comparison of CTC, Merocyanine 540, and FITC-PNA Staining
In a separate experiment, the proportion of viable cells with positive FITC-PNA staining were evaluated for three stallions (three ejaculates per stallion analyzed). The FITC-PNA staining was analyzed under bicarbonate-enriched (15 mM) and bicarbonate-depleted (0 mM) conditions using either a flow cytometer or a confocal microscope (as described above). The labeled sperm samples were analyzed in an unfixed state as well as after 2% (w/v) paraformaldehyde fixation. The results obtained were compared with microscopic evaluations of CTC staining of the same incubated sperm samples (i.e., after fixation with 1.1% w/v glutaraldehyde) and both stainings were also compared with merocyanine 540 staining (unfixed cells only).
Hyperactivation
In order to examine the motility pattern of incubated spermatozoa, samples from each treatment and time point were examined using a Hamilton Thorne Research Motility Analyzer (HTM IVOS; model 8020, version 8.1, Hamilton Thorne, Beverly, MA). The Hamilton Thorne CASA system was programmed to the following settings: frames acquired, 20; frame rate, 30/sec; minimum contrast, 8; minimum size, 6; low/high size, 0.5–1.8; low/high intensity gates, 0.5–1.8; nonmotile head size, 13; nonmotile intensity, 25; medium VAP value, 25; low VAP value, 9; slow cells motile, No; and threshold STR, 80. These values were selected on the basis of previous experience, and their suitability was confirmed using the playback option of the motility analyzer. A 20-µm counting chamber (Cell Vision, Anthos Labtec BV, The Netherlands) maintained at 37°C was used for analysis, and samples were tested within 1 min of being taken out of the incubator. It took less than 5 min to analyze a sample on the motility analysis machine, and particular attention was paid to the classic parameters of sperm hyperactivation; namely, curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) [21, 22]. A spermatozoon was designated as being hyperactive if it had a VCL 
180 µm/sec and an ALH 12 µm.
Statistical Analysis
Analysis was centered around identifying decreases in the percentages of noncapacitated or acrosome-intact spermatozoa. Statistical analysis was carried out using repeated measures ANOVAs (n = 3) to examine the effects of staining method, time, and stallion. Because the sample size was only three, normality could not be properly assessed and the data were therefore analyzed untransformed. As an additional check the data were reanalyzed after a normalizing sine transformation, and because this did not alter the outcome, the assumption of normality was strengthened. In the case of the hyperactivation data, a one-way ANOVA was used to compare the motility of the six ejaculates. The statistical package used was SPSS 8.0 (SPSS for Windows 1996; SPSS Inc., Chicago, IL). Differences were taken to be statistical significant when P < 0.05.
RESULTS
CTC Staining Patterns
Viable spermatozoa (EthD-1 negative) stained with CTC showed three major fluorescence patterns. In the first of these, pattern A, the entire sperm head showed bright fluorescence, with or without a brighter equatorial band; this pattern was representative of noncapacitated spermatozoa (Fig. 1A). In pattern B, the acrosomal region of the sperm head fluoresced brightly, whereas the postacrosomal segment was nonfluorescent; this pattern was indicative of capacitated but acrosome-intact spermatozoa (Fig. 1B). Finally, in pattern C, the acrosomal region did not fluoresce, whereas the postacrosomal segment either was or was not fluorescent; this pattern indicated capacitated and acrosome-reacted spermatozoa (Fig. 1C). For the effect of time, incubation in Tyr+bic medium for 5 h resulted in a significant decrease in the average percentage of spermatozoa showing pattern A (i.e., viable, noncapacitated sperm; 50.2% ± 0.8% at 0 h of incubation and 22.8% ± 4.9% at 5 h), and an increase in the percentage of spermatozoa displaying pattern C (i.e., viable, capacitated and acrosome-reacted sperm; 9.5% ± 3.2% at 0 h and 14.6% ± 2.85% at 5 h).
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Merocyanine Staining Pattern
Merocyanine 540 staining gave rise to two basic fluorescent patterns (Fig. 2A) in viable (Yo-Pro-1 negative) sperm cells, one of which was characterized by cells with poor fluorescence and the other by relatively higher fluorescence. Sperm cells with poorly fluorescent heads were considered noncapacitated, whereas a highly fluorescent sperm head was considered to be characteristic of a capacitated spermatozoon. These patterns were analyzed with flow cytometry and the resulting data presented in quadrants (Fig. 2, B and C) in which the X-axis represents increasing merocyanine 540 fluorescence, and the Y-axis shows increasing Yo-Pro-1 fluorescence. When incubated in Tyr+bic medium, 49.6% ± 2.9% (mean ± SD) of the spermatozoa showed low merocyanine 540 fluorescence at 0 h. After the 5-h incubation, the proportion of poorly fluorescent sperm had decreased to 8.7% ± 2.8% (Fig. 2, B and C), thereby indicating a significant time-dependent decrease in the percentage of noncapacitated spermatozoa. Conversely, the percentage of spermatozoa with high merocyanine 540 fluorescence increased from 6.4% at 0 h to 25.6% at 5 h, demonstrating a corresponding increase in the percentage of capacitated, live spermatozoa (Fig. 2, B and C). The remaining sperm cells were labeled with Yo-Pro-1, and were therefore nonviable.
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FITC-PNA Staining Pattern
FITC-PNA staining differentiated the viable (PI negative) sperm cells into two distinct groups (Fig. 3A). The sperm were either not labeled with FITC-PNA, thereby demonstrating that their acrosomes were intact, or they showed acrosomal FITC-PNA staining, which indicated that their acrosome was either reacting or had reacted. Absence or presence of FITC-PNA labeling was also analyzed flow cytometrically and the resulting data presented in quadrants (Fig. 2, B and C) in which the X-axis shows increasing FITC-PNA fluorescence and the Y-axis shows increasing PI fluorescence (Fig. 3, B and C). At the onset of incubation in Tyr+bic medium (0 h), 53.8% ± 3.2% (mean ± SD) of the spermatozoa were not labeled with FITC-PNA. After 5 h of incubation, the proportion of unlabeled sperm cells had decreased to 38.9% ± 4.1%, indicating a time-dependent decrease in the percentage of acrosome-intact spermatozoa. On the other hand, the increase in the percentage of viable spermatozoa that were labeled with FITC-PNA between 0 h (2.2%) and 5 h (4.0%), demonstrated that only a slight increase in the percentage of acrosome-reacted live spermatozoa occurred. The remaining cells were labeled with PI, and thus were shown to be nonviable.
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Comparison Between Capacitation State Detected with Merocyanine 450 and CTC
At the onset of incubation in Tyr+bic medium (0 h), merocyanine 540 staining demonstrated that 49.6% ± 2.9% of the spermatozoa had not undergone capacitation. Thereafter, the percentage of noncapacitated spermatozoa decreased rapidly, at least initially, to reach 21.5% ± 5.0% after 0.5 h, and then more gradually, to reach a final value of 8.7% ± 2.8% after 5 h of incubation (Fig. 4B). On the other hand, when capacitation status was monitored using CTC staining, a less dramatic and more gradual decrease in the number of noncapacitated sperm was observed, from 50.2% ± 0.8% at 0 h to 22.8% ± 4.9% at 5 h (Fig. 4A).
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Thus, although the proportion of noncapacitated sperm detected at the onset of incubation did not differ between the two techniques, the apparent rate of loss of noncapacitated sperm differed significantly (P < 0.05). Therefore, if it is accepted that merocyanine 540 and CTC staining both follow changes in capacitation status, then it is clear that CTC staining is very much slower to recognize these changes.
In the presence of Ca2+ ionophore and bicarbonate, a similar decrease in the percentage of noncapacitated spermatozoa was observed with merocyanine 540 staining, from 48.2% ± 5.0% at time 0 h, to 22.7% ± 3.5% at the 0.5 h time point, and 7.7% ± 4.9% at 5 h (Fig. 4B). In these latter conditions, CTC staining gave similar results to those of merocyanine 540 for the rate of decrease in the percentage of noncapacitated sperm because the values went from 45.2% ± 3.5% at time 0 h, to 25.8% ± 4.9% at 0.5 h, and to 2.4% ± 2.1% at 5 h (Fig. 4A). Repeated measures ANOVA did not show any significant differences between these two staining methods for the proportion of noncapacitated sperm detected at the various time points, when both bicarbonate and Ca2+ ionophore were included in the incubation medium.
In bicarbonate-free Tyrode medium (i.e., Tyr), only a very slight decrease in the percentage of noncapacitated sperm over time was observed with merocyanine 540 (51.5% ± 2.9% at 0 h to 42.2% ± 8.9% at 5 h; Fig. 4B) and CTC (52.3% ± 3.1% at 0 h and 45.6% ± 7.5% at 5 h; Fig. 4A) staining. Furthermore, incubation with Ca2+ ionophore had no apparent effect on the capacitation status of sperm, as detected with the merocyanine 540 and CTC stain (Fig. 4, A and B). In contrast, the change in capacitation status of sperm was only fully (i.e., comparable to the effects monitored by the merocyanine 540 staining) detectable in the presence of Ca2+ ionophore with the CTC stain (Fig. 4A).
Comparison of CTC and FITC-PNA Staining for Detecting Acrosomal Integrity
The percentage of acrosome-intact cells as determined by CTC staining was taken to be the sum of the percentages of noncapacitated and capacitated acrosome-intact sperm (see Fig. 1, A and B). At the onset of incubation in Tyr+bic medium, the percentage of acrosome-intact cells as detected by CTC staining was 55.2% ± 1.6%. During incubation the proportion of acrosome-intact sperm cells decreased gradually, to 30.4% ± 2.7% after 5 h (Fig. 4C). These two values were similar to those observed with FITC-PNA staining, for which the percentage of acrosome-intact cells dropped from 53.8% ± 3.2% at 0 h to 38.9% ± 4.1% after 5 h of incubation.
The addition of Ca2+ ionophore to the Tyr+bic incubation medium resulted in a more rapid and pronounced decrease of acrosome-intact sperm cells as detected by CTC staining and, to a lesser extent, by FITC-PNA staining (Fig. 4, C and D), thereby indicating that Ca2+ ionophore induces the AR in stallion sperm incubated in a bicarbonate-containing medium. Although the decrease in the proportion of acrosome-intact cells was more prominent in CTC-stained samples (55.6% ± 2.0% at 0 h to 5.6% ± 3.7% at 5 h) than in those stained with FITC-PNA (53.3% ± 4.4% at 0 h to 24.6% ± 8.8% at 5 h), there was no significant difference between the data.
In the absence of bicarbonate, however, there was no time-dependent decrease in the percentage of acrosome-intact spermatozoa as detected by either CTC or FITC-PNA staining (Fig. 4, C and D), and even addition of Ca2+ ionophore to the Tyr medium did not induce the AR in incubated spermatozoa (Fig. 4, C and D).
In a separate experiment, FITC-PNA staining was assessed in bicarbonate-primed and control cells prior to and after 2% (w/v) paraformaldehyde fixation using flow cytometry and confocal laser scanning microscopy (Table 1). Both methods gave similar results. This was also the case with merocyanine 540 staining of the same sperm samples (only unfixed samples were analyzed). However, it was clear that fixation allowed FITC-PNA/PI binding to an extended sperm subpopulation both for control and bicarbonate-stimulated samples. The CTC acrosome staining gave lower relative numbers of acrosome-intact cells when compared with the FITC-PNA staining of unfixed sperm cells. This effect may be attributed to fixation because FITC-PNA staining of paraformaldehyde-fixed stallion sperm gave similar results as CTC staining of glutaraldehyde-fixed cells. Finally, the CTC capacitation staining gave lower relative amounts of capacitated cells when compared with merocyanine 540 staining.
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Assessing the Viability of Sperm Cells
In this study the sperm cells stained with CTC, FITC-PNA, or merocyanine 540 for the assessment of capacitation or acrosome status were counterstained with EthD-1, PI, and Yo-Pro-1, respectively, for the simultaneous detection of sperm viability. At the onset of incubation in Tyr+bic, PI and Yo-Pro-1 staining identified approximately 43% of the cells as dead, whereas EthD-1 staining recorded only 37% of the cells as dead. However, at all other times and for all treatments, the three viability stains gave nearly identical results for the proportions of dead cells. Thus, after 5 h of incubation, the percentage of nonviable cells as recorded by all three staining methods had increased to approximately 55% (the three methods gave nonsignificant differences), whereas the addition of Ca2+ ionophore to the Tyr+bic medium induced even greater cell death, to the extent that by 5 h of incubation, more than 70% of the cells were nonviable.
In the absence of bicarbonate, however, minimal cell death was observed, so that by the end of the 5-h incubation period, only 45% of cells were nonviable, irrespective of the presence or absence Ca2+ ionophore. This demonstrated that in the absence of bicarbonate, sperm cells do not lose their membrane integrity, even in the presence of Ca2+ ionophore. By contrast, when exposed to the capacitating agent, bicarbonate, the sperm became vulnerable to membrane disruption and cell death during the 5-h incubation period, and these processes were facilitated by Ca2+ ionophore.
Sperm Motility and Hyperactivation
Sperm motility with specific regard to hyperactivation was analyzed in two stallions (three ejaculates each) incubated in Tyr+bic medium. The average motility at 0 h was approximately 60% in all cases, and had decreased to around 30% after 5 h of incubation. The addition of Ca2+ ionophore had a negative effect on sperm motility, and no spermatozoa were scored as motile after 3.5 h of incubation, event though approximately 35% of the cells were still viable at this time point, as determined with counterstaining using EthD-1 as supra vital dye.
Hyperactivation was determined objectively by plotting the VCL and ALH of the motile cells on a two-dimensional scatter graph [22]. Spermatozoa were considered hyperactive if their VCL was 180 and their ALH was 12 (i.e., they were in the upper right quadrant on Figure 5, A and B). Using these parameters, 5.3% ± 2.4% of the motile spermatozoa were considered hyperactive, and the proportion of hyperactive cells did not change significantly with time (Fig. 5C). However, the presence of Ca2+ ionophore in the incubation medium resulted in the loss of about all hyperactive spermatozoa by 2 h, and an almost complete motility loss within 3.5 h of incubation.
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It was clear then, that at any given time the percentage of hyperactivated spermatozoa was lower than the percentage of capacitated spermatozoa, as detected by either merocyanine 540 or CTC staining.
While assessing sperm motility visually it was observed that hyperactivated sperm (identified visually using Yanagimachi's criteria [1]) showed a biphasic motility pattern. In short, hyperactive sperm tended to make vigorous, nonprogressive movements, but then became static for a while before moving again with the same vigorous, nonprogressive movement.
DISCUSSION
Capacitation is a vital phenomenon that a spermatozoon must undergo before it can fertilize an oocyte. However, the lack of a reliable and easy method for assessing sperm capacitation has resulted in an incomplete understanding of this important process. At present, the only generally accepted capacitation assay is CTC staining, in which CTC is a fluorescent antibiotic that binds to the surface of sperm cells in a Ca2+-dependent manner [23]. In short, it appears that the CTC-Ca2+ complex binds preferentially to hydrophobic regions, such as the cell membrane [23, 24], and that capacitation-induced changes in the sperm cell plasma membrane result in changes of the CTC labeling pattern that are widely considered to reflect the attainment of the capacitated state [4–7]. However, the molecular basis of the interaction between CTC, Ca2+, and the sperm plasma membrane are far from clear and, moreover, CTC staining is a laborious technique, particularly because it cannot be analyzed using a flow cytometer (Table 1). The reason for this flow cytometer incompatibility is that although the distribution of the CTC dye clearly changes (Fig. 1), the total amount of CTC staining does not change between noncapacitated and capacitated cells, and it is this absolute change that a FACS machine would require in order to differentiate the cells. Furthermore, assessment of CTC staining implies fixation of cells, and it is therefore important that the cells should be labeled with a membrane-impermeable DNA stain for the discrimination of live and dead (fluorescent) cells before fixation, because this latter process introduces artifacts such as cellular degeneration, acrosomal degeneration, or both (Table 1). A further drawback of CTC staining is its Ca2+ dependency, which renders it useless for detecting Ca2+-independent capacitation changes in sperm cells. This latter point was illustrated clearly in the present study because the capacitation-induced changes detected by merocyanine 540 staining were only fully detectable by CTC staining when Ca2+ ionophore was present (Fig. 4). These drawbacks to the CTC technique underline the importance of developing flow cytometric assays to monitor capacitation-dependent changes in, for example, membrane fluidity [11] and acrosome status [17, 18] in live cells.
Flow cytometric detection of capacitation-related changes in membrane architecture using merocyanine 540 as a reporter probe, and acrosome status using FITC-PNA staining, have some clear advantages over the all-compassing CTC staining technique. First, given the clear differences in the intensity of fluorescence between control and capacitated or acrosome-reacted cells, flow cytometry allows for the very rapid and objective discrimination of the status of large numbers of sperm cells. For example, in the current study we analyzed 10 000 sperm cells per data point in only a few seconds. Second, prior to analysis, the sperm suspension requires only simultaneous addition of appropriate amounts of PI and FITC-PNA or Yo-Pro-1 and merocyanine 540, followed by a 10-min incubation for the completion of labeling. Third, the cells can be analyzed in a flow cytometer in the unfixed state and under relatively physiological conditions (i.e., at 37°C and 5% CO2). This ability to control the ambient conditions minimizes the risk of cell deterioration, especially for the notoriously delicate capacitated sperm cells.
In the current study, we analyzed the results in terms of the decrease in the percentage of viable noncapacitated or acrosome-intact spermatozoa, rather than by the increase in the percentage of viable capacitated or acrosome-reacted cells. It was considered that this represented a more realistic approach because of the greater likelihood that the more delicate capacitated or acrosome-reacted cells would die for reasons other than incubation condition or treatment per se, and thus be missed from the analysis.
The results of this study demonstrate clearly that CTC differs significantly to merocyanine 540 in terms of its ability to detect changes in the capacitation state of spermatozoa incubated in Tyr+bic medium. Merocyanine 540, a marker for increased membrane fluidity, detected a rapid decrease in the number of noncapacitated sperm during the first 0.5 h of incubation, after which numbers tended to plateau. By contrast, CTC detected the apparent loss of noncapacitated status much more slowly, and a similar level of apparent capacitation was not seen until the end of the 3-h incubation.
It is proposed that these differences relate to the probability that the membrane fluidity related changes detected by merocyanine 540 precede the Ca2+ influx on which CTC binding depends. This conclusion is supported by the CTC and merocyanine 540 staining patterns in the presence of Ca2+ ionophore. That is, a more pronounced and rapid decrease in the proportion of live, noncapacitated sperm cells as detected by CTC staining, but no change detected by merocyanine 540 staining when Ca2+ ionophore was present. In summary, the ability of CTC staining to detect changes in capacitation status equaled that of merocyanine 540 only when Ca2+ ionophore was included in the incubation medium; otherwise, CTC was very slow at detecting changes in sperm membrane status.
It was also interesting that in the absence of bicarbonate, capacitation was minimal, whether measured by CTC or merocyanine 540 staining. In total then, the data presented in this paper indicate that bicarbonate induces a change in the lipid packaging of the plasma-membrane of stallion sperm that can be monitored by merocyanine 540, a marker for lipid ‘scrambling’ [11]. A further experiment (data not shown) has demonstrated that only a subpopulation of the merocyanine 540-responsive cells show the CTC capacitation response, and this presumably indicates that the rise in intracellular Ca2+ concentration, which is necessary for CTC binding, occurs later in sperm capacitation than bicarbonate-mediated lipid scrambling. Results obtained for two-staining methods when Ca2+ ionophore was included in the incubation medium were similar, presumably because the ionophore allows the merocyanine 540-responsive cells to take up Ca2+ in a fashion that allows appropriate CTC binding, because the ionophore makes the plasma membrane permeable to Ca2+ (2 mM).
The other parameter commonly assessed using CTC is the degree to which the acrosome is intact [7]. The AR is a Ca2+-dependent process [1] and high intracellular Ca2+ concentration is required for fusion of the sperm plasma membrane to the outer acrosome membrane. Thus, the AR can proceed only after the intracellular Ca2+ levels of capacitated sperm cells have increased, and it is only after this secretory event has occurred that FITC-PNA is able to bind to the appropriate epitope on the outer acrosomal membrane [12, 18]. Therefore, like CTC, FITC-PNA staining is Ca2+-dependent, albeit indirectly, and this may explain why AR assessments were similar for the two techniques. It is interesting that a moderate induction of the AR was observed when sperm were incubated in Tyr+bic, whereas the AR induction was not observed in the absence of bicarbonate. Addition of Ca2+ ionophore resulted in an even more widespread induction of the AR in Tyr+bic-incubated sperm suspensions (but not significant for FITC-PNA-stained sperm), probably by facilitating the rise in intracellular Ca2+ concentration required for this secretory event. Thus, it appears that Ca2+ ionophore synergizes the effect of bicarbonate regarding the AR induction and, whereas bicarbonate alone is sufficient to induce the AR, Ca2+ ionophore alone is not. It must be presumed that the bicarbonate-mediated increase in plasma membrane fluidity allows a subsequent increase in the permeability of that membrane to Ca2+.
With regard to sperm hyperactivation, it has been reported previously that this phenomenon occurs spontaneously in the majority of spermatozoa incubated under capacitating conditions in a medium that contains bicarbonate [25] and Ca2+ (see [16]). The suggestion was that bicarbonate activates adenylate cyclase, either directly or indirectly, by causing Ca2+ influx (see [16]), and thereby elevates intracellular cAMP concentrations, which in turn, induce sperm hyperactivation [26]. For this reason, we expected that in the current experiment the incubation of sperm in a medium containing bicarbonate and Ca2+ would lead to sperm hyperactivation, a phenomenon that is characterized by an increase in flagellar bending amplitude that can be detected in a CASA system by an increase in the amplitude of ALH [27] and increased VCL [28]. However, the results of our study indicated that only a small proportion of the spermatozoa demonstrated characteristics of hyperactivity at any given time and, while it is likely that the percentage of hyperactive spermatozoa was underestimated because of the typical stop-start biphasic motility pattern recorded for hyperactive cells, it was clear that hyperactivity is not a suitable parameter for accurately estimating the percentage of capacitated cells.
In conclusion, we have compared several methods for detecting sperm capacitation, acrosome status, sperm viability, and particular characteristics of motility, under conditions in which either sperm membrane fluidity was affected or the AR was induced. The major aim of the study was to relate the CTC staining patterns obtained with fixed sperm cells, and those used for simultaneous assessment of capacitation and AR status, with the results of novel flow cytometric assays that detect membrane changes in a Ca2+-independent manner [10, 11, 18]. It is concluded that the merocyanine 540 and FITC-PNA assays are preferable to CTC staining because flow cytometric assays are easier to perform, quicker, more objective, and more accurate than fluorescence microscopy assays (i.e., CTC), and, in addition, because the washing and fixation steps that may interfere with apparent cell viability and integrity are not required. Moreover, the mechanisms of the molecular interaction between merocyanine 540/FITC-PNA and the biomembranes and their relationship to the occurrence of capacitation and the AR are clear, whereas the same cannot be said for CTC. It is, of course, important to simultaneously detect sperm viability using a membrane-impermeable DNA stain to ensure that detected membrane changes are biologically relevant. In this respect, all three viability stains examined in the current study gave equally satisfactory results.
ACKNOWLEDGMENTS
We thank Dr. T.A.E. Stout for his critical reading of the manuscript, A. Zandee for collecting stallion semen, and C. Kruitwagen for help with statistics.
FOOTNOTES
First decision: 24 August 2000.
1 Supported in part by the Graduate School of Animal Health to R.R. Supported by Royal Dutch Academy of Sciences and Arts (KNAW) to B.M.G. 
2 Correspondence: B.M. Gadella, Department of Farm Animal Health, Faculty
of Veterinary Medicine, Yalelaan 7, 3584 CL Utrecht, The Netherlands.
FAX: 31 30 2535492; b.gadella{at}vet.uu.nl
Accepted: March 20, 2001.
Received: July 28, 2000.
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|
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|
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|
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|
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|
|
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|
|
|
|
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|
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|
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|
|
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|
|
|
|
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, Tyrode medium with 15 mM HCO3-; broken line 
, Tyrode medium with 1 µM Ca2+ ionophore; broken line 
, Tyrode medium with 15 mM HCO3- and 1 µM Ca2+ ionophore