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Kinetics of the Progesterone-Induced Acrosome Reaction and Its Relation to Intracellular Calcium Responses in Individual Human Spermatozoa¹

School of Biosciences,⁴ Reproductive Biology and Genetics Research Group,⁵ The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom Assisted Conception Unit,⁶ Birmingham Women's Hospital, Birmingham B15 2TG, United Kingdom

ABSTRACT

Progesterone at 3 µM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca²⁺]_i) in human sperm, which is believed to be a prerequisite for progesterone-induced acrosome reaction (AR). As very little is known about how AR occurrence, latency, and completion relate to the characteristics of the progesterone-induced [Ca²⁺]_i signal, we examined these events using fluorescence microscopy of individual living human sperm. Direct assessment of acrosomal status after calcium imaging showed no differences in kinetics or amplitude of the preceding progesterone-induced calcium responses in acrosome-reacted and acrosome-intact cells, which indicates that the amplitude of the [Ca²⁺]_i signal is not the critical determinant of AR. Chelation of extracellular calcium to arrest AR at varying times after progesterone stimulation revealed that maximal AR occurred immediately following progesterone stimulation, during the initial transient calcium influx rather than during the sustained calcium response. Attempts to follow acrosomal dispersal in real-time by staining with the acidic organelle probes LysoTracker DND-99 and dapoxyl (2-aminoethyl) sulphonamide (DAES) proved inconclusive due to heterogeneous labeling of the cell population. Surprisingly, the dye was often not confined to the acrosome but stained the whole sperm head, which suggests that only a subpopulation of human sperm cells contains a sufficiently acidic acrosome.

acrosome reaction, calcium, human, progesterone, signal transduction, sperm, spermatozoa

INTRODUCTION

The acrosome reaction (AR) involves the fusion of the outer acrosomal membrane with the overlying plasma membrane [1] and consequent exocytosis of acrosomal contents [2, 3]. The zona pellucida (ZP) is generally considered to be the primary physiological initiator of the AR in mammalian sperm [4]. ZP-induced AR involves a complex signal transduction pathway that is mediated by an increase in intracellular calcium ([Ca²⁺]_i; [5]). The addition of EGTA, to remove Ca²⁺ from the extracellular medium, abolishes both the rise in [Ca²⁺]_i and the induction of the AR, which shows that the response is dependent upon Ca²⁺-influx [2].

The steroid hormone progesterone (P), which is secreted at micromolar levels by the oocyte and steroidogenic cumulus cells that surround it [6, 7], can also initiate AR in vitro [8–14]. Due to the difficulty in obtaining human ZP, P is the only well-characterized physiological agonist of AR in human sperm. Data exist for other pseudo-agonists, such as various BSA-neoglycoproteins (e.g., [15, 16]), although these data are sparse, and their relevance is unclear. The action of P on human spermatozoa is via a non-genomic pathway which, like ZP, induces AR via a rise in [Ca²⁺]_i [17]. The [Ca²⁺]_i response to micromolar concentrations of progesterone occurs as a transient event followed by sustained elevation, with both phases being dependent upon Ca^2+-influx [8, 18]. The response to P occurs in approximately 98% of selected, motile human spermatozoa [14] but shows marked heterogeneity [18].

If P acts as an inducer or coinducer of AR in vivo, loss of the acrosome and its contents must occur at the appropriate time and place within the female reproductive tract. However, in the extensive literature regarding the responses of human spermatozoa to P, the kinetics of AR in human spermatozoa has received little attention. Neither the relationship between membrane fusion and agonist-induced [Ca²⁺]_i signaling nor the kinetics of the loss of acrosomal contents have been established. Meizel and colleagues [12] stained individual sperm after [Ca²⁺]_i imaging but only 35 cells in total were assessed and of these, only six were successfully scored for acrosomal status. Previously, it has been reported by the same group that human sperm AR occurs within 30 sec of P addition [19], during the initial [Ca²⁺]_i transient phase and before the slowly developing sustained phase. Considering the heterogeneity of [Ca²⁺]_i signaling in human sperm populations, further studies are required. Not only are much larger sample sizes required to understand the kinetics of AR, but it is vital to address the significance of the variation between cells in terms of P-induced [Ca²⁺]_i signaling in determining the probability that a cell will undergo AR.

An exciting new approach to investigating the kinetics of AR and acrosome dispersal in mice is the creation of acrosome-specific GFP knock-ins [20–22]. However, although dispersal occurs within 3 sec of AR onset [20] the sperm do not respond to ZP and must be treated with ionophore to induce AR. Although these mice are fertile, there are no data on the rates of AR induced by physiological stimuli. An alternative approach to monitor acrosomal loss is to stain live cells with acrosome-specific dyes. The mouse acrosome is an acidic compartment [21, 23, 24; predicted mouse intra-acrosomal pH of 5.3], and the pH change that occurs during the early stages of AR, before acrosomal loss, is potentially useful for monitoring the kinetics of AR. Using the fluorescent probes N-(n-dodecyl) amino acridine (NDAA) and dapoxyl (2-aminoethyl) sulfonamide (DAES), AR initiated by both intact and solubilized ZP has been monitored in mouse sperm [23, 25, 26]. This loss of pH gradient that occurs before acrosomal dispersal [27] is potentially part of the earlier second-messenger signaling and is possibly not due to fenestration of the membrane during actual acrosomal loss [21].

Ideally, we would use human sperm with GFP-labeled acrosomes. However, since this is not yet possible with the rapidly emerging stem cell technologies, we have investigated alternative techniques to examine acrosomal status and its relationship to calcium signals in individual cells. First, to investigate the significance of variation in P-induced [Ca²⁺]_i signaling, we have developed a technique for in situ scoring of the acrosomal status of large numbers of cells, immediately after completion of single cell [Ca²⁺]_i imaging. Second, we have used the technique of stopped AR, achieved by the addition of excess EGTA to sperm populations, to assess the time-course of acrosomal loss in P-stimulated cells. Third, we have attempted to follow in real-time the loss of the acrosomal pH gradient using LysoTracker DND 99, which is a pH-sensitive, membrane, permeant dye that accumulates in organelles with low internal pH. Finally, we have attempted to use dyes that label the plasma membrane leaflet, to observe the loss of the acrosomal membranes.

MATERIALS AND METHODS

Preparation and Capacitation of Spermatozoa

All donors were recruited at the Birmingham Women's Hospital (HFEA Centre 0119), in accordance with the Code of Practice of the Human Fertilisation and Embryology Authority. Approval was obtained from the South Birmingham Local Ethical Committee (Ref: 2003/239), All donors gave informed consent for this study. Highly motile spermatozoa were harvested into sEBSS (0.3 % BSA, 1 mM NaH₂PO₄, 5.4 mM KCl, 0.8 mM MgSO₄.7H₂0, 5.6 mM glucose, 2.7 mM sodium pyruvate, 25 mM D,L-lactic Acid, 1.8 mM CaCl₂.H₂O, 26 mM NaHCO₃, 116 mM NaCl), as described previously [14]. Aliquots of 2 ml (for fluorimetry) and 200 µl (for AR assays and single-cell imaging) were incubated under capacitating conditions for 6 h at 37°C in 5% CO₂.

Imaging of Sperm [Ca²⁺]_i

Aliquots of capacitated cells were incubated with 12 µM Oregon Green BAPTA 1-AM (OGB1; Molecular Probes), which was diluted from a stock solution of 50 µg OGB1 in 20 µl of a solution that contained 0.01 g of pluronic acid F127 dissolved in 50 µl of freshly made DMSO (dimethyl sulfoxide, cell culture grade), transferred to the microscope and imaged under fluorescence using a 40x objective on an inverted Nikon TE200 microscope with continuous perfusion of sEBBS, as described previously [14]. Progesterone (3 µM; Sigma) was applied via the perfusion header. All experiments were carried out at 25°C.

Data acquisition and storage were performed using a PC running AQM Orca 2001 (Kinetic Imaging Ltd., Nottingham, UK). Image series were analyzed offline using AQM Orca 2001, as described previously [14]. The raw intensity values from the head of each sperm were imported into Microsoft Excel and normalized to the prestimulus values.

Endpoint Acrosomal Labeling

To assess acrosomal status and relationship to preceding [Ca²⁺]_i responses, we adopted the following method. Immediately following the collection of a series of fluorescent images to monitor P-induced changes in [Ca²⁺]_i (approximately 30 min), the same field of cells was labeled for acrosomal status using FITC-labeled Pisum sativum agglutinin (FITC-PSA; Sigma) and a modified method that allows the chamber to remain in place on the microscope. This method was carried out at 25°C. After collection of a series of Oregon green BAPTA-1 images for the assessment of [Ca²⁺]_i signaling, the chamber was perfused with 100% methanol for 30 sec, and then washed through with medium to remove traces of methanol. The field was then bleached by exposure to direct fluorescent illumination (no neutral density filters) for 30 sec; controls were carried out to confirm that this process did not increase significantly the number of acrosome-reacted cells. FITC-PSA was infused into the chamber and left for 45 min, and distilled water was then perfused through the chamber for 10 min. An image of the field-of-view was captured and the cells were assessed for acrosomal status. Approximately 15% of cells were not scored for acrosomal status either because the cells were washed off during the staining or it was not possible to assess acrosomal status from the labeling pattern.

Time-Course Assay of AR by Staining with PSA

Assessment of acrosomal status via FITC-PSA staining of the inner leaflet of the outer acrosomal membrane and the acrosomal content of fixed cells was performed essentially as described previously [14]. Briefly, 200-µl aliquots were incubated with 3 µM P for 10 sec, 30 sec, 1 min, 2 min, 5 min, 10 min, 30 min, and 1 h. At the end of each incubation period, an excess of EGTA (20 mM) was added to the tube, to chelate Ca²⁺ ions and prevent further AR. The cells were centrifuged at 300 x g for 5 min and smeared on duplicate microscope slides that were precoated with 10% poly-L-lysine solution. To assay cell viability in the populations incubated for 10 min or less, the tubes were treated with propidium iodide (PI; 0.2 µg/ml) 10 min before P treatment. For the populations incubated for more than 10 min, propidium iodide was added 10 min before the end of P treatment. Exclusion of PI by the cell was employed as a measure of membrane integrity. Separate tubes were incubated with A23187 (10 µM) under the same conditions as for P. Solvent controls (0.05% DMSO) were treated for 10 sec, 30 sec, and 1 h under the same conditions as for P. A blank control (no additions) and a tube with excess EGTA added for 10 sec were run in parallel. The slides were evaluated for acrosomal status using FITC-PSA according to the previously described methods [28], using a Leitz microscope with FITC filters and a 63x oil immersion lens, with 400 cells per treatment being scored over two slides. Only viable cells, as assessed by PI exclusion, were scored.

Fluorimetric Measurement of Sperm [Ca²⁺]_i

For the fluorimetric studies, 2-ml aliquots (6 x 10⁶ cells/ml) were labeled with 7.5 µM Fura-2 AM for 12 min at 37°C in 5% CO₂, centrifuged at 300 x g for 5 min, resuspended in sEBSS, and incubated for an additional 17 min. Experiments were carried out at 37°C using the Perkin-Elmer LS50B fluorescence spectrofluorimeter and Fl-Winlab version 2.01, as described previously [14]. A K_D of 224 nm was assumed, as in other relevant studies [16, 29], and calibration was performed using ionomycin and excess EGTA according to the equation of Grynkiewicz et al. [30]. The resting [Ca²⁺]_i and amplitude of the transient and sustained responses were determined as described previously [14].

Real-Time Acrosomal Labeling

Staining with LysoTracker DND-99. Aliquots of 200 µl of capacitated cells were incubated with 100 nM of 2 µM LysoTracker DND-99 (Molecular Probes) for 30 min at 37°C in 5% CO₂. A 100-µl droplet of labeled cell suspension was transferred into an open imaging chamber, in which the lower surface was a poly-L-lysine-coated coverslip (10% poly-L-lysine solution [Sigma]), incubated for a further 10 min (37°C) to enable the cells to adhere, and then imaged at 25°C. The cells were observed under phase-contrast and fluorescence using the inverted Nikon TE200 microscope described previously [14, and above]. In some experiments, the chamber was perfused with sEBSS. A23187 (5 µM) was added directly to the open chamber and images were taken at intervals before and after stimulation.

The methods used for DAES (Molecular Probes) staining were as described by Rockwell and Storey [26], with the modification and observations for human sperm described above. Since the cells were not clearly observable using conventional filter sets, the Perkin Elmer LS50B fluorimeter was employed to generate the excitation/emission spectral characteristics of the labeled sperm (see the Results section).

Real-Time Acrosomal Labeling via Membrane Labeling

Staining with DiI. Cells were prepared as described above and BSA-free EBSS was used to allow to dissolution of the dye. The cells were incubated with 1–20 µM DiI (Molecular Probes; diluted in a 20% solution of pluronic F-127 [Sigma] in 50% ethanol) for 1 h at 37°C in 5% CO₂. Aliquots of 100 µl were transferred to the imaging chamber and observed as described above.

Staining with FM1–43. Aliquots of 200 µl of the capacitated cells were incubated with 1–100 µM FM1–43 (Molecular Probes) for 15 min at 37°C in 5% CO₂, and 100-µl samples were then transferred to an imaging chamber and observed as described above.

Data Analysis

Prior to the construction of amplitude-frequency histograms, the mean (population) response of all cells in the field-of-view was used to identify the three timepoints that straddled the [Ca²⁺]_i transient peak. For each cell, the values (change in fluorescence percentages) of these three points were averaged, and the means were then sorted into amplitude classes. The mean amplitudes of the transient and sustained responses were analyzed by the paired t-test (two-tailed) using Microsoft Excel, with statistical significance set at P < 0.05. All the data are presented as mean values ± SEM. The AR rate is presented as rate per minute (obtained from the increment in percentage AR divided by the time over which that increment occurred), normalized to the maximum rate for that treatment.

RESULTS

Characteristics of Progesterone-Induced [Ca²⁺]_i Response in Acrosome-Reacted Cells

Cells were initially imaged for [Ca²⁺]_i in response to 3 µM P stimulation, and the same field of cells was then assessed for acrosomal status (see the Materials and Methods section). The total duration of P treatment was approximately 30 min (20 min during the image series and 10 min after completion of the image series). Figure 1a shows clear labeling of a field of cells for the assessment of endpoint acrosomal status. The mean rate of P-induced AR in seven experiments was 25.2 ± 1.5 % (of the cells with recordable acrosomal status; see the Materials and Methods section). To ensure that the same field of cells was being assessed for the P-induced [Ca²⁺]_i responses and AR, a phase-contrast image from the start of the Ca²⁺ imaging experiment was overlaid on the fluorescence image for acrosomal staining (Fig. 1b). Detailed analysis of the [Ca²⁺]_i data revealed that there were no significant differences between the responses of cells that underwent AR and those of cells that remained intact after 30 min of P stimulation. The amplitude distributions of the transient [Ca²⁺]_i responses in acrosome-intact (AI) and acrosome-reacted (AR) cells were very similar (Fig. 2a; seven experiments, 757 cells assessed for acrosomal status). Comparison of the mean amplitudes of the transient and sustained [Ca²⁺]_i responses showed no difference (P > 0.05), and when the mean (R_tot) [Ca²⁺]_i response for the AI and AR cells was plotted, the traces in each of the seven experiments were showed strong overlap, which indicates that the amplitude and kinetics of the [Ca²⁺]_i responses are virtually identical (Fig. 2).

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   FIG. 1. a) Cells within the perfusion chamber that are fixed and labeled with FITC-PSA to assess the endpoint acrosomal status after completion of the Ca2+ image series. b) Phase-contrast image taken at the start of the Ca2+ image series, overlaid with a fluorescence FITC-PSA image of the same field of cells following the image series. It is apparent that not all the cells can be assessed for acrosomal status due to inconclusive labeling patterns. Bar = 20 µm.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   FIG. 2. a) Distribution of amplitudes for P-induced [Ca2+]i transient signaling in cells subsequently identified as acrosome-intact (AI; white square) or acrosome-reacted (AR; black square). Bars show the mean percentage frequency (± SEM) of seven experiments for each amplitude class. b) Overlaid plots of the mean Ca2+ response data from one experiment. Cells that are undergoing AR (black square) and cells that remain acrosome-intact (white square) after 30 min of P stimulation (AR = 32 cells; AI = 107 cells) are shown. Similar results were obtained in six other experiments (total of 757 cells).

Relating AR to the Kinetics of the [Ca²⁺]_i Response

Biphasic calcium response to progesterone. Previously reported data from our laboratory using population fluorimetry have shown that 3 µM P generates a transient peak of duration around 50 sec with an average time to peak of 11.7 ± 0.5 sec. A sustained plateau follows the transient [Ca²⁺]_i increase at around 200 sec after application of P (twenty experiments; [14]). Stopping AR (by the removal of Ca²⁺ using an excess of EGTA) at various times after the application of P allowed comparison of the progress of AR with the biphasic [Ca²⁺]_i response that triggers AR.

Timing of AR induced by progesterone and ionophore. The kinetics of AR was investigated by the addition of excess EGTA to quench membrane fusion events. There was no increase in cell death after the addition of excess EGTA, as assessed using propidium iodide (data not shown), and the cells that were stopped after incubation for 1 h (completion of agonist-induced AR) gave similar AR rates for all treatments, with or without EGTA (P = 0.46, n = 6) (Fig. 3a), which demonstrates that EGTA has no direct effect on the AR rate.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   FIG. 3. a) The effect of incubation time on percentage AR during incubation with 3 µM P (black bars), 10 µM ionophore (white bars), solvent (0.05% DMSO; striped bars), or medium alone (spotted bars). The incubations were terminated by the addition of excess EGTA (20 mM), except where stated. Bars represent the mean ± SEM of six experiments. b) Time-course showing the effect of incubation time on percentage AR in response to P (white squares) and for comparison, a fluorimetric trace showing [Ca2+]i after P stimulation (black squares). The [Ca2+]i is scaled (nm/25) to allow both datasets to be shown on the same axis. P-induced AR is corrected for solvent-induced AR. Incubation was terminated by the addition of excess EGTA (20 mM) at the timepoints shown. c) P-induced AR represented as rate values (percentage of final AR per min; obtained from the increment in percentage AR divided by the time over which that increment occurred, normalized to the level of AR after 1 h of P stimulation; white triangles). P-induced [Ca2+]i is presented for comparison (black squares). The [Ca2+]i is scaled (nm/10) to allow both datasets to be shown on the same axis. The rate of AR is plotted at the midpoint of each time interval. d) Ionophore-induced rate of AR per min, represented as described in c.

In solvent (DMSO)-treated cells, AR was 4.1 ± 0.8% (n = 5) after a 10-sec incubation, and this did not increase significantly with longer incubation periods (for 10 min, P = 0.3, n = 5; for 1 h, P = 0.35, n = 5). The spontaneous AR rates of the untreated media controls were similar to those of the DMSO-treated cells (Fig. 3a). When cells stimulated with 3 µM of P were stopped by the addition of EGTA after 30 sec, the rate of AR was double that of the solvent controls (P = 0.002, n = 6) and the level of P-induced AR continued to increase with the duration of incubation (Fig. 3a). Figure 3b shows the fluorimetric measurements of [Ca²⁺]_i (nM) and AR at intervals after stimulation with 3 µM P. Clearly, the majority of the AR-competent cells have undergone AR during the first 300 sec after P stimulation. Figure 3c shows the same data but with AR presented as the AR rate (obtained from the increment in percentage AR divided by the time over which that increment occurred) normalized to the percentage AR at 1 h. P-induced AR follows [Ca²⁺]_i, although with a slight lag, AR occurred most rapidly between 10 and 30 sec after stimulation, as compared with [Ca²⁺]_i, which peaked at 11 sec (see above) and subsequently fell to very low levels.

A23187 at 10 µM induced almost instantaneous AR, doubling the mean percentage of acrosome-reacted cells after 10 sec of incubation (Fig. 3a; P < 0.01, n = 6). A23187-induced AR then progressed more slowly throughout the 60-min incubation. Plotting the rate of AR (as described above) showed that two bursts of AR occurred in response to A23187 stimulation, the first being almost instantaneous (within the first 10 sec of incubation) and the second occurring between 100 and 300 sec after application of the ionophore (Fig. 3d). However, despite these early responses, the majority of the A23187-induced AR occurred later, accumulating at a relatively slow rate during the final 50 min of the 60-min incubation (Fig. 3a). The kinetics of A23187- and P-induced AR are clearly different.

Visualization of AR by Labeling of Live Cells

Acidic organelle tracking. Exposure of human sperm to 500 nM LysoTracker caused labeling of the sperm heads of approximately 80% of the cells (eight experiments, 942 cells; Fig. 4a), although this rate varied between experiments (79 ± 4%). The location of labeling was also variable, with localization seen in the acrosome, post-acrosomal region, and throughout the head in different cells (Fig. 4b). Labeling was rapidly reversible. Perfusion of the chamber with medium that lacked LysoTracker resulted in rapid dye loss from all the cells and many of the cells lost their staining even when the medium contained dye at the labeling concentration (22 experiments, dye concentrations from 100 nM to 2 µM). This indicates that the dye is extremely sensitive to changes in concentration.

View larger version (104K): [in this window] [in a new window] [Download PPT slide]   FIG. 4. Labeling of live human spermatozoa with 500 nM LysoTracker DND-99. a) Merged phase-contrast and fluorescence images showing a field of adhered live cells. b) The labeling pattern is heterogeneous within the field, with localization within the acrosome, post-acrosomal region, and whole head, as indicated. Bar = 20 µm.

The addition of 5 µM A23187 (a Ca²⁺ ionophore) directly into the imaging chamber (without medium perfusion) to induce AR had complex effects on cells that were labeled with 500 nM LysoTracker. Images of the labeled field of cells taken before and at various times after ionophore stimulation showed that two variables changed as the duration of stimulation increased. First, there was a rapid fall in the number of labeled cells (around 25%) during the first 5 min after stimulation with A23187, apparently reflecting loss of the dye (Fig. 5a, black line, average of 32 cells from three experiments). This may reflect the occurrence of AR in a subset of cells, although loss of label was often from the post-acrosomal region. Second, we observed an increase in fluorescence in the remaining labeled cells over time (Fig. 5a, gray line) with many cells reaching maximal fluorescence within 6 min and subsequently decreasing, occasionally falling below the prestimulation levels (Fig. 5b; 12 individual cell traces from two experiments, represented by black and gray lines). As staining was not confined to an acrosomal localization and the results produced were variable and inconclusive, this method was not pursued further.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   FIG. 5. a) Effect of ionophore stimulation on individual human spermatozoa labeled with 500 nM LysoTracker DND-99. The gray line shows the increase after stimulation with 5 µM A23187 in fluorescence intensity of the dye over time compared to the control (recorded from –4 to 0 min) levels (three experiments, average of 32 randomly chosen cells). The black line represents the percentage of labeled cells at spaced time intervals after ionophore stimulation (three experiments, 308 cells). Both variables are calculated from the same three experiments. b) Individual cell traces showing changes in intensity of 500 nM LysoTracker DND-99 in the heads of the human spermatozoa after stimulation with A23187. The data are from two experiments (black traces show six cells that were randomly selected from an experiment involving 221 cells; the gray traces show six cells randomly selected from an experiment involving 87 cells). All the traces are corrected for changes in background intensity.

We utilized other acidic organelle probes, including DAES, as acrosomal labels [26] to study AR in the mouse. None of these probes proved any more successful, and in the case of DAES, the large difference between the published methanol excitation/emission spectra and those we obtained when the dye was loaded into human spermatozoa (excitation spectral shift from 370 to 390 nm; emission shift from 490 to 590 nm) rendered the dye unsuitable for use with standard microscopy filter sets.

Membrane labeling. The fluorophore DiI comes from a group of lipophilic dyes that become fluorescent when incorporated into membranes [31]. We predicted that upon induction of AR by the ionophore, the label would be lost around the front of the head, indicative of fusion of the plasma membrane and outer acrosomal membranes. Trial experiments using DiI produced high background levels of fluorescence with bright spots of fluorescence in the medium, which indicated that the dye was not in solution. Subsequent attempts using BSA-free medium showed a lower level of background fluorescence and increased labeling of the sperm, indicating that the dye was in solution. Although the majority of the stained cells were immotile and therefore not part of the fertilizing population, some of these cells showed clear acrosomal labeling (Fig. 6; 37 experiments, concentrations from 1 µM to 20 µM). However, acrosomal staining was inconsistent and unpredictable. In many cases, only immotile cells retained the dye. Furthermore, when labeling was achieved, no change or loss of dye was seen upon the addition of ionophore to the medium.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   FIG. 6. Labeling of human spermatozoa with 10 µM DiI. Merged phase-contrast and fluorescence images. a) The live sperm often show labeling of the acrosome, whereas non-viable sperm are strongly labeled at the rear of the head and in the midpiece (b). Bar = 20 µm.

FM1–43 is believed to insert into the outer leaflet of the surface membrane, resulting in intense fluorescence. It has been used to investigate the endocytosis of vesicles at neuromuscular junctions, whereby the dye becomes internalized within the vesicles [32]. This dye predominantly stained the midpieces of non-viable sperm, as assessed by PI staining. It appeared that the dye was unable to penetrate the lipid bilayer while the cells were alive (24 experiments, concentrations from 1 µM to 100 µM; data not shown).

DISCUSSION

Various potential methods exist for following the progress of the AR. Acrosomal presence or absence can be assessed via the detection of acrosomal contents both in live cells (e.g., by GFP) or after fixation and permeabilization. Alternatively, dyes may be used to monitor associated events, such as loss of pH gradient or changes in membrane status (directly by leaflet labeling or indirectly by antibody staining). All of these approaches run the risk of modifying the process itself and creating an artifact. The diversity of approaches may explain the variability in the data reported by different laboratories studying the occurrence and kinetics of AR. In the present study, we have used a number of these methods in parallel to clarify certain issues, although in certain respects we have raised further questions.

The Progesterone-Induced [Ca²⁺]_i Response and AR

Developing a method of recording [Ca²⁺]_i responses and acrosomal status in the same cells has enabled us to study the particular P-induced [Ca²⁺]_i responses that precede the acrosome reaction. In previous studies, we and others have shown that the amplitude of the P-induced [Ca²⁺]_i signal (by fluorimetric population measurement or imaging) shows dose-dependence similar to that seen for AR [9, 14]_.. We conclude for cells that are competent to undergo AR that the amplitude of the [Ca²⁺]_i signal in each cell determines the probability that AR will occur [14]. Surprisingly, we have found that sorting cells according to acrosomal status at the end of [Ca²⁺]_i imaging reveals no detectable difference for the [Ca²⁺]_i signal induced by 3 µM P between acrosome-reacted and acrosome-intact cells (Fig. 2). Single-cell analysis of human sperm stimulated with 3 µM P shows that the [Ca²⁺]_i response varies greatly between cells, with the amplitude distribution extending from the maximum down to the limits of detection [14]. Thus, if the probability of AR is dependent upon the amplitude of the [Ca²⁺]_i response, AR should occur primarily in those cells at the upper end of the amplitude distribution, which clearly is not the case (Fig. 2a). These findings are consistent with the view that other factors, such as tyrosine phosphorylation status, render many cells incompetent to undergo AR despite the generation of a normal [Ca²⁺]_i response. They also suggest that the [Ca²⁺]_i transient response, if it is detectable by imaging, is usually adequate to induce AR.

Time-Course of Stopped AR

The use of EGTA to stop further AR by chelation of extracellular Ca²⁺ shows that the rate of AR largely follows the biphasic [Ca²⁺]_i signal. The majority of AR occurred during the first 300 sec, with the rate peaking during the initial transient phase. This analysis suggests that the rate of AR lags slightly (5 to 15 sec) behind the elevation of [Ca²⁺]_i. After completion of the transient phase, AR continued during the sustained response, albeit at a low rate (Fig. 3c). This contrasts with AR in mouse sperm stimulated by ZP3 [33] or in sea urchin spermatozoa stimulated by egg jelly factors [34, 35]. In these studies, it was concluded that AR occurred after the initial transient Ca²⁺ influx and depended primarily on the activation of store-operated influx. The timings of P- and ZP-induced AR have also been analyzed in caprine sperm populations; higher levels of AR were observed after stimulation with P (2 µg/ml) compared to human sperm (partly due to a spontaneous rate of > 15%), and the highest rate of P-induced AR occurred in the first 6 min after stimulation, whereas the response to solubilized ZP was much slower [36]. Recently, we have shown that when P is applied in an ascending concentration gradient (in an attempt to represent more closely the stimulus experienced in vivo), rather than induce an initial, large transient [Ca²⁺]_i response, it causes a slow rise in [Ca²⁺]_i upon which the oscillations are superimposed. This response does not induce AR but modifies flagellar activity [37]. The synchronization of P-induced AR with the initial large transient response that occurs upon application of 3 µM P may be due to spill-over of the P-induced [Ca²⁺] signal into areas in which the AR machinery is activated.

A23187-induced AR occurred more rapidly than P-induced AR, with the maximum rate occurring immediately upon stimulation. Nakanishi and colleagues [20] have observed that A23187-induced AR occurs within 3 sec by dispersal of the acrosomal-EGFP marker. This instant effect is probably a reflection of the fact that A23187 does not require activation of calcium mobilization. It is known that some sperm samples that are unable to undergo AR in response to P are capable of ionophore-induced AR [38], whereby the persistently high [Ca²⁺]_i directly mediates membrane fusion. It is possible that defective cells undergo AR almost instantaneously after A23187 exposure. The burst of AR between 100 and 300 s after ionophore exposure and the subsequent slow but extended period of AR (600 sec to 1 h after the ionophore; Fig. 5d) may relate to additional events caused by this large non-physiological calcium load. At 1 h, the apparent ongoing occurrence of AR possibly reflects the early stages of cell death (Harper, unpublished data).

Visualization of AR

The ability to visualize P-induced AR in real time would provide valuable insights into the length and nature of the required stimulus, the duration of the reaction process, and the characteristics of the sperm after AR (e.g., Do the sperm remain motile/viable and for how long? Do any observable types or changes in motility occur in cells that undergo the acrosome reaction). Unfortunately, this has proven to be impossible with the acidic organelle probe LysoTracker DND-99, although results have previously been attained with mouse sperm using this dye [39]. The heterogeneity of staining may reflect differences in the intracellular and acrosomal pH values in the population of cells. Populations of rodent cells are generally more homogenous in their attributes. This species difference deserves further investigation, since it suggests the possibility that the human sperm acrosome is not maintained at a strongly acidic pH. The observation that A23187 causes a temporary increase in fluorescence of the probe, followed by a decrease in some cells, may relate to the properties of A23187. The ionophore not only transports calcium, but its action can be modulated by and possibly modulates pH [40], causing a change in accumulation of the dye.

Overall, the results of the present study suggest that there are significant variations in acrosomal status and competence for acrosome reactivity within a population of motile human spermatozoa. Whether this reflects varying degrees of capacitation (including intracellular Ca²⁺ concentration) or viability remains an open question. The ability to visualize AR should be a very useful tool in the future. Research on humans and other species is required to reveal more clearly the coupling and timing between the sperm-egg vestment interaction and acrosome dispersal events and to clarify the significance of when and how induction occurs.

ACKNOWLEDGMENTS

We acknowledge the assistance of the Birmingham Women's Healthcare NHS Trust, the Assisted Conception Unit, and our sperm donors in facilitating this study.

FOOTNOTES

³Current address: School of Biological Sciences, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, United Kingdom.

¹Supported by a Wellcome Trust Showcase Grant (060843). C.V.H. was a recipient of a Biotechnology and Biological Sciences Research Council quota studentship.

Correspondence: ² FAX: +44 151 795 4404; e-mail: claire.harper{at}liv.ac.uk

Received: 12 June 2006.

First decision: 29 June 2006.

Accepted: 1 September 2006.

REFERENCES

1. Yudin AI, Gottlieb W, Meizel S. Ultrastructural studies of the early events of the human sperm acrosome reaction as initiated by human follicular fluid. Gamete Res 1988; 20:11–24[CrossRef][Medline]
2. The Physiology of Reproduction. Yanagimachi R. Mammalian fertilization. 1994:New York: Raven Press;135–185. In:
3. Sabeur K, Edwards DP, Meizel S. Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction. Biol Reprod 1996; 54:993–1001[Abstract]
4. Elements of Mammalian Fertilization, vol. 1. Kopf GS and Gerton GL. The mammalian sperm acrosome and the acrosome reaction. 1991:Boston: CRC Press;153–203. In:
5. Florman HM. Sequential focal and global elevations of sperm intracellular Ca²⁺ are initiated by the zona pellucida during acrosomal exocytosis. Dev Biol 1994; 165:152–164[CrossRef][Medline]
6. Osman RA, Andria ML, Jones AD, Meizel S. Steroid-induced exocytosis: The human sperm acrosome reaction. Biochem Biophys Res Commun 1989; 160:828–833[CrossRef][Medline]
7. Hartshorne GM. Steroid production by the cumulus: relationship to fertilization in vitro. Hum Reprod 1989; 4:742–745[Abstract/Free Full Text]
8. Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and 17-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 1990; 265:1376–1380[Abstract/Free Full Text]
9. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human sperm. J Androl 1991; 12:323–330[Abstract/Free Full Text]
10. Oehninger S, Sueldo C, Lanzendorf S, Mahoney M, Burkman LJ, Alexander NJ, Hodgen GD. A sequential analysis of the effect of progesterone on specific sperm functions crucial to fertilization in vitro in infertile patients. Hum Reprod 1994; 9:1322–1327[Abstract/Free Full Text]
11. JBFS 2: Hum Reprod 1997; 12 Natl. Aitken RJ. The extragenomic action of progesterone on human spermatozoa. suppl:38–42
12. Meizel S, Turner KO, Nuccitelli R. Progesterone triggers a wave of increased free calcium during the human sperm acrosome reaction. Dev Biol 1997; 182:67–75[CrossRef][Medline]
13. Bronson RA, Peresleni T, Golightly M. Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining. Mol Hum Reprod 1999; 5:507–512[Abstract/Free Full Text]
14. Harper CV, Kirkman-Brown JC, Barratt CLR, Publicover SJ. Encoding of progesterone stimulus intensity by intracellular [Ca²⁺] ([Ca²⁺]_i) in human spermatozoa. Biochem J 2003; 372:407–417[CrossRef][Medline]
15. Benoff S, Hurley IR, Mandel FS, Cooper GW, Hershlag A. Induction of the human sperm acrosome reaction with mannose-containing neoglycoprotein ligands. Mol Hum Reprod 1997; 3:827–837[Abstract/Free Full Text]
16. Blackmore PF and Eisoldt S. The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa. Mol Hum Reprod 1999; 5:498–506[Abstract/Free Full Text]
17. Thomas P and Meizel S. Phosphatidylinositol 4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent upon Ca²⁺ influx. Biochem J 1989; 264:539–546[Medline]
18. Kirkman-Brown JC, Bray C, Stewart PM, Barratt CLR. Biphasic elevation of [Ca²⁺]_i in individual human spermatozoa exposed to progesterone. Dev Biol 2000; 222:326–335[CrossRef][Medline]
19. Fertilization in Mammals. Norwell: Serono Symposia Meizel S, Pillai M, Diaz-Perez E, Thomas P. Initiation of the human sperm acrosome reaction by components of human follicular fluid and cumulus secretions including steroids. 1990:205–222. In:
20. Nakanishi T, Ikawa M, Yamada S, Parvinen M, Baba T, Nishimune Y, Okabe M. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett 1999; 449:277–283[CrossRef][Medline]
21. Nakanishi T, Ikawa M, Yamada S, Toshimori K, Okabe M. Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation. Dev Biol 2001; 237:222–231[CrossRef][Medline]
22. Kim KS and Gerton GL. Differential release of soluble and matrix components: evidence for intermediate states of secretion during spontaneous acrosomal exocytosis in mouse sperm. Dev Biol 2003; 264:141–152[CrossRef][Medline]
23. Lee MA and Storey BT. Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca²⁺ permeability: population and single cell kinetics. Gamete Res 1989; 24:303–326[CrossRef][Medline]
24. Cross NL and Razy-Faulkner P. Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone. Biol Reprod 1997; 56:1169–1174[Abstract]
25. Santi CM, Santos T, Hernandez-Cruz A, Darszon A. Properties of a novel pH-dependent Ca²⁺ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function. J Gen Physiol 1998; 112:33–53[Abstract/Free Full Text]
26. Rockwell PL and Storey BT. Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca²⁺ changes monitored by fluo-3. Mol Reprod Dev 2000; 55:335–349[CrossRef][Medline]
27. Amin AH, Bailey JL, Storey BT, Blasco L, Heyner S. A comparison of three methods for detecting the acrosome reaction in human spermatozoa. Hum Reprod 1996; 11:741–745[Abstract/Free Full Text]
28. Cross NL, Morales P, Overstreet JW, Hanson FW. Induction of acrosome reactions by the human zona pellucida. Biol Reprod 1988; 38:235–244[Abstract]
29. Luconi M, Bonaccorsi L, Maggi M, Pecchioli P, Krausz C, Forti G, Baldi E. Identification and characterization of functional nongenomic progesterone receptors on human sperm membrane. J Clin Endocrinol Metab 1998; 83:877–885[Abstract/Free Full Text]
30. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescent properties. J Biol Chem 1985; 260:3440–3450[Abstract/Free Full Text]
31. Gan WB, Bishop DL, Turney SG, Lichtman JW. Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye. J Neurosci Methods 1999; 93:13–20[CrossRef][Medline]
32. Betz WJ and Bewick GS. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 1992; 255:200–203[Abstract/Free Full Text]
33. O'Toole CM, Arnoult C, Darszon A, Steinhardt RA, Florman HM. Ca²⁺ entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction. Mol Biol Cell 2000; 11:1571–1584[Abstract/Free Full Text]
34. Gonzalez-Martinez MT, Galindo BE, de De La Torre L, Zapata O, Rodriguez E, Florman HM, Darszon A. A sustained increase in intracellular Ca²⁺ is required for the acrosome reaction in sea urchin sperm. Dev Biol 2001; 236:220–229[CrossRef][Medline]
35. Hirohashi N and Vacquier VD. Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle. Biochem Biophys Res Commun 2003; 304:285–292[CrossRef][Medline]
36. Somanath PR, Suraj K, Gandhi KK. Caprine sperm acrosome reaction: promotion by progesterone and homologous zona pellucida. Small Ruminant Research 2000; 37:279–286[CrossRef][Medline]
37. Harper CV, Barratt CLR, Publicover SJ. Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca²⁺]_i oscillations and cyclical transitions in flagellar beating. J Biol Chem 2004; 279:46315–46325[Abstract/Free Full Text]
38. Tesarik J and Mendoza C. Defective function of a nongenomic progesterone receptor as a sole sperm anomaly in infertile patients. Fertil Steril 1992; 58:793–797[Medline]
39. Moreno RD, Ramalho-Santos J, Chan EK, Wessel GM, Schatten G. The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations. Dev Biol 2000; 219:334–349[CrossRef][Medline]
40. Erdahl WL, Chapman CJ, Taylor RW, Pfeiffer DR. Effects of pH conditions on Ca²⁺ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophys J 1995; 69:2350–2363

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