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Biology of Reproduction 60, 683-690 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Maintenance of Motility in Mouse Sperm Permeabilized with Streptolysin O1

Linda R. Johnsona, Stuart B. Moss2,a, and George L. Gerton2,3,a

a Center for Research on Reproduction & Women's Health and the Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6080


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [{gamma}-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Permeabilization of intact sperm and the subsequent reactivation of motility is a powerful experimental approach for studying the regulatory mechanisms that govern sperm movement. In previous studies of sperm motility, permeabilization has been accomplished primarily by treating the sperm with non-ionic detergents [1–14]. Although this method has been successfully used to study protein phosphorylation as it relates to motility, detergent treatment has the major disadvantages that membranes, membrane proteins, and many intracellular proteins are solubilized.

An alternative approach to permeabilization with detergents is the use of bacterial toxins. Streptolysin O (SLO) is a cytolytic streptococcal secretory protein with a molecular weight of 69 000 [15]. The SLO protein permeabilizes cells by interacting with cholesterol in the plasma membrane [16–19], aggregating within the lipid bilayer, and forming stable pores [20]. The pores give continuous access to the intracellular milieu without stripping the membrane. Since SLO forms pores in the membranes, the membrane remains associated with the sperm, and signal transduction pathways may be initiated through constituents of the membrane such as cell-surface receptors [21]. As in detergent-treated models, relatively large molecules can be introduced into the cells, and the cells can be stimulated with specific agents [21]. Recently, SLO treatment of mouse sperm has proven to be a potential model system for studying the signal transduction pathways that mediate the acrosome reaction [22].

The purpose of this study was to develop a method to permeabilize mouse sperm plasma membranes while maintaining flagellar movement. Optimal permeabilization of the mouse sperm plasma membrane occurred with 0.6 U/ml of SLO. Plasma membrane integrity was assessed by the effects of the toxin on sperm motility, the accumulation of fluorescent dyes within the cells, and the increase in overall protein phosphorylation. Permeabilization by SLO was dependent on the preincubation of sperm in MJB medium containing Ca2+. If permeabilization occurred in the presence of a medium containing 1 mM ATP, sperm treated with SLO maintained flagellar movement such that the percentage of motile SLO-treated sperm was similar to the levels seen in the nontreated controls. The permeability of the sperm treated with SLO in the presence of 1 mM ATP was confirmed by fluorescence staining patterns indicative of permeabilized plasma membranes. This study demonstrates that permeabilization of sperm with SLO is an attractive method for studying sperm motility by introducing reagents into cells without the indiscriminate extraction of membranes that occurs with non-ionic detergents.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Spermatozoa preparation

Caudae epididymides from (C57/B6xC3H) F1 males (The Jackson Laboratory, Bar Harbor, ME) were excised, minced, and incubated in MJB medium (109 mM NaCl; 25 mM HEPES; 25 mM sodium lactate; 25 mM NaHCO3; 5.6 mM glucose; 5 mM KCl; 1.2 mM MgCl2; 1 mM sodium pyruvate; 0.85 mM CaCl2; 50 µg/ml gentamicin; pH 7.5) for 15 min at 37°C in a humidified incubator with 5% CO2:95% air. In some experiments, the CaCl2 concentration was varied. After a 15-min swim-out period, the tissue was removed and the percentage of motile sperm and the sperm concentration were determined with a hemocytometer. All animal experimentation was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC).

Spermatozoa Permeabilization

Each bottle containing 40 U of SLO (Murex Diagnostics, Dartford, UK) was resuspended in 20 ml of ice-cold 1 mM dithiothreitol (DTT). A stock solution of 80 U/ml was obtained by reducing the 20-ml sample to 500 µl with a Centriprep-30 ultrafiltrater (Amicon, Beverly, MA). The solution in the ultrafiltrater was centrifuged in 10-min increments at 4°C, and the ultrafiltrater was decanted until the sample was reduced to 500 µl. Aliquots of the SLO stock solution were quick-frozen in a dry ice-ethanol bath and stored at -80°C for up to 1 mo before using.

To permeabilize the sperm membranes, an aliquot of the stock SLO solution was diluted with MJB medium to a final concentration of 10 U/ml. Sperm samples that had been incubated at 37°C were counted using a hemocytometer. The samples were diluted to approximately 5 x 106 cells/ml and then treated with various concentrations of SLO. For the Ca2+-dependence experiments, sperm were isolated and incubated in MJB medium containing various concentrations of CaCl2.

Fluorescence Microscopy

Sperm membrane permeabilization was assessed by fluorescence microscopy using the membrane-impermeant red ethidium homodimer nuclear dye (Molecular Probes, Inc., Eugene, OR) and the membrane-permeant green 2'7'-bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein acetoxymethyl ester dye (BCECF-AM; Sigma, St. Louis, MO), which becomes highly fluorescent and membrane-impermeant when cleaved by intracellular esterases. To stain the cells, 20-µl sperm samples were incubated with 2 µl of PBS containing 4 µM ethidium homodimer dye and 40 µM BCECF-AM dye for 20 min in the dark. Aliquots of the stained samples were diluted 1:1 with PBS and transferred to a slide. Slides were viewed with a Zeiss Photomicroscope III (Carl Zeiss, Inc., Thornwood, NJ) equipped with epifluorescence and photographed with Kodak T-Max P3200 (Eastman Kodak Co., Rochester, NY).

Indirect Immunofluorescence

One-milliliter aliquots of control and SLO-treated sperm were washed in MJB, resuspended to 5 x 106 cells/ml, and transferred to coverslips. After settling for 15 min, the cells were fixed in 4% (w:v) paraformaldehyde, washed, and blocked in normal goat serum as previously described [23]. The sperm samples were incubated with monoclonal antibody HS19 (a gift from Katie Bechtol, Genentech Inc., South San Francisco, CA) diluted 1:1000 (v:v) in 10% goat serum for 1 h at 37°C. The samples were washed in PBS and then incubated with secondary antibody (fluorescein isothiocyanate [FITC]-conjugated goat anti-mouse IgG; Life Technologies Inc., Gaithersburg, MD). The coverslips were washed in PBS before being mounted on slides with Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL). Slides were viewed with a Zeiss Photomicroscope III equipped with epifluorescence. Photographs were taken with Kodak T-Max Film, 3200 ASA.

Motility Assessment and Maintenance

Sperm samples treated with SLO, and the appropriate controls were observed microscopically. A hemocytometer was used to count at least 100 cells to calculate the percentage of motile sperm. Any flagellar movement was scored as motile.

To maintain sperm motility during the permeabilization step, sperm were permeabilized with SLO in the presence of motility maintenance medium (MMM: MJB containing 1 mM DTT; 1 mM MgCl; 2 mM EGTA; 1 mM or 5 mM ATP; 10 µM cAMP). Sperm with any flagellar movement were scored as motile.

Phosphorylation of Endogenous Sperm Proteins

Permeabilization of sperm for protein phosphorylation experiments was carried out as described for the motility maintenance experiments except that the concentration of ATP in the MMM was reduced to 200 µM and permeabilization occurred in the presence of [{gamma}-32P]ATP. After incubating for various times, the reactions were stopped by treating the samples with SDS sample buffer that contained 40 mM DTT and 100 µM sodium vanadate. The samples were boiled for 5 min and separated by SDS-PAGE on a 10% polyacrylamide gel. The gel was silver-stained, dried, and then exposed to Reflection film (New England Nuclear, Boston, MA).

To determine the total incorporation of 32P, sperm proteins from SLO and [{gamma}-32P]ATP-treated samples and controls were precipitated with trichloroacetic acid (TCA) [24]. Ten microliters of sample were transferred to 10 x 10-mm squares of Whatman (Clifton, NJ) filter paper. The filters were treated for 5 min with 10% TCA. While under the hood, the filters were treated for 5 min with 5% TCA at 90°C and then for 5 min at room temperature. They were sequentially washed for 5 min each at room temperature with 95% ethanol, 95% ethanol:acetone (1:1), and acetone. The filters were air-dried, transferred to vials containing 1 ml of scintillation fluid, and counted in a scintillation counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Percentage of Motile Sperm Decreased after Treatment with Streptolysin O

SLO binds to plasma membranes and forms stable pores [16, 17, 20]. Pore formation within the sperm plasma membrane will subsequently allow the release of ATP and cAMP, the components necessary for sperm motility, and other small molecules as well as soluble proteins capable of passing through pores with an internal radius of up to 26 nm [25, 26]. In the case of sperm, the effect should be manifested as a decrease in motility.

To determine whether SLO treatment affects sperm motility, mouse sperm were treated for 0, 15, 30, and 45 min at 37°C with 0.2, 0.6, 1.0, and 5.0 U/ml of SLO in MJB containing 1.7 mM CaCl2 (5 U/ml of SLO and the 500 µM DTT control were incubated for 30 min). The samples were diluted 1:20 with MJB medium, and the percentages of motile sperm were determined (Fig. 1, A–D). No effect on motility was observed with 0.2 U/ml SLO (Fig. 1A). After 5 min of treatment, there was a 50% decrease in the percentage of motile sperm in samples that had been treated with 0.6 U/ml of SLO (Fig. 1B). In these samples, the percentage of motile sperm was maximally reduced after 30 min of incubation with SLO. However, maximal inhibition of motility occurred at 5 min, when sperm were treated with 1 U/ml and 5 U/ml of SLO (Fig. 1, C and D).



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  		FIG. 1. The effects of SLO on motility as a function of the length of toxin exposure. A) 0.2 U/ml SLO and 20 µM DTT control; B) 0.6 U/ml SLO and 60 µM DTT control; C) 1 U/ml SLO and 100 µM DTT control; D) 5 U/ml SLO and 500 µM DTT control. While 0.2 U/ml had no effect on the percentages of motile sperm, with 0.6 U/ml, the percentages of motile sperm dramatically declined over the time course of the experiment. Maximal inhibition occurred after 30 min of incubation with SLO (B). By 5 min of treatment, 1 U/ml (C) or 5 U/ml (D) of SLO maximally inhibited motility. Squares, no DTT; circles, DTT as indicated, no SLO; diamonds, SLO as indicated and DTT as indicated in DTT-alone control. n = 4; mean ± SEM; no error bar indicates SEM less than 2.

To maintain SLO in the active reduced form, DTT was present in all samples. DTT (without SLO) at concentrations of 20–500 µM had no effect on the percentage of motile sperm (Fig. 1, A–D). However, the high DTT concentrations of 100 µM and 500 µM present in the 1 U/ml and 5 U/ml SLO samples, respectively, caused the flagellum of some of the sperm to fold back on itself (data not shown). This folding was not observed in sperm that were treated with solutions containing 20 µM or 60 µM DTT. Since 0.6 U/ml of SLO (containing 60 µM DTT) had profound inhibitory effects on sperm motility but not flagellar folding, this concentration was chosen for further study.

Inhibitory Effects of SLO on Sperm Motility Required a Preincubation Period

In some experiments, after a 5-min incubation, concentrations as high as 5 U/ml of SLO had no effect on sperm motility. However, inhibition of motility was observed when the sperm were exposed to SLO for a more extended period of time (data not shown). This inconsistency suggested that either the SLO stock solution became inactive, or events occurring during the incubation affected the sperm plasma membrane and were important for SLO activity. It is unlikely that the SLO stock solution was inactive since there was a reduction in the percentage of motile sperm after further incubation. For this reason, we focused on events occurring during the incubation period.

To determine whether a preincubation in MJB medium affected the ability of the SLO to inhibit sperm motility, mouse sperm were preincubated for 60 min in MJB lacking toxin. They were then treated with 0.2 U/ml and 0.6 U/ml of SLO for 5 min at the times indicated in Figure 2. After 5 min of treatment, with 0.2 U/ml of SLO (Fig. 2A) or a control containing 20 µM or 60 µM DTT (Fig. 2, A and B), there were no effects on the percentage of motile sperm. The percentage of motile sperm remained unaffected even after an extended preincubation period. However, after treatment with 0.6 U/ml of SLO for 5 min, there was a consistent and dramatic decrease in the percentage of motile sperm (Fig. 2B). Extended treatment with SLO did not increase the inhibition of sperm motility. Thus, 0.6 U/ml of SLO was sufficient to inhibit sperm motility after a 60-min preincubation in MJB medium containing 1.7 mM Ca2+. In addition, the results suggest that the preincubation period facilitates SLO permeabilization.



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  		FIG. 2. The effects of SLO on sperm that had been preincubated in MJB medium. After a 1-h preincubation, there were no effects when the samples were treated for 5 min with 0.2 U/ml of SLO (A) or with DTT alone (20 µM; 60 µM) (A, B). However, at 0.6 U/ml of SLO, maximal inhibition of sperm motility occurred after treating for only 5 min (B). Squares, no DTT; circles, DTT as indicated, no SLO; diamonds, SLO as indicated and DTT as indicated in DTT-alone control. n = 4; mean ± SEM; no error bar indicates SEM less than 2.

Effects of SLO Were Ca2+-Dependent

Pore formation by some cytolytic proteins is inhibited by Ca2+ [27, 28], so we sought to determine whether the effects of SLO on sperm motility were Ca2+-sensitive. Sperm were preincubated for 1 h in MJB medium containing 0 mM, 0.25 mM, 0.5 mM, 0.85 mM, 1 mM, and 1.7 mM CaCl2 and then treated with 0.6 U/ml of SLO for 5 min. When samples were preincubated in Ca2+-free medium, the SLO had only a slight effect on sperm motility (Fig. 3). However, the percentage of motile sperm decreased as the Ca2+ concentration increased. Maximal inhibition occurred when samples were preincubated in MJB medium containing at least 0.85 mM Ca2+.



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  		FIG. 3. Ca2+ dependency of the SLO effect. Samples that were preincubated in MJB medium for 1 h in the presence of various concentrations of Ca2+ and then treated with 0.6 U/ml of SLO for 5 min demonstrated maximal inhibition of sperm motility with concentrations of exogenous Ca2+ exceeding 0.85 mM. n = 4; mean ± SEM; no error bar indicates SEM less than 2.

Ca2+ appeared to act directly on the sperm and not via an association with the SLO. When sperm were preincubated in MJB medium containing CaCl2 and then washed in Ca2+-free medium before SLO treatment, the toxin inhibited motility (data not shown). Likewise, when samples were preincubated in medium containing low Ca2+ concentrations and then were treated with a higher SLO concentration (1.2 U/ml), sperm motility was greatly affected. However, sperm motility was never affected when exogenous Ca2+ was not present in the preincubation medium. These data demonstrate that SLO effects on sperm motility were Ca2+ dependent.

Fluorescence Microscopy Detected SLO Permeabilization of the Sperm Plasma Membrane

To determine whether SLO permeabilized the sperm membranes, samples were preincubated in MJB medium containing 0.85 mM Ca2+ and then treated with 0.6 U/ml of SLO for 5 min. The percentages of motile sperm were determined, and then the samples were treated with fluorescent dyes. Sperm with intact plasma membranes did not stain with the ethidium homodimer; however, the head and tail regions accumulated the membrane-impermeant, green fluorescent product when the permeant BCECF-AM ester was cleaved by cytoplasmic esterases (Fig. 4, A and B). Sperm with permeabilized plasma membranes accumulated the membrane-impermeant red ethidium dye in the head region (Fig. 4A, arrowhead). Sperm with permeabilized plasma membranes (red heads) but intact mitochondrial membranes stained green in the midpiece (Fig. 4B). Under the conditions described, less than 20% of the sperm population treated with 0.6 U/ml of SLO remained motile (Fig. 4C). Concurrently, less than 20% of the population remained intact on the basis of their inability to exclude the ethidium dye or accumulate BCECF-AM (Fig. 4C). These data indicate that SLO permeabilized the sperm membrane, presumably through pore formation.



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  		FIG. 4. Assay of sperm plasma membrane integrity by fluorescence microscopy. Aliquots of the samples were processed for either fluorescence microscopy (A, B) or for determining the percentage of motile sperm (C). Intact sperm accumulated the green dye in both the head and tail regions (A, B). Permeabilized cells exhibited two staining patterns: an accumulation of the red dye only in the head region (A, arrowhead), or an accumulation of the red dye in the head region and an accumulation of the green dye in the midpiece region (B, arrowhead). An accumulation of the green dye in the midpiece was indicative of intact mitochondrial membranes. At least 100 cells from each sample were counted, and the percentages of intact sperm and the percentage of motile sperm were determined (C). The percentage of intact cells corresponded to the percentage of motile sperm. Less than 20% of the sperm population that was treated with SLO had intact membranes and were motile. n = 4; mean ± SEM. x500 (reproduced at 89%).

To determine whether the permeabilization was limited to the plasma membrane, the integrity of the acrosome was assessed. Sperm samples were preincubated for 1 h in MJB medium containing 0.85 mM Ca2+. The samples were then treated with 0.6 U/ml of SLO for 5 min, centrifuged to remove the SLO, and then processed for immunofluorescence with the anti-acrosomal monoclonal antibody HS19 (Fig. 5, A and B; arrowheads indicate a sperm lacking an intact acrosome). Under these conditions, approximately 75% of the SLO-treated sperm maintained intact acrosomes (Fig. 5C). This was similar to the level of intact sperm seen in the control sample. However, when samples were treated with SLO for 15 min, 44% of the sperm population lacked intact acrosomal membranes as determined by the lack of immunoreactivity with HS19 (Fig. 5C). These results demonstrate that a brief exposure to SLO did not greatly affect the acrosomal membranes such that the acrosomal contents were released.



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  		FIG. 5. The effects of SLO on acrosomal membrane integrity. Immunofluorescence with an acrosomal monoclonal antibody, HS19 (A), and the corresponding phase-contrast (B) views of epididymal sperm that were treated with 0.6 U/ml of SLO for 0 min, 5 min or 15 min after a preincubation in MJB medium containing 0.85 mM CaCl2. An arrowhead indicates a sperm lacking an intact acrosome. At least 100 cells were counted, and the percentages of sperm with intact acrosomes were determined (C). After a 5-min treatment with SLO, the majority of the sperm retained their acrosomal contents. However, after a 15-min treatment with SLO, there was a decrease in the percentage of intact acrosomes. n = 3; mean ± SEM. x1000.

Sperm Permeabilized by SLO in the Presence of Exogenous ATP Maintained Flagellar Movement

To determine whether the sperm samples that were permeabilized by SLO could maintain flagellar function, samples were simultaneously treated with SLO and MMM. As shown above, when sperm were treated with SLO alone, the percentage of motile sperm was reduced (Fig. 6). However, when the sperm were simultaneously treated with SLO and MMM, the percentage of motile sperm was maintained at a level similar to that seen in untreated sperm. Sperm rendered immotile by SLO treatment were reactivated when treated with MMM (data not shown). MMM in the absence of SLO had no effect on the percentage of motile, intact sperm.



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  		FIG. 6. Maintenance of sperm motility in the presence of exogenously added ATP. Without the MMM (containing 1 mM exogenous ATP), less than 20% of the sperm population remained motile. Sperm treated with both SLO and MMM maintained a percentage of motile sperm similar to that observed in the control. n = 4; mean ± SEM.

To confirm that plasma membrane permeabilization occurred when sperm were treated simultaneously with SLO and MMM, samples were assayed by fluorescence microscopy. While over 70% of the population in the control samples (no SLO treatment) maintained intact membranes, only 20% of the SLO-treated samples maintained intact membranes (Fig. 7). These data indicate that the MMM did not interfere with the action of SLO and that flagellar activity was maintained by providing access to exogenous ATP through the SLO pores.



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  		FIG. 7. The effects of the MMM on membrane permeabilization. In the presence of 0.6 U/ml of SLO, motility was not maintained in the absence of exogenous ATP, but in the presence of MMM (containing 1 mM exogenous ATP), more than 70% of the sperm population maintained flagellar movement. MMM alone had no effect on the percentage of intact cells; however, in the presence of SLO, less than 20% of the sperm populations exhibited intact membranes. n = 4; mean ± SEM.

Although sperm motility was maintained, the quality of movement was different from that in the control samples. Sperm that had been treated with SLO and MMM were slower, and the flagellar waveform was attenuated. The amplitude of the wave was not as pronounced as that seen in the controls. Nonetheless, any flagellar movement is evidence of axoneme and accessory structure function.

Total Macromolecular Incorporation of 32P from Exogenous [{gamma}-32P]ATP Increased When Sperm Were Permeabilized by SLO

Demembranated sperm systems frequently have been used to determine the phosphorylation status of proteins under various conditions [1–3, 5–10]. To determine whether SLO stimulated an increase in the overall sperm protein phosphorylation, control sperm and sperm that were treated with 0.6 U/ml of SLO were exposed simultaneously to [{gamma}-32P]ATP (5 Ci/mmol; final ATP concentration approximately 200 µM) for 0–5 min at 37°C. Under these conditions, there was little flagellar movement. To determine total 32P incorporation into macromolecules, the samples were precipitated with TCA in the presence of 100 µM sodium vanadate. As shown in Figure 8, there was an essentially linear rate of increase in the overall level of 32P incorporation as a function of time in SLO-treated sperm. Sperm that were not treated with SLO did not show a time-dependent increase of 32P incorporation. The incorporation of 32P into TCA-precipitable products in SLO-treated sperm was reflected in the quantitative increase in the phosphorylation of many proteins as shown by SDS-PAGE (data not shown).



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  		FIG. 8. The effects of SLO on the total incorporation of 32P from [{gamma}-32P]ATP. There was a continuous increase in 32P incorporation with time in the samples treated with SLO. The level of 32P incorporation in the control samples remained relatively constant. Experiment was repeated twice. Shown is a representative figure.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Permeabilization of intact cells provides a powerful experimental approach for studying cell function. Conventional methods for permeabilizing sperm membranes involve treating the sample with non-ionic detergents such as Triton X-100 [1–14]. Although these reagents have been used successfully, the membranes as well as their integral and surface-associated components are stripped from the sperm. Such protocols may not be appropriate for studying some sperm functions. Often it is the surface or membrane components that are triggered by extracellular factors to initiate signal transduction pathways that stimulate or inhibit cell function [29–38]. In demembranated systems, it is impossible to study the roles that the membrane components play in sperm function since membrane proteins and lipids are removed by the detergent extraction.

In other cell types, an effective permeabilization agent has been SLO [21]. This toxin gives access to the intracellular milieu but still allows signal transduction events to be initiated by extracellular stimulation via an association with the cell surface components, i.e., receptors [21]. By creating pores as large as 26 nm, SLO treatment can allow relatively large molecules such as antibodies or Fab fragments into the cell [25, 26, 39]. This approach would also be useful for introducing small molecules such as activators or inhibitors of specific enzyme activities.

Since our goal was to develop a method to permeabilize mouse sperm while maintaining intact membranes, we examined the use of SLO permeabilization of mouse sperm. Permeabilization was assessed by the effects of SLO on the percentage of motile sperm and by fluorescence microscopy with dyes sensitive to the integrity of the plasma membrane. Optimal permeabilization was dependent on SLO concentration, Ca2+ levels, sperm preincubation, and the length of SLO treatment.

Maximal and consistent permeabilization was achieved with 0.6 U/ml of SLO with samples that were preincubated in MJB medium containing 0.85 mM Ca2+. Although the percentage of motile sperm was reduced in samples that were treated with higher SLO concentrations, the high DTT concentrations (100 µM and 500 µM) in these samples caused the flagellum to fold back upon itself. Such a deleterious effect of DTT on the flagellum has been previously described by Cornwall and Chang [40]. However, we did not see this effect in samples that contained lower concentrations (20 µM or 60 µM) of DTT.

The Ca2+ concentration dependency on pore formation by SLO was unexpected. Although pore formation by some cytolytic proteins can be inhibited by Ca2+ [41–43], in our experimental system, the SLO effect on sperm directly correlated with Ca2+ concentrations. When sperm were preincubated in medium lacking Ca2+ and then were treated with SLO, they remained motile and accumulated the BCECF-AM esterolysis product, indicating that the sperm plasma membranes were intact. SLO treatment in the presence of Ca2+ rendered the sperm immotile; furthermore, the sperm accumulated the ethidium dye, which indicated that the plasma membranes were permeabilized. In other systems, Ca2+ is carefully removed with chelating agents and also is used to stop the formation of pores by some cytolytic proteins such as alpha toxin [28]. One might argue that sperm treated with SLO in the absence of calcium are actually permeabilized and remain motile, but if the permeabilization is carried out in the presence of exogenously added Ca2+, the sperm become immotile due to the detrimental effects of increased intracellular calcium concentrations on sperm motility. However, it is unlikely that sperm that were preincubated in Ca2+-free medium are truly permeabilized because these cells excluded the ethidium dye and accumulated the BCECF-AM esterolysis product, a finding that is indicative of an intact plasma membrane.

The requirement for a preincubation in a Ca2+-containing medium could indicate that there are changes at the plasma membrane level that allow the SLO to interact with cholesterol and form pores [44–46]. Although Díaz et al. [22] demonstrated that mouse sperm can be permeabilized by SLO in the absence of a preincubation, the sperm in their protocol were washed with a hypertonic buffer prior to SLO treatment. Under those conditions, membrane-associated factors could have been removed or the membrane could have been modified in a substantial way. In our experiments using 0.6 U/ml, sperm that had not been preincubated in MJB containing Ca2+ were not affected by SLO treatment. However, when sperm were preincubated in MJB containing Ca2+ and then were washed free of Ca2+, they were permeabilized by SLO and became immotile. Thus, it is likely that Ca2+ affects sperm directly and does not cause these effects via an association with SLO.

The acrosomal status of SLO-treated sperm was investigated to determine the extent to which the cell membranes were permeabilized. After a 5-min incubation with 0.6 U/ml of SLO, more than 78% of the sperm preincubated in MJB medium containing 0.85 mM Ca2+ maintained intact acrosomes as assessed by immunofluorescence with the HS19 monoclonal antibody (Fig. 5). However, when the samples were incubated for 15 min with SLO, there was an decrease in the percentage of sperm (~44%) that immunoreacted with the HS19 antibody. This finding suggests that with increasing times of incubation with SLO, sperm begin to lose their acrosomes as a consequence of the plasma membrane permeabilization. Díaz et al. [22] reported similar results when Ca2+ was added to their system.

In our system, the majority of the sperm population remained acrosome-intact after 5 min of SLO treatment in the presence of 0.85 mM Ca2+, suggesting that although the sperm were permeabilized, permeabilization was limited to the plasma membrane. In addition, the data suggest that 5 min of incubation was not sufficient to trigger the acrosome reaction. In other studies, pretreatment of cells at 4°C followed by washing and shifting to a higher temperature limited permeabilization to the plasma membrane [17, 20, 22, 47]. At this temperature, SLO binds to the plasma membrane without aggregation, a prerequisite for pore formation. As a result, only the plasma membrane is affected. Unfortunately, both cold temperatures and centrifugation can be deleterious to mouse sperm. In our system, a brief exposure to SLO at 37°C limited permeabilization to the plasma membrane. This is significant since those deleterious treatments that can affect sperm function can be avoided.

The ability of SLO-treated sperm to maintain flagellar movement was investigated to determine whether this system could be used as a functional assay in studies of the regulatory mechanisms that govern sperm motility. In many demembranated systems, sperm are treated with detergents and then motility is reactivated by the addition of exogenous ATP [1–14]. To maintain flagellar movement, preincubated sperm were simultaneously treated with SLO and MMM containing 1 mM ATP. Under these conditions, the percentage of motile sperm remained comparable to that of the control samples. Similar results were observed when SLO-treated sperm were subsequently treated with MMM (data not shown). The ability of these SLO-treated sperm to be reactivated or to maintain flagellar movement is significant. An experimental model in which sperm are permeabilized without losing the membrane components provides an alternative to detergent extraction in studies of the mechanisms regulating sperm motility.

Movement of SLO-treated sperm flagella in the presence of exogenously added ATP was qualitatively different from that of the controls. The flagella of treated sperm beat slowly, and the waveform was attenuated. This type of movement has been previously described as partial activation in Ciona sperm [3]. It is not surprising that there was only partial reactivation of the flagellar apparatus since many ATPases are present in the membrane [48]. In detergent demembranation systems, ATPases are undoubtedly removed and rendered inactive. In our system, since the membrane components were probably present, the exogenously added ATP was probably quickly hydrolyzed. In support of this theory, when the concentration of MgATP- was increased 2-fold by adding an increased amount of exogenous ATP (5 mM) to sperm that were permeabilized with SLO, flagellar movement approached that of the controls. This response to increasing ATP concentrations is similar to that described by Ishijima and Witman [12]. An ATP regenerating system may remove the need to add such high ATP concentrations. Increasing the efficiency of protein phosphorylation with 32P under conditions whereby motility is maintained will enable us to identify and characterize proteins regulating flagellar movement. Studies in progress are examining the quantitative aspects of flagellar movement and reactivation that occurs in the presence of exogenous ATP.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Drs. Bayard T. Storey, Regina M. O. Turner, and Tayyab Rahil for critically reviewing the manuscript prior to submission.


   FOOTNOTES
 
1 Supported by NIH P01 HD-06274 (S.B.M. and G.L.G.) and T32 HD-07305 (L.R.J.). Back

2 Authors contributed equally to this work. Back

3 Correspondence: George L. Gerton, Center for Research on Reproduction and Women's Health, John Morgan Building, Room 306, University of Pennsylvania Medical Center, Philadelphia, PA 19104–6080. FAX: 215 349 5118; gerton{at}mail.med.upenn.edu Back

Accepted: October 14, 1998.

Received: August 20, 1998.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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