Molecular Human Reproduction, Vol. 5, No. 10, 941-949,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of sperm function |
An important role of actin polymerization in the human zona pellucida-induced acrosome reaction
1 Department of Obstetrics and Gynaecology and 2 Department of Medicine, University of Melbourne, 3 Andrology Laboratory and 4 Reproductive Biology Unit, Royal Women's Hospital, 132 Gratten Street, Carlton, Victoria 3053 and 5 Melbourne IVF, Melbourne, Australia
Abstract
The effects of inhibitors of actin polymerization and depolymerization, cytochalasins and phalloidin, on the human zona pellucida (ZP)-induced acrosome reaction (AR) were investigated. Motile spermatozoa, selected by swim-up technique from normozoospermic men, were incubated in medium with or without the actin modulators. Oocytes (four per test) which had failed to fertilize in vitro were added and incubation continued for 2 h. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small-bore pipette and the status of the acrosomes was determined by fluorescein-labelled Pisum sativum agglutinin (PSA). Double immunofluorescent staining with PSA and an anti-actin monoclonal antibody illuminated the acrosomal region of acrosome-intact spermatozoa. In calcium ionophore-induced AR spermatozoa, actin staining was confined to the equatorial segment, post-acrosomal region and tail. Cytochalasins B and D significantly inhibited ZP-induced AR in a dose-dependent manner (P < 0.001). Both inhibitors had no effect on the acrosome of spermatozoa in the insemination medium. Cytochalasin B or D (10–40 µmol/l) had no effect on total percentage motile spermatozoa but decreased sperm velocity and hyperactivation. Phalloidin had no effect on the ZP-induced AR or sperm motility. In conclusion, actin polymerization plays an important role in human ZP-induced AR.
acrosome reaction/actin/cytochalasin B and D/phalloidin
Introduction
The acrosome and the physiological acrosome reaction (AR) are very important for sperm–oocyte interaction during the process of human fertilization. Human spermatozoa without an acrosome do not bind to or penetrate the zona pellucida (ZP) or fuse with the oocyte plasma membrane in vitro (von Bernhardi et al., 1990
; Bourne et al., 1995). It is believed that acrosome-intact spermatozoa bind to the ZP and that the physiological AR occurs subsequently on the surface of the ZP, being triggered by ZP glycoproteins, such as ZP3 (Bleil and Wassarman, 1988; Tesarik, 1989; Yanagimachi, 1994; Wassarman, 1999). Artificially inducing the AR with the calcium ionophore A23187 reduces sperm–ZP binding (Liu and Baker, 1990).
That the AR on the ZP is important for subsequent ZP penetration is indicated by the finding of a highly significant correlation between the ZP-induced AR and sperm–ZP penetration in vitro (Liu and Baker, 1996a,b). Sperm–ZP penetration is blocked by inhibiting acrosin activity with trypsin inhibitors and the AR of spermatozoa bound on the ZP is reduced (Liu and Baker, 1993; Llanos et al., 1993). The requirement for the ZP-induced AR for human fertilization is further supported by our discovery of patients with disordered ZP-induced AR. In this condition there is severe infertility with failure of sperm–ZP penetration and failure of fertilization in vitro despite normal semen analysis and normal sperm–ZP binding (Liu and Baker, 1994). Preliminary studies indicate that up to 30% of patients with idiopathic infertility may have disordered ZP-induced AR (D.Y.Liu and H.W.G.Baker, unpublished data).
Research on the AR has mainly involved studies of the spontaneous AR and effects of chemical and biological inducers such as calcium ionophore A23187, human follicular fluid and progesterone (Tesarik, 1985; Aitken, 1993; Brucker, 1995). There are only a few studies of the AR induced by solubilized or intact human ZP (Cross et al., 1988; Coddington et al., 1990; Hoshi et al., 1993; Liu and Baker, 1993, 1996a, Liu and Baker, b; Bielfeld et al., 1994; Morales et al., 1994). We found that there was no correlation between calcium ionophore A23187 and human ZP-induced AR in vitro (Liu and Baker, 1996a,b). Therefore human ZP is needed to study the mechanisms of the human AR.
The human AR is a complex exocytotic process. The biochemical mechanisms are not fully understood. Although calcium influx plays a central role in the AR, many other factors including protein kinases A, G and C (PKA, PKG and PKC) and G-proteins have been implicated in the human AR (De Jonge et al., 1991; Breitbart et al., 1992; Rotem et al., 1992; De Jonge, 1995; Doherty et al., 1995; Tollner et al., 1995; Breitbart and Spungin, 1997; Liu and Baker, 1997). However, many studies of factors regulating the human AR were performed using non-physiological inducers such as A23187 or other biological materials such as progesterone. Our recent study using intact human ZP suggests that PKC plays a major role in the ZP-induced AR (Liu and Baker, 1997).
The subplasmalemmal actin cytoskeleton is involved in exocytosis in many cells including chromaffin, mammary epithelial, and mast cells (Koffer et al., 1990; Burgoyne et al., 1991). A role for the cytoskeleton in the human AR induced by mannose and progesterone has been reported (Benoff et al., 1996). Actin has been identified, mainly in the monomeric form, in the spermatozoa of many species: human, boar, bull and hamster (Clarke et al., 1982; Ochs and Wolf, 1985; Camatini et al., 1986; Flaherty et al., 1988). However, polymerized actin is also present in the plasma and outer acrosomal membranes of boar spermatozoa (Peterson et al., 1990; Castellani-Ceresa et al., 1993). A variety of studies implicate actin polymerization and depolymerization in fertilization. Inhibition of actin polymerization with cytochalasin D blocks guinea-pig and human sperm penetration into zona-free hamster eggs (Rogers et al., 1989). Cytochalasin D also inhibits boar fertilization in vitro (Castellani-Ceresa et al., 1993). Inhibition of actin depolymerization with phalloidin blocks isolated sperm membrane fusion, loss of polymerized actin from the membranes and the AR of spermatozoa exposed to A23187 (Spungin et al., 1995). The aim of this study was to determine if actin polymerization and depolymerization plays an important role in the ZP-induced AR in humans.
Materials and methods
Chemicals and culture medium
Cytochalasins B and D, anti-mouse IgG labelled with fluorescein isothiocyanate (FITC), dimethylsulphoxide (DMSO), A23187, phalloidin and Pisum sativum agglutinin labelled with FITC (PSA–FITC) or with tetramethylrhodamine B isothiocyanate (PSA–TRITC) were obtained from Sigma Chemical Company (St Louis, MO, USA). An anti-actin monoclonal antibody which binds to all forms of actin was purchased from ICN Pharmaceuticals Inc. (Costa Mesa, CA, USA). Human tubal fluid (HTF; Irvine Scientific, Irvine, CA, USA) medium supplemented with 0.5% bovine serum albumin (BSA; Commonwealth Serum Laboratory, Melbourne, Australia) was used for all the experiments.
Cytochalasin and phalloidin stock solutions (2 mmol/l) were prepared by dissolving them in DMSO. Small aliquots were stored at –70°C. On the day of the experiment, the stock solution was diluted 2 times with HTF medium and then 5–30 µl of the diluted cytochalasin or phalloidin was added into 0.5 ml sperm suspension to achieve final concentrations of 10–60 µmol/l. Similarly diluted DMSO was used for controls. Both cytochalasins B and D were used because cytochalasin B inhibits glucose uptake as well as actin polymerization and may reduce energy-dependent cell functions (Burgoyne et al., 1991).
Gamete preparation
Semen was obtained by masturbation after 2–3 days abstinence from men with normal semen analysis according World Health Organization criteria (WHO, 1992). Motile spermatozoa were selected by a swim-up technique as described previously (Liu and Baker, 1997). The motile spermatozoa were washed once with fresh HTF medium by centrifugation and then the sperm pellet was resuspended in fresh HTF medium to a sperm concentration of 2x106/ml for the experiments.
Oocytes which showed no evidence of two pronuclei or cleavage at 48–60 h after insemination in a clinical in-vitro fertilization (IVF) programme were used. Most of the oocytes displayed regular shape and had lost the cumulus and corona cells. In the remainder, the cumulus and corona cells were removed by aspiration with a glass pipette. If the oocyte had spermatozoa bound to the ZP from IVF, these were removed by aspiration of the oocyte with a glass pipette with an inner diameter (120 µm) slightly smaller than the oocyte diameter. Most of the oocytes were obtained from patients with partial failure of fertilization and more than 50% these unfertilized oocytes had a few spermatozoa penetrating into the ZP from IVF. We have shown previously that oocytes with spermatozoa in the ZP had an ability for sperm–ZP binding and ZP-induced AR similar to those without spermatozoa penetrating into the ZP (Liu and Baker, 1996b). Some of the oocytes were stored in 1 mol/l ammonium sulphate at 4°C (Yanagimachi et al., 1979; Liu and Baker, 1997). The salt-stored oocytes were washed in HTF medium with four changes of the medium at least 12 h before being used for sperm–ZP interaction tests.
All patients signed consent forms permitting use of their unfertilized oocytes for research. The patients and donors gave permission for their semen to be used for research. The project was approved by the Royal Women Hospital Research and Ethics Committees.
Experimental design
For actin antibody immunofluorescence, motile spermatozoa (2x106/ml) were incubated with 3 µmol/l ionophore A23187 to induce the AR. Dual-fluorescent stains of the actin antibody and PSA labelled with TRITC were performed on the same sperm smear to determine if there was a different anti-actin immunofluorescent pattern for acrosome-intact and -reacted spermatozoa. A23178 was used to ensure a high proportion of AR spermatozoa to aid examination of both acrosome-intact and -reacted spermatozoa in the same microscopic field of view.
For the ZP-induced AR studies, motile spermatozoa (2x106) were incubated in 1 ml HTF medium with or without (control) cytochalasins B or D or phalloidin at 37°C in 5% CO2 in air for 30 min before the addition of four fresh or salt-stored oocytes and incubation continued for 2 h. For each experiment, the same types of oocytes (fresh or salt-stored) were used for both inhibitor and control experiments. Four oocytes were used for each dose of the cytochalasins and phalloidin. The sperm–ZP binding, ZP-induced AR and the AR of spermatozoa in the insemination medium were assessed as described below. Sperm–ZP penetration was not determined since most of the oocytes used in this study had spermatozoa penetrating the ZP from the IVF inseminations.
Dual-fluorescent stains for actin and acrosome status
After incubation of motile spermatozoa without or with 3 µmol/l A23187 to induce the AR, spermatozoa were washed with 0.9% NaCl, smeared on a glass microscope slide and allowed to air-dry. Slides were then fixed in 95% ethanol for 30 min. The fixed sperm smear was placed in PSA–TRITC for 1 h and then incubated with the actin antibody (1:50) diluted in PBS with 1% BSA for 2 h in a humidified box at 37°C. Slides were washed in PBS to remove any excess actin antibody and then incubated with anti-mouse IgG–FITC for 1 h in a humidified box. Finally the slide was rinsed and mounted with distilled water. Immunofluorescent patterns of the anti-actin antibody and acrosomal status were examined with a fluorescence microscope (Dialux 20; Leitz, Wetzlar, Germany) using excitation at 450–490 nm for FITC and 546 nm for TRITC. Controls omitting the actin antibody or substituting it with an irrelevant monoclonal antibody resulted in no FTIC fluorescence on the spermatozoa.
Sperm–ZP binding and ZP-induced AR
After 2 h incubation of spermatozoa with oocytes, each group of four oocytes exposed to sperm suspensions with modulator or control conditions were transferred to PBS, pH 7.4, containing 2 mg/ml BSA and washed by aspiration in and out of a glass pipette (inside diameter ~250 µm) to dislodge spermatozoa loosely adhering to the surface of the ZP. Because a high concentration (2x106/ml) of spermatozoa was used in the insemination medium, the number of spermatozoa bound tightly to the ZP was usually >100 per oocyte. The actin modulators had no effect on sperm-ZP binding.
All spermatozoa bound to the surface of the ZP of each oocyte were then removed by vigorous aspiration in and out of a narrow gauge glass pipette with an inner diameter (~120 µm) slightly smaller than that of the oocyte (Liu and Baker, 1993, 1996b). This was performed on a glass slide with ~5 µl PBS containing 0.2% BSA and the zona-bound spermatozoa smeared in limited area (~4 mm2) and then the smear was marked with a glass pen to help find the spermatozoa under the microscope for acrosome assessment. This pipetting procedure removed all spermatozoa bound on the surface of the ZP and >1000 ZP-bound spermatozoa were obtained from the four oocytes. Our previous study confirmed that this pipetting procedure did not affect acrosome status or damage the spermatozoa severely as many remain motile after removal from the ZP (Liu and Baker, 1993).
Assessment of acrosome status
The acrosome status of spermatozoa was determined with fluorescein-labelled Pisum sativum agglutinin (PSA; Sigma Co., St Louis, MO, USA) as described previously (Cross et al., 1986; Liu and Baker, 1993). Sperm smears were fixed in 95% ethanol for 30 min after air-drying and then stained in 25 µg/ml PSA in PBS for 2 h. The slide was washed and mounted with distilled water and 200 spermatozoa per sample were counted with a fluorescence microscope and oil immersion at a magnification of x400. When more than half the head of a spermatozoon was brightly and uniformly fluorescing, the acrosome was considered to be intact. Spermatozoa with a fluorescing band at the equatorial segment or without fluorescence (a rare pattern) were considered to be acrosome-reacted. Acrosome status was scored blindly by one technician who did not know which samples had been exposed to modulators and which were controls.
Assessment of sperm motility and movement characteristics
After 2 h incubation of spermatozoa with cytochalasins B and D, or phalloidin, sperm motility, straight line velocity (VSL) and hyperactivation were measured by Hamilton–Thorn Motility Analyser (IVOS 10, 60 Hz; Hamilton–Thorn Research, Danvers, MA, USA). Because the sperm concentration was only 2x106/ml when the spermatozoa were incubated in culture medium, the sperm concentration was adjusted to ~20x106/ml by centrifugation at 800 g for 5 min and resuspension in 100 µl of the same medium. A sample of 5 µl was placed in a Microcell (20 µm depth) for computer-assisted sperm analysis (CASA) assessment. Criteria for hyperactivation were as follows: curvilinear velocity (VCL) 
100 µm, linearity (LIN) <65 and amplitude of lateral head displacement (ALH) 7.5 µm (Burkman et al., 1991). For each sperm sample, an average of five (four to six) fields with 200 spermatozoa (mean total number 300, range 200–477) were assessed.
For all ZP-induced AR experiments, percentage motility of spermatozoa in the insemination medium after 2 h incubaton was also assessed manually on 200 spermatozoa in each sample.
Statistical analysis
The significance of differences in percentages of motility, acrosome-reacted spermatozoa, hyperactivation with dose was examined by paired t-test or two-way analysis of variance and linear regression analysis. The relative effectiveness of cytochalasins B and D were determined as for a parallel line bioassay. For each sperm sample the zero-dose result was subtracted from that of the sperm exposed to the various doses of cytochalasin.
Results
Actin in the acrosomal area
Dual-fluorescent stains of actin and PSA were performed on three sperm samples before and after induction of AR with calcium ionophore A23187. In all samples the two-fluorescent labels produced the same pattern in the acrosomal region: the fluorescein-labelled actin antibody stained the acrosomal region of acrosome-intact spermatozoa but only the equatorial segment of AR spermatozoa (Figure 1). The fluorescein-labelled actin antibody also stained the post-acrosomal region midpiece and tail of all spermatozoa. No FITC fluorescence was observed on the control spermatozoa which had not been incubated with the actin antibody.
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Effect of cytochalasins B and D on sperm motility and the AR
At doses of <60 µmol/l, cytochalasins B and D had no effect on percentage motility or the spontaneous AR of spermatozoa in the insemination medium (Table I
). However, cytochalasins B and D inhibited ZP-induced AR in dose-dependent manner (Figure 2). Cytochalasin B was more active than cytochalasin D in inhibiting the ZP-induced AR (Figure 2). For half-maximal inhibition of ZP-induced AR, approximate concentrations for cytochalasin B and D were ~10 and ~20 µmol/l. Eight different sperm preparations were exposed to 40 µmol/l concentrations of both cytochalasins. The suppression of ZP-induced AR was significantly greater with cytochalasin B (Figure 3).
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Effect of phalloidin on sperm motility, spontaneous and ZP-induced AR
Phalloidin (10–40 µmol/l) had no effect on percentage motility, spontaneous AR of spermatozoa in insemination medium, sperm–ZP binding or ZP-induced AR (Table II
and Figure 4).
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Effect of cytochalasins B and D on sperm velocity and hyperactivation
At concentrations of 10–40 µmol/l cytochalasins B and D had no effect on percentage motility after 2 h incubation assessed by either manual or CASA methods (Table I
). However, VSL and hyperactivation were significantly reduced when concentrations of 20 µmol/l cytochalasin B or D were used, and cytochalasin B was more effective than cytochalasin D (Figure 5). Cytochalasin D had half the potency of cytochalasin B (0.5 95% confidence limits 0.3–0.9). For hyperactivation, the regression equation was y = –14 log dose + 6 (regression P = 0.10) for cytochalasin B and y = –10 log dose + 6 (regression P = 0.14) for cytochalasin D. The combined slope was significant (P = 0.030) and there was no significant non-linearity or non-parallelism but the potency estimate of cytochalasin D with respect to cytochalasin B is imprecise (0.3 95% confidence limits 0–86).
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Discussion
In general, the physical state of actin in the cell goes through a reversible cycle. First, monomeric G-actin polymerizes to form filamentous F-actin and the F-actin depolymerizes to regenerate G-actin (Aderem, 1992). This cycle is regulated by actin-binding proteins such as: cofilin (actin depolymerizing factor), profilin, villin, fragmin, ß-actinin, geoslin and depactin (Aderem, 1992; Carlier et al., 1997; Didry et al., 1998). Actin is complexed with membrane proteins and, as in other cells, contributes to surface regional specialization in spermatozoa (Virtanen et al., 1984). The subcortical actin network has been implicated in the regulation of exocytosis in some endocrine and secretory cells (Koffer et al., 1990; Burgoyne et al., 1991; Dudani and Ganz, 1996). The human physiological AR occurs on the surface of the ZP and involves multiple fusion between plasma and outer acrosomal membranes resulting in vesiculation and release of acrosomal enzymes. The AR is considered to be an exocytotic process and should therefore have mechanisms common with exocytosis in other cells. The present study confirms that actin is present in the acrosomal region of human spermatozoa, and that inhibition of actin polymerization by cytochalasins B and D blocks the ZP-induced AR. Inhibition of actin depolymerization with phalloidin had no effect on ZP-induced AR. The cytochalasins also inhibited hyperactivation at concentrations that did not have a general inhibitory effect on sperm motility.
The presence of actin has been demonstrated in many mammalian spermatozoa including human, bull, rabbit, mouse, guinea-pig, hamster and boar, but with variable localization and distribution (Clarke et al., 1982; Ochs and Wolf, 1985; Camatini et al., 1986; Flaherty et al., 1988; Virtanen et al., 1984; Fouquet and Kann, 1992; Moreno-Fierros, 1992). In guinea-pig spermatozoa, actin is mainly in the acrosome and tail regions (Moreno-Fierros, 1992). In contrast, in human spermatozoa, actin is present in most regions: acrosomal, post-acrosomal, neck and tail (Clarke et al., 1982; Virtanen et al., 1984; Flaherty et al., 1988; Peterson et al., 1990). In the sperm head most actin is in monomeric form and F-actin was difficult to demonstrate in the early studies. In some lower species G-actin present between the inner acrosomal and nuclear membranes polymerizes to form an acrosomal process (Tilney et al., 1973). Commonly used methods of fixation destroyed the plasma and outer acrosomal membranes, thus the presence of actin in this region was not clear initially (Fouquet and Kann, 1992). However, Peterson et al. (1990) demonstrated the presence of polymeric or oligomeric actin in the plasma and outer acrosomal membranes of boar spermatozoa by electron microscopy and by assay or blotting of membrane extracts. The presence of F-actin in these membranes has also been reported in bovine spermatozoa (Spungin et al., 1995; Yagi et al., 1995). The present study using dual-fluorescent stains for both actin and components of the acrosome confirms that actin is present between the plasma and outer acrosome membranes of human spermatozoa where it could be involved in the AR. The identical patterns with both PSA and the anti-actin monoclonal antibody localizing in the acrosomal region of acrosome-intact spermatozoa with loss of fluorescence in the anterior part of the acrosome region of acrosome-reacted spermatozoa indicates that the AR results in actin being lost or rendered unable to react with the antibody.
Previous studies of the role of actin in fertilization have produced an array of results. Inhibition of actin polymerization with 10–30 µmol/l cytochalasin D blocked guinea-pig and human sperm penetration into zona free hamster eggs, with an ED50 of 5 µmol/l (Rogers et al., 1989). However, no effect on the spontaneous AR or sperm motility assessed by CASA was seen (Rogers et al., 1989). These results suggest that actin polymerization was involved in sperm-oocyte fusion after the AR had occurred or, possibly, in intravitelline sperm head decondenzation. Castellani-Ceresa et al. (1993) showed that incubation of spermatozoa with 20 µmol/l cytochalasin D inhibited penetration of in-vitro matured porcine oocytes. They suggested that actin polymerization occurring during capacitation was important for fertilization because F-actin was not detectable using phalloidin immunoelectron microscopy when boar spermatozoa were incubated with cytochalasin D during capacitation and the A23187-induced AR. Also, by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, the loss of G-actin from post-AR detergent extracts of sperm membranes was blocked in the presence of cytochalasin D (Castellani-Ceresa et al., 1993). Spungin et al. (1995) reported that exposure of bovine spermatozoa to 10 µmol/l phalloidin inhibited the release of acrosin induced by exposure to A23187. Using a model system of plasma and outer acrosomal membrane fusion, they showed that phalloidin or exogenous actin blocked membrane fusion. In addition, assays of membrane extracts with FITC-labelled phalloidin indicated that F-actin increased with capacitation and decreased upon exposure to high calcium concentrations. The capacitation-related actin polymerization was blocked by phalloidin added during incubation. They also reported that phalloidin blocks A23187-induced fusion but not calcium uptake in bovine sperm membranes (Spungin et al., 1995; Spungin and Breitbart, 1996; Breitbart and Spungin, 1997). They suggested that polymerization of actin to form an F-actin network between the plasma and outer acrosomal membranes may hold phospholipase C at the membrane surface and that depolymerization of the F-actin network between the two membranes is essential for the AR (Breitbart et al., 1992; Spungin et al., 1995; Breitbart and Spungin, 1997). Moreno-Fierros et al. (1992) reported that F-actin may play a role in calmodulin translocation during the AR of guinea-pig spermatozoa.
The present results are consistent with and could explain the results of Castellani-Ceresa et al. (1993). Although both cytochalasins B and D are potent inhibitors of polymerization of actin, D is more specific than B for inhibition of actin polymerization. Cytochalasin B also inhibits glucose uptake and may reduce energy-dependent cell functions (Burgoyne et al., 1991). This may explain why B is more effective than D in inhibition of ZP-induced AR, VSL and hyperactivation in the present study. However, taken together the cytochalasin results indicate that actin polymerization plays an important role in the physiological AR in humans.
There is a difference between the present results on actin depolymerization and those of Spungin et al. (1995). We found no effect of phalloidin on the human ZP-induced AR whereas they showed inhibition of sperm membrane fusion. It is reported for other cells that although phalloidin binds to F-actin and stabilizes it, phalloidin may not be capable of preventing cortical actin network disassembly with increased calcium concentration or the effect of gelsolin (Verkhovsky et al., 1984; Koffer et al., 1990). Furthermore, another actin-binding protein, cofilin, is known to be a potent regulator of actin filament dynamics through induction of depolymerization (Moon and Drubin, 1995; Carline et al., 1997; Therriot, 1997). Both calcium influx and cofilin can stimulate depolymerization of F-actin, and rapid turnover of F-actin induced by cofilin is important for motility and exocytosis (Hawkins et al., 1993; Hayden et al., 1993; Carlier et al., 1997; Lappalainen and Drubin, 1997; Didry et al., 1998). Therefore, the apparent discrepancy with the Spungin et al. (1995) results — lack of effect of phalloidin on ZP-induced AR in the present study — may be related to insufficient inhibition of depolymerization of F-actin by phalloidin. Alternatively, depolymerization may be less important than the initial polymerization for the human AR. Further study on the dynamics of actin polymerization and depolymerization is needed to determine if actin depolymerization is important for the human ZP-induced AR.
In the present study, cytochalasins B or D at concentrations 
40 µmol/l had no effect on percentage motility or sperm–ZP binding but significantly decreased sperm VSL and percentage hyperactivation. In contrast, it has been shown that 10–30 µmol/l cytochalasin D had no effect on motility and velocity of spermatozoa from four subjects (Rogers et al., 1989). However, the experimental conditions were different. They used a high sperm concentration and overnight incubation in capped plastic tubes in an incubator without CO2. This might explain the low (30–40%) sperm motility in both control and test samples. The similar dose–response effects of cytochalasins B or D on both ZP-induced AR and the hyperactivation link the two processes and are consistent with the belief that both are markers of the completion of capacitation (Yanagimachi, 1994). It is possible that percentage hyperactivation may reflect the proportion of spermatozoa able to undergo AR on the ZP, but this requires further study.
The finding that inhibition of actin polymerization will block ZP-induced AR suggests that cytochalasins would also decrease sperm–ZP penetration. Our previous studies showed that in humans the AR of sperm bound to the ZP is closely related to sperm–ZP penetration and that impairment of the human ZP-induced AR with trypsin inhibitor blocks sperm–ZP penetration (Liu and Baker, 1993, 1996a; Llanos et al., 1993). As actin polymerization appears to be critical for the AR during the human fertilization process, it is important to determine if defective actin polymerization occurs in the spermatozoa of patients with disordered ZP-induced AR. PKC, plays a key role in the ZP-induced AR. Some patients with disordered ZP-induced AR fail to respond to stimulation of PKC suggesting defects of pathways downstream of PKC (Liu and Baker, 1997). In other cells, PKC is an important signal to the actin cytoskeleton. It has been reported that two actin-binding proteins, MARCKS (myristoylated alanine-rich C kinase substrate) and profilin, are PKC dependent (Aderem, 1992). MARCKS is an F-actin cross-linking protein that appears to function as an integrator of PKC and calcium calmodulin signals in the regulation of actin–membrane interactions (Aderem, 1992; Nairn and Aderem, 1992). Profilin stabilizes F-actin and is activated by the coordinated action of a receptor tyrosine kinase and phospholipase C-r1 (Aderem, 1992). In addition, PKC may also regulate cofilin, the ubiquitous actin-binding protein that is essential in the regulation of actin dynamics (Arber et al., 1998). A pool of active cofilin is controlled by phosphorylation. Activation of PKC leads to inhibition of LIM-kinase and dephosphorylation of cofilin (Arber et al., 1998).
In conclusion: actin is present in the human acrosomal area and is lost with the AR; inhibition of actin polymerization with cytochalasins B or D blocks the ZP-induced AR and sperm hyperactivation in humans. These results suggest that actin polymerization plays an important role in these processes.
Acknowledgments
We thank Mingli Liu for technical assistance, the scientists in both Royal Women's Hospital and Melbourne IVF Laboratories for collecting the oocytes and spermatozoa, and Melbourne IVF for funding.
Notes
6 To whom correspondence should be addressed 
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Submitted on February 17, 1999; accepted on July 8, 1999.
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