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BOR - Papers in Press, published online ahead of print October 23, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.009233
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BIOLOGY OF REPRODUCTION 68, 837–845 (2003)
DOI: 10.1095/biolreprod.102.009233
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Remodeling of the Actin Cytoskeleton During Mammalian Sperm Capacitation and Acrosome Reaction1

Ephraim Brenera, Sara Rubinsteina, Gili Cohena, Keren Shternalla, Joel Rivlina, and Haim Breitbart2,a

a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-ß-cyclodextrin, Ca2+, or NaHCO3, components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO3, cAMP, epidermal growth factor, H2O2, and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.

acrosome reaction, gamete biology, in vitro fertilization, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian species, sperm-egg interaction and mutual activation are mediated by the zona pellucida (ZP), the glycoprotein coat of the egg (reviewed in [1]). After the spermatozoon has penetrated the cumulus oophorus of the ovum, it binds to the ZP with its plasma membrane intact. Sperm binding to the ZP occurs via specific receptors that are localized over the anterior head region of the sperm. ZP binding stimulates the sperm to undergo the acrosome reaction, an exocytotic event in which the outer acrosomal membrane fuses with the overlying plasma membrane (reviewed in [2]). This multiple fusion results in the release of the acrosomal contents and exposure of new membrane domains, which are essential for fertilization to proceed.

The acrosome reaction will follow ZP binding only if the sperm has previously undergone a poorly defined maturation process known as capacitation. Capacitation occurs in vivo when sperm are exposed to the female reproductive tract or in vitro after incubation in a defined medium. We recently reviewed the known signal transduction events occurring during capacitation and during the acrosome reaction [3]. The possible changes in the cell architecture and actin cytoskeleton as part of the mechanisms of capacitation and the acrosome reaction remain open to investigation. In spermiogenic cells, actin filaments have been described primarily in the subacrosomal space between the nucleus and the developing acrosome of spermatids of certain species [4]. In mature spermatozoa, however, the structure and location of actin filaments have not been made clear. In most cases, actin seems to be present in its monomeric form, although filamentous (F)-actin has been described in several mammalian species [5–10]. The sperm regions reported to contain actin include the acrosomal space, the equatorial and postacrosomal regions, as well as the tail. In all species studied, actin was found in the tail, although its role in flagellar motion remains unclear. In addition, several reports suggest that actin expressed in the sperm head may be important for the fertilization process.

The assembly of monomers to form F-actin is controlled by several actin-binding proteins. The presence of proteins in spermatozoa such as thymosin ß10, destrin, testis-specific actin capping protein [6], gelsolin [10], the capping protein ß3 [11], and scinderin [12] suggest that actin polymerization and depolymerization processes may be involved in sperm function. It was shown that inhibition of actin polymerization in boar spermatozoa by cytochalasin D reduces the fertilization rate [13]. Actin polymerization plays an important role in the ZP-induced acrosome reaction in human sperm [14]. Moreover, phalloidin, which blocks F-actin depolymerization, inhibits Ca2+-ionophore-induced acrosomal exocytosis [15]. These observations are consistent with studies that implicate F-actin in regulating exocytosis in other cell types [16–19].

In our studies with isolated bovine sperm membranes we have demonstrated that the F-actin network located between the plasma and the outer acrosomal membranes forms a scaffold that immobilizes phospholipase C{gamma}, which is involved in the acrosome reaction at the membrane surface (reviewed in [20]). Inhibition of actin depolymerization by phalloidin inhibits the acrosome reaction [15], indicating that the dispersion of F-actin is necessary for the acrosomal reaction to occur. The observation that both actin depolymerization [21] and membrane fusion [15] require relatively high calcium concentrations (in the millimolar range), supports the notion that actin filaments constitute the final barrier to fusion [20]. In the present study, we demonstrate the rearrangement of F-actin in intact ram and bull sperm during capacitation and acrosomal exocytosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Methyl-ß-cyclodextrin (MBCD), BSA (Fraction V), tetramethylrhodamine isothiocyanate (TRITC)-phalloidin, fluorescein isothiocyanate (FITC)-phalloidin, and monoclonal antiphosphotyrosine (clone PT-66) were purchased from Sigma Chemical Company (St. Louis, MO). Horseradish peroxidase (HRP)-linked goat anti-mouse immunoglobulin G (IgG) was from Bio-Rad Laboratories (Hercules, CA).

Sperm Preparation

Ejaculated bull and ram spermatozoa were obtained using an artificial vagina. The semen was washed three times by centrifugation (780 x g, 10 min at 25°C) in NKM buffer containing 110 mM NaCl, 5 mM KCl, and 10 mM N-morpholino propanesulfonic acid pH 7.4. The washed cells were suspended in NKM buffer to a concentration of 109 cells/ml and were maintained at room temperature until use. Human semen samples were donated after 2 or 3 days of abstinence by healthy donors with normozoospermia. The samples were ejaculated into sterile containers and left for at least 30 min to liquefy before being processed by discontinuous Percoll gradient centrifugation [22] and suspended at a final concentration of 20 x 106/ml in Hepes-buffered Biggers, Whitten, Whittingham medium [23]. The Percoll-purified sperm suspensions used in this study were more than 80% motile.

Mouse sperm cells were recovered from epididymides of mature male BALB/C mice. The excised epididymides were minced in 0.4 ml of modified Krebs-Ringer bicarbonate medium containing (in mM): 119.4 NaCl, 4.8 KCl, 1.7 CaCl2, 1.2 MgCl2, 25.1 NaHCO3, 1 sodium pyruvate, 25 sodium lactate, and 5.56 glucose; and 20 mg/ml BSA, 0.001% phenol red, and 10 IU/ml penicillin. Epididymis pieces were then incubated at 37°C in 5% CO2 for 15 min to allow motile sperm to escape into the medium before the pieces of tissue were removed. The spermatozoa were incubated for 1 h at 37°C in 5% CO2 at a final concentration of 5 x 106 cells/ml.

Immature (4–6 wk old) female BALB/C mice were injected with 7 IU eCG, followed 48–56 h later with 7 IU hCG. After 13–16 h, the oocyte-cumulus complexes were recovered from the oviducts and pooled. At least eight female mice were used in each experiment.

Fertilization Assay

Unfertilized mouse eggs were pooled and loaded with Hoechst 33342 (5 µM) for 30 min in 5% CO2 at 37°C. This fluorochrome stains the chromatin of the eggs, thus enabling assessment of its distribution (metaphase II-arrested unfertilized eggs and a round pronuclei) in the fertilized eggs. The oocytes were then washed three times in fresh medium, and groups of 15–20 oocytes were mixed with spermatozoa (5 x 106 cells/ml) and incubated in 250 µl droplets under mineral oil for 24 h in 5% CO2 at 37°C. The eggs were then removed, washed in fresh medium, fixed in 5% formaldehyde, and the percentage of the fertilized eggs in each group was counted using a fluorescence microscope.

Capacitation and Acrosome Reaction

In vitro capacitation of bull sperm was induced according to the method described by Parrish et al. [24]. Briefly, sperm pellets were resuspended to a final concentration of 108 cells/ml in glucose-free TALP medium containing (in mM): 100 NaCl, 3.1 KCl, 1.5 MgCl2, 25 NaHCO3, 0.29 KH2PO4, 21.6 sodium lactate, 0.1 sodium pyruvate, 2 CaCl2, 20 Hepes pH 7.4; and 50 µg/ml BSA, 10 U/ml penicillin, and 20 µg/ml heparin. The cells were incubated in this capacitation medium for 4 h at 39°C with 5% CO2. In vitro capacitation of ram sperm was induced by resuspending washed sperm pellets to a final concentration of 108 cells/ml in cTyH medium containing (in mM): 119.37 NaCl, 4.78 KCl, 1.71 CaCl2, 1.19 MgSO4, 1.19 KH2PO4, 25.0 NaHCO3, 5.56 glucose, 0.5 pyruvic acid, 20 Hepes pH 7.4, and 0.75 methyl-ß-cyclodextrin; and 60 µg/ml penicillin and 1 mg/ml polyvinyl alcohol. The cells were incubated in this medium for 3 h at 37°C with 5% CO2. Following capacitation, the acrosome reaction was induced with 10 µM Ca2+-ionophore A23187, which was added for an additional 20 or 60 min of incubation for bull or ram sperm, respectively. The percentage of acrosome reacted sperm was determined microscopically using a biotin-conjugated Pisum sativum agglutinin (PSA) procedure described by Mendoza et al. [25]. Samples of cells treated to induce the acrosome reaction were smeared on microscope slides. After air-drying, sperm smears were dipped in absolute methanol for 30 sec and allowed to dry rapidly. Methanol-fixed smears were incubated with blocking solution (PBS containing 1% BSA) for 10 min, then with biotin-conjugated PSA (50 µg/ml) in PBS containing 1% BSA for 30 min, and finally with peroxidase-conjugated extravidin (1:400) for 10 min. All incubations were performed in a humid chamber. The slides were washed between incubations by dipping them in PBS for 5 min. The substrate (aminoethyl carbazole from the Histostain-SP kit, Zymed Laboratories, Burlingame, CA) was then added for 10 min, followed by washing with distilled water. Hematoxylin was usually used for counterstaining (3 min). The slides were mounted with GVA (Zymed) and examined with a brightfield microscope. Cells with red staining over the acrosomal cap were considered acrosome intact; equatorial red staining or no staining at all were considered acrosome-reacted; 200 cells were counted per slide.

Whole Cell Lysates

Washed sperm cells (109 cells) were solubilized in SDS-lysis buffer consisting of 125 mM Tris pH 7.5, 4% SDS, 1 mM sodium orthovanadate, 1 mM benzamidine, and 1 mM PMSF added just before use. Cells were lysed for 10 min at room temperature and centrifuged at 12 930 x g for 5 min at 4°C. The supernatant was supplemented with 0.05% bromophenol blue, 5% glycerol, and 2% ß-mercaptoethanol and boiled for 5 min.

Immunoblot Analysis

For immunoblotting, proteins derived from equivalent cell numbers were separated on 7.5% SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (200 mAmp for 1 h) using a buffer composed of 25 mM Tris pH 8.2, 192 mM glycine, and 20% methanol. For Western blotting, nitrocellulose membranes were blocked with 5% BSA in Tris-buffered saline (TBS) pH 7.6 containing 0.1% Tween-20 (TBST) for 30 min at room temperature. The membranes were incubated overnight at 4°C with the antibody diluted 1:10 000. Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific HRP-linked secondary antibody diluted 1:10 000 in TBST. The membranes were washed three times with TBST and visualized with an enhanced chemiluminescence kit (Amersham, Little Chalfont, U.K.).

Fluorescence Staining of Actin Filaments

Samples of cells were spread on microscope slides. After air-drying, sperm were fixed in 5% formaldehyde for 10 min, washed with 25 mM TBS pH 7.6, then dipped in cold acetone (-20°C) for 10 min and air-dried. The slides were incubated with TBS containing 1% BSA for 10 min, then with TRITC-phalloidin (1.5 µM in TBS) or FITC-phalloidin (3 µM in TBS) for 1 h, washed with TBS, and mounted with GVA. Actin polymerization was determined by fluorescence microscopy and the fluorescence intensity was determined quantitatively using MetaMorph "Image J" (Universal Imaging Corp., West Chester, PA) and the Adobe PhotoShop software programs (Adobe Systems Inc., San Jose, CA).

Statistical Analysis

Statistical analyses were performed using ANOVA and a t-test with multiple comparisons. Statistical significance is indicated in the figure legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rearrangement of F-Actin During Sperm Capacitation and the Acrosome Reaction

To evaluate the role of actin polymerization in the sperm capacitation reaction, changes in the cell content of F-actin were quantified fluorimetrically using FITC-phalloidin or TRITC-phalloidin. Incubation of ram, bull, mouse, or human spermatozoa in capacitating conditions resulted in an increase in polymerized actin mainly in the sperm head (Fig. 1). In uncapacitated sperm, no F-actin was observed in the tail of human sperm, very little in bull or ram sperm, and quite high amounts were observed in mouse sperm tails. The postacrosomal region of uncapacitated human and mouse sperm also contained F-actin. In capacitated cells, high concentrations of F-actin were observed in the head of the four species, including a great increase in the upper head region of human and mouse sperm. Figure 2 shows the dynamics of the increase in ram sperm F-actin formation during incubation in capacitating conditions, which initiated in the midpiece and then propagated toward the sperm head. The induction of acrosomal exocytosis by the Ca2+-ionophore A23187 at the end of capacitation resulted in a rapid disappearance of F-actin (Fig. 2). The addition of A23187 (Fig. 3) or the intracellular Ca2+-ATPase blocker thapsigargin (not shown) after 3 h of capacitation caused a fast disappearance of F-actin followed by the acrosome reaction, which began after the complete depolymerization of F-actin (Fig. 3). The capacitation status of sperm was assessed by following protein tyrosine phosphorylation and after the sperm underwent the acrosome reaction or fertilized a mouse egg in vitro. Figure 4 shows that the acrosome reaction was fully expressed only after 3 h of incubation, when actin was maximally polymerized. No increase in acrosome reaction was seen during the first 2 h of incubation, suggesting that the maximal level of actin polymerization correlates with the onset of the capacitated state of the sperm cells. Moreover, incubating the spermatozoa under noncapacitating conditions (in medium lacking MBCD or heparin) revealed no increase in actin polymerization and a relatively poor induction of the acrosome reaction by A23187 (Fig. 5 and Table 1).



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  		FIG. 1. Polymerization of actin during capacitation of bull, mouse, human, and ram spermatozoa. Bull, mouse, human, and ram sperm were capacitated for 4, 2, 3, and 3 h, respectively; the cells were stained with TRITC-phalloidin, and photographed with a fluorescence microscope before and after capacitation. Magnification: bull x1000, mouse x400, human x1500, and ram x1000. The photos are representative of at least three experiments



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  		FIG. 2. Polymerization of actin during capacitation and depolymerization in the acrosome reaction. Ejaculated ram sperm were incubated in capacitation medium and the amount of polymerized F-actin was determined at various times using a fluorescence microscope after staining with TRITC-phalloidin. After 180 min of incubation, the acrosome reaction was induced by adding the Ca2+-ionophore A23187 (arrow). A) Images from fluorescence microscopy. B) Quantitative presentation of the relative fluorescence of F-actin–TRITC-phalloidin complex. The photos are representative of two experiments



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  		FIG. 3. Time curves of F-actin depolymerization and acrosome reaction in capacitated cells treated with Ca2+ ionophore. Cpacitated ram sperm (following 3 h of incubation in capacitation medium) were treated with the Ca2+-ionophore A23187, and the amount of F-actin and the percentage of acrosome reacted cells were determined at various times. The data represent the mean ± SEM of three experiments; P < 0.0001



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  		FIG. 4. Time curves for F-actin status and the ability of the cells to undergo acrosome reaction following different incubation times in capacitation medium. Ram spermatozoa were incubated in capacitation medium and the amount of F-actin and the ability of the cells to undergo an A23187-induced acrosome reaction were determined at various times. The data represent the mean ± SEM of three experiments; P < 0.0001



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  		FIG. 5. Effect of cytochalasin D on actin polymerization and capacitation-dependent acrosome reaction. Ram and bull spermatozoa were incubated in capacitation medium in the presence or absence of 20 µM cytochalasin D, followed by determination of the F-Actin and Ca2+-ionophore-induced acrosome reaction rate. A) F-Actin in ram sperm: uncapacitated cells incubated for 3 h in medium without MBCD, capacitated cells incubated for 3 h in capacitation medium, and capacitation + CD incubation in capacitation medium plus 20 µM cytochalasin D. B) F-Actin in bull sperm: uncapacitated cells inoculated for 4 h without heparin, capacitated cells incubated for 4 h in capacitation medium, and capacitated + CD cells incubated for 4 h in capacitation medium plus 20 µM cytochalasin D. C) A231870-induced acrosome reaction rate in ram or bull sperm incubated in capacitation medium in the presence or absence of 20 µM cytochalasin D. F-Actin was determined using TRITC-phalloidin. The photos are representative of three experiments. In C, the data represent the mean ± SEM of three experiments; P < 0.001


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  		TABLE 1. Effect of different incubation conditions on sperm actin polymerization and acrosome reaction

It is well known that bicarbonate and heparin are essential components in the medium for in vitro capacitation of bovine spermatozoa to occur, whereas glucose inhibits sperm capacitation [24]. Removal of Ca2+, bicarbonate, or heparin from the capacitation medium or the addition of glucose prevented capacitation-dependent actin polymerization as well as the acrosome reaction (Table 1).

The increase in polymerized actin during sperm capacitation is further supported by showing that cytochalasin D, a known inhibitor of actin polymerization, blocked capacitation-dependent F-actin formation as well as the acrosome reaction in ram and bull spermatozoa (Fig. 5). The acrosomal reaction induced by the Ca2+-ionophore A23187 in capacitated cells (in the presence of MBCD or heparin) was almost completely blocked by treating the cells with cytochalasin D during sperm capacitation. We also show that cytochalasin D inhibited the acrosome reaction in mouse sperm and reduced the fertility of sperm in vitro (Table 2). The relationship between actin polymerization and sperm capacitation is further supported by the requirement for the small GTPase, Rho, in these processes. Rho is involved in rearrangement of actin cytoskeleton in many cell types [26]. Rho protein is present in sperm cells, and its ADP-ribosyl-transferase (C3) inhibits sperm motility [27]. Because cells have a relatively low permeability to C3, the sperm cells were preincubated for 1 h with the inhibitor before starting capacitation. C3 caused complete inhibition of capacitation-induced F-actin formation and 80% inhibition of sperm capacitation as revealed by following the occurrence of the capacitation-dependent acrosome reaction (Fig. 6). A spontaneous acrosome reaction in noncapacitated cells (in the absence of MBCD) was not affected by C3 (not shown).


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  		TABLE 2. Effect of cytochalasin D on F-actin formation, acrosome reactions and in vitro fertilization (IVF) in mouse sperm



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  		FIG. 6. Effect of EGF and the Rho inhibitor C3 or actin polymerization and acrosome reaction. Ram spermatozoa were incubated for 3 h in capacitation medium (containing MBCD) or in noncapacitating conditions (without MBCD) in the presence or absence of EGF or the ADP-ribosyl transferase exotoxin (C3). A) F-Actin in sperm cells: uncapacitated, capacitation medium without MBCD; capacitated, capacitation medium; EGF, capacitation medium with 10 ng/ml EGF but without MBCD; C3, capacitation medium with 10 µg/ml C3. B) A23187-induced acrosome reaction in ram sperm incubated in the described capacitation conditions. F-Actin was determined using TRITC-phalloidin. Panel A represents three experiments and in B the data represent the mean ± SEM of three experiments; P < 0.001

Protein Tyrosine Phosphorylation and Actin Polymerization

Protein tyrosine phosphorylation stimulated by the cAMP/protein kinase A (PKA) pathway during capacitation was shown to occur in mammalian spermatozoa [28, 29]. We show here that factors that trigger protein tyrosine phosphorylation stimulate actin polymerization, whereas inhibitors of tyrosine phosphorylation block F-actin formation. Several laboratories have demonstrated the importance of bicarbonate in the completion of capacitation. It was shown that bicarbonate activates sperm soluble adenylyl cyclase to produce cAMP, leading to protein tyrosine phosphorylation [28, 30]. Here we show that in the absence of bicarbonate, there is no increase in protein tyrosine phosphorylation (Fig. 7), actin polymerization, or sperm capacitation (Table 1). These activities could be restored by adding the permeable dibutyryl cAMP to the incubation medium (Fig. 7 and Table 1). Inhibition of protein tyrosine phosphorylation using genestein, which blocks tyrosine kinases, or H-89, which blocks PKA-dependent tyrosine phosphorylation, resulted in inhibition of actin polymerization as well as the capacitation-dependent acrosome reaction (Fig. 7 and Table 1).



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  		FIG. 7. Protein tyrosine phosphorylation during sperm capacitation. Bull spermatozoa were incubated in TALP medium for 4 h in the indicated conditions, proteins were extracted, and protein tyrosine phosphorylation was determined by Western blot analysis. The blot shown is representative of at least three experiments. The concentrations used (in mM): dbcAMP, 1.0; H-89, 0.05; H2O2, 0.1; sodium vanadate, 0.1; and EGF, 10 ng/ml

The correlation between protein tyrosine phosphorylation and actin polymerization is further supported by showing the effects of the tyrosine phosphorylation enhancers epidermal growth factor (EGF), H2O2, and sodium vanadate on actin polymerization (Table 1). A receptor for the EGF receptor, a tyrosine kinase involved in the sperm acrosome reaction, was identified in the head of bovine sperm [31, 32]. Moreover, when EGF is present during sperm capacitation, it stimulates tyrosine phosphorylation of several proteins (Fig. 7) and causes a significant increase in the binding of F-actin and phospholipase C{gamma} (PLC{gamma}) to the sperm plasma membrane during the capacitation process [15]. Incubation of ram or bull sperm with EGF in the absence of the capacitating compound MBCD or BSA shows a high increase in actin polymerization and reduced capacitation rate as revealed by following the acrosome reaction (Fig. 6 and Table 1). MBCD, BSA, or EGF induce F-actin production to similar extents, whereas MBCD or BSA are better inducers of the acrosome reaction.

Hydrogen peroxide is another enhancer of protein tyrosine phosphorylation in mammalian spermatozoa [33]. Here we demonstrate that H2O2 enhances tyrosine phosphorylation of several sperm proteins (Fig. 7) and stimulates actin polymerization (Table 1). However, in the absence of heparin, H2O2 has no significant effect on sperm capacitation as determined by a sperm's ability to undergo the acrosome reaction (Table 1). To further support these data, we incubated the cells in the presence of sodium vanadate, a known phosphatase inhibitor. We found a significant elevation of protein tyrosine phosphorylation in sperm cells in its presence (Fig. 7). Similarly to H2O2, sodium vanadate stimulated actin polymerization but it did not affect the rate of the acrosome reaction (Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanisms involved in mammalian sperm capacitation and acrosome reaction are not well understood. In the present study we examined the changes in the actin cytoskeleton during these events. In vitro capacitation of ram, bull, mouse, or human spermatozoa revealed a time-dependent increase in F-actin formation first in the midpiece and later in the sperm head. This did not occur when sperm were incubated in noncapacitating conditions or when capacitation was blocked. The increase in F-actin during capacitation of sperm from four species suggests that this might be a general phenomenon that occurs during mammalian sperm capacitation. The appearance of F-actin in the head during capacitation suggests that it may be involved in the sperm acrosome reaction. Induction of the acrosome reaction in capacitated sperm that contain high levels of F-actin resulted in a fast disappearance of this F-actin. Thus, it seems that G-actin is polymerized to F-actin during capacitation and depolymerized by enhancing intracellular Ca2+ concentrations ([Ca2+]i), resulting in acrosomal exocytosis. In our previous studies with membranes extracted from bovine sperm, we showed an increase of F-actin binding to the plasma membranes and to the outer acrosomal membranes during capacitation [15]. Moreover, we found that in order to fuse between the two extracted membranes, F-actin must be released from the membranes [15]. In addition, inhibition of F-actin dispersion by phalloidin blocks the acrosome reaction [15, 34]. The localization of actin between the plasma and outer acrosome membrane in human spermatozoa might also indicate actin involvement in the acrosome reaction [14]. Moreover, we show here that a significant acrosome reaction occurs only after 180 min of incubation, when actin polymerization is maximal (see Fig. 4). This indicates that actin polymerization precedes the acrosome reaction. In the kinetics of the acrosome reaction, significant enhancement of acrosome-reacted cells can be seen only after complete depolymerization of F-actin (Fig. 3). In our previous studies with plasma membranes isolated from capacitated sperm, we showed that F-actin release from the membranes depends on the elevation of [Ca2+]i and PLC{gamma} activation [15]. Here we show using Ca2+-ionophore that F-actin depolymerization in intact cells depends on raising [Ca2+]i.

Our notion regarding F-actin formation during capacitation is further supported by showing that inhibition of actin polymerization by cytochalasin D blocks the capacitation-dependent acrosome reaction (see Fig. 5) and in vitro fertilization (Table 2). A previous study demonstrated that cytochalasin D inhibits in vitro fertilization of boar spermatozoa, suggesting that actin polymerization is essential for the fertilization process [13]. To further support this notion, we showed that inhibition of the small G-protein, Rho by ADP ribosylation using the C3 exoenzyme blocks capacitation-induced actin polymerization as well as the acrosome reaction (see Fig. 6). Similar results were obtained with brefeldin, which inhibits the small G-protein, ARF (not shown). It has been well established in other cell types that Rho GTPase interacts with cellular target proteins or effectors to trigger the reorganization of the actin cytoskeleton [26, 35]. Here we demonstrate for the first time the possible involvement of Rho GTPase in sperm capacitation in a mechanism that involves actin polymerization. In sea urchin sperm, a 25-kDa Rho protein is localized in the flagellum and acrosomal region, suggesting its possible involvement in regulating motility and the acrosome reaction [36].

Stimulation of protein tyrosine phosphorylation is an important event that occurs during mammalian sperm capacitation [28, 29, 32]. In order to establish whether protein tyrosine phosphorylation correlates with F-actin formation, we attempted to modulate intracellular protein tyrosine phosphorylation using various mediators.

We show here that bicarbonate or dibutyryl cAMP, which trigger tyrosine phosphorylation [28, 29], can stimulate actin polymerization, whereas genestein or H-89, which inhibit tyrosine phosphorylation, can block F-actin formation as well as the capacitation-dependent acrosome reaction.

Furthermore, we demonstrate that EGF, which is found in the female reproductive tract, stimulates protein tyrosine phosphorylation (Fig. 7) and actin polymerization (Fig. 6 and Table 1). Moreover, we have found that EGF strongly stimulates the fast binding of F-actin followed by PLC{gamma} to the sperm plasma membrane during capacitation [15]. EGF may exert its action by binding to specific tyrosine kinase receptors localized in the plasma membrane of the upper sperm head region [31]. It is known from other cell types that EGF causes actin polymerization [37, 38]. Here we show that EGF can induce F-actin formation similarly to polymerization, which occurred under capacitating conditions; however, the acrosome reaction is not fully stimulated under these conditions (Fig. 6 and Table 1). The stimulation of protein tyrosine phosphorylation by EGF is also very high; in fact, it is even higher compared with the phosphorylation that occurs under capacitating conditions. Thus, our data suggest that protein tyrosine phosphorylation and actin polymerization during sperm capacitation are related processes. Enhancement of protein tyrosine phosphorylation and actin polymerization by the phosphatase inhibitors hydrogen-peroxide or sodium vanadate further support this notion. Phosphatase inhibitors cannot by themselves induce sperm capacitation, although they enhance tyrosine phosphorylation and actin polymerization (Table 1). These data indicate that actin polymerization and protein tyrosine phosphorylation are necessary but insufficient processes for sperm capacitation. It is known that other processes such as cholesterol efflux from the sperm plasma membrane must occur to achieve capacitation [39]. This process is accomplished in vitro by MBCD or BSA in capacitating medium for ram or bull, respectively. Thus, in their absence, tyrosine kinase activation or phosphatase inhibition can stimulate protein tyrosine phosphorylation and actin polymerization, but not full capacitation.

Moreover, the stimulation of actin polymerization by EGF, H2O2, or sodium vanadate and its inhibition by genestein or H-89 suggest that protein phosphorylation occurs before actin polymerization. This notion is further supported by showing the involvement of tyrosine phosphorylation in the formation of actin stress fibers in fibroblasts [40]. In human platelets, activation of protein tyrosine phosphatase inhibits actin polymerization [41]. It was also shown that phosphorylation of threonine 560 of p85MBS regulates protein phosphatase 1{delta} activity, leading to dephosphorylation of myosin light chain 2 and subsequent actin-myosin disassembly [42]. Among actin-binding proteins, ADF/cofilin is the most defined mediator of actin dynamics [43–45]. Phosphorylation of ADF/cofilin on Ser-3 by the Rho-GTPase dependent LIM-kinase [46–48] or by testicular protein kinase [49, 50] inhibits its filament-severing activity. We show here that Rho-GTPase is probably involved in sperm actin polymerization; thus it is possible that LIM-kinase or testicular protein kinase, or both, are involved in ADF/cofilin phosphorylation/inactivation, thus allowing actin polymerization. We found during the acrosome reaction a rapid dephosphorylation of tyrosine phosphorylated proteins (not shown). Thus, it is possible that before the acrosome reaction occurs, that phosphorylated ADF/cofilin or related actin severing proteins might undergo rapid dephosphorylation, which could result from the downregulation of the kinases, up-regulation of one or more phosphatases, or both [43]. It was recently shown that the phosphatase slingshot dephosphorylates ADF/cofilin and reactivates its actin-severing activity [51].

However, it seems that this is not always the case. It was previously shown in other cell types that neuropeptides and growth factors require the integrity of the actin cytoskeleton to cause tyrosine phosphorylation of focal adhesion kinase (p125FAK) and the cytoskeleton-associated paxillin [40, 52–55]. It is possible that protein tyrosine phosphorylation and actin polymerization are coupled reactions in which actin polymerization is initiated by one or more tyrosine phosphorylated proteins, while further progress of phosphorylation depends on the promotion of actin polymerization.

In conclusion, our data suggest that actin polymerization occurs during sperm capacitation, followed by fiber breakdown during the acrosome reaction. The small GTPase Rho and tyrosine phosphorylated proteins are probably involved in the mechanism leading to actin polymerization. The elevation of [Ca2+]i at the end of the capacitation process might activate one or more phosphatases to dephosphorylate one or more actin-severing proteins, resulting in F-actin dispersion. Our previous observation that both actin depolymerization [21] and membrane fusion [15] require supramicromolar calcium, support the notion that the actin filaments constitute the final barrier to membrane fusion and acrosomal exocytosis.


   FOOTNOTES
 
1 This research was supported by the Ihel Research Fund. Back

2 Correspondence: Haim Breitbart, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900 Israel. FAX: 972 3 5344766; e-mail: breith{at}mail.biu.ac.il Back

Received: 10 July 2002.

First decision: 30 July 2002.

Accepted: 16 September 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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