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BOR - Papers in Press, published online ahead of print May 9, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.056705
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BIOLOGY OF REPRODUCTION 77, 263–273 (2007)
DOI: 10.1095/biolreprod.106.056705
© 2007 by the Society for the Study of Reproduction, Inc.

Role of PI3-Kinase and PI4-Kinase in Actin Polymerization During Bovine Sperm Capacitation1

Nir Etkovitz , Sara Rubinstein , Limor Daniel , and Haim Breitbart 2

The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

ABSTRACT

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP2(4,5), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 {alpha} regulatory subunit of PI3K) and PIKCB (PI4K ß) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 µM, respectively. PI4K activity and PIP2(4,5) production were blocked by 10 µM WT but not by 10 nM WT, whereas PI3K activity and PIP3(3,4,5) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP2(4,5) and F-actin formation. The increases in PIP2(4,5) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP2(4,5), PIP3(3,4,5), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP2(4,5) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP3(3,4,5) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.

developmental biology,, gamete biology,, kinases,, signal transduction,, sperm capacitation

INTRODUCTION

Mammalian spermatozoa undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, where it undergoes the acrosome reaction, enabling it to penetrate the egg. We have recently demonstrated that actin polymerization occurs during mammalian sperm capacitation and that the F-actin polymers are dispersed prior to the acrosome reaction [1]. The formation of F-actin during capacitation occurs mainly in the sperm head but also in the tail [1]. Over 30 years ago it was shown in echinoderm sperm that actin can be polymerized and that actin is localized in the microfilaments during the acrosomal process [2]. Later, it was suggested that sperm motion is affected by the rapid polymerization of actin [3]. In our studies with isolated bovine sperm membranes, we have suggested that the F-actin network located between the plasma membrane and the outer acrosomal membrane forms a scaffold that immobilizes phospholipase C-{gamma} (PLCG1), which is involved in the acrosome reaction (reviewed in [4]). The observation that both actin depolymerization [5] and membrane fusion [6] require relatively high calcium concentrations (in the mM range) supports the notion that actin filaments constitute the final barrier to fusion [4]. In a recent publication, we have provided evidence that translation of nuclear-encoded proteins occurs in sperm mitochondria during capacitation [7]. It has been shown in other cell types that mRNA can be translocated on actin filaments to the translation locus in the cell. Thus the formation of F-actin during sperm capacitation may be important for the translocation of nuclear mRNA to the sperm midpiece, where the mitochondria are located.

We have previously demonstrated that the process of actin polymerization depends on phospholipase D (PLD) activity and is regulated by cross-talk between protein kinases A and C (PKA/PKC) [8]. Phosphatidylinositol 4,5-bisphosphate (PIP2(4,5)) is required as a cofactor for the activation of PLD in many cell types [9–12], although there are no solid data regarding its production in sperm. Furthermore PIP2(4,5) serves as a precursor for two well-defined second messengers produced by its phospholipase C-catalyzed hydrolysis [13], i.e., diacylglycerol (DAG), which activates PKC [14], and inositol 1,4,5-triphosphate (IP3), which mobilizes Ca2+ from intracellular stores [15]. PKC is involved in the sperm acrosome reaction [16] and actin polymerization [8] and IP3 is involved in intracellular calcium regulation in sperm [16–18]. Therefore information regarding PIP2(4,5) production is essential in order to understand the regulation of their activities.

PIP2(4,5) can be phosphorylated by phosphatidylinositol 3-kinases (PI3K) to phosphatidylinositol 3,4,5-triphosphate (PIP3(3,4,5)) [19]. Although PIP2(4,5) and PIP3(3,4,5) represent less than 1% of membrane phospholipids, they function in several crucial cellular processes (reviewed in [20]). In other cell types, PIP2(4,5) directly controls actin-binding proteins and PLD activation, thereby regulating actin cytoskeletal rearrangement [21–23]. PIP3(3,4,5) is also known to participate in actin cytoskeletal rearrangement by recruiting members of the small G-protein ADP-ribosylation-factor (ARF) exchange factor family, leading to actin polymerization [24, 25].

PI3Ks have been identified and categorized into three main classes (I, II, and III) [26]. Although multiple forms of PI3Ks exist in higher eukaryotes, the class IA enzymes are primarily responsible for the production of PIP3(3,4,5) in response to growth factors [27, 28]. The Class IA PI3Ks are heterodimers, consisting of a 85-kDa or 55-kDa regulatory subunit (PIK3R1, PIK3R2, and PIK3R3) and a 110-kDa catalytic subunit (PIK3CA, PIK3CB, and PIK3CD) [26–29]. The PIK3R1 (p85 {alpha} regulatory subunit of PI3K) mRNA can be alternatively spliced to generate three new regulatory subunits, termed p55, p45, and p85+8aa, (55 kDa, 45 kDa, and 100 kDa, respectively), in addition to p85 (PIK3R1) [29]. PIK3R1 contains two SH2 domains, one SH3 domain, a Bcr homology domain, and proline-rich sequences [26, 29]. When activated, the two subunits bind and translocate to the plasma membrane where they become catalytically active.

In mammalian cells, phosphatidylinositol 4-phosphate (PI4P) is synthesized by PI4-kinases (PI4K), which phosphorylate phosphatidylinositol (PI) at the D-4 position of the inositol ring. PI4P is further phosphorylated at the D-5 position of the inositol ring by PI4P5-kinase (PI4P5K), to produce PIP2(4,5) [30]. Several isoforms of PI4Ks have been characterized based on their catalytic properties, including the type II and type III enzymes [31]. Type III enzymes have been cloned, and two forms of ~200 kDa (PIK4CA) and ~100 kDa (PIK4CB), which are the mammalian homologues of the yeast stt4 and pik1 gene products, have been characterized [32–34]. The molecular identity of PI4K type II has also been characterized [35, 36]. In mammalian cells, PIK4CB is localized to the Golgi [37] and can be regulated by Arf [38]. The two isoforms of type III (PIK4CA and PIK4CB) are inhibited by a relatively high concentration (10 µM) of the PI3K inhibitor Wortmannin (WT), whereas PI3K is inhibited at a lower concentration of WT (10 nM) [39]. The type II PI4Ks are not inhibited by WT [40]. PI4K activity has not been described as yet in sperm, while PI3K has been demonstrated in mouse, hamster, boar, and human spermatozoa [41–45].

In the present study, we investigated the involvement of PI4K and PI3K in the regulation of the intracellular levels of PIP2(4,5), the cofactor for PLD, which is a key enzyme in sperm actin polymerization during capacitation. Therefore, we examined the roles of these kinases in F-actin formation during sperm capacitation.

MATERIALS AND METHODS

Materials

The [{gamma}-32P]ATP was obtained from Amersham (Little Chalfont, UK). Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bis-phosphate were obtained from Cal-Biochem (San Diego, CA). Mouse anti-PIP2(4,5) and anti-PIP3(3,4,5) (biotin-conjugated) antibodies were purchased from Echelon (Salt Lake City, UT). Antibodies against PIK4CB and PIK3R1 (clone U15) were purchased from BD Bioscience, Transduction Labs (San Diego, CA). Anti-mouse rhodamine RED-X was obtained from Jackson ImmunoResearch (West Grove, PA). Texas Red-conjugated streptavidin was obtained from Oncogene (La Jolla, CA). The polyclonal antibody against phospho-PIK3R1 (Tyr 458) was from Cell Signaling (Beverly, MA). All other chemicals were purchased from Sigma (Sigma-Aldrich Israel Ltd., Rehovot, Israel) unless otherwise stated.

Sperm Preparation

Ejaculated bull spermatozoa were obtained by using artificial vagina, and the ‘swim up' technique was applied to obtain motile sperm. Bovine sperm was supplied by the SION Artificial Insemination Center (Hafetz-Haim, Israel). Sperm cells were washed three times by centrifugation (780 x g for 10 min at 25°C) in NKM buffer that contained 110 mM NaCl, 5 mM KCl, and 20 mM 3-N-morpholino propanesulfonic acid (Mops) (pH 7.4) and the sperm were allowed to swim up after the last wash. The washed cells were counted and maintained at room temperature until use. Only sperm preparations that contained at least 80% motile sperm were used in the experiments, and the motility was not significantly reduced at the end of the incubations.

Sperm Capacitation

In vitro capacitation of bovine sperm was induced as described previously [46]. Briefly, sperm pellets were resuspended to a final concentration of 108 cells/ml in mTALP (modified Tyrode solution) medium that contained 100 mM NaCl, 3.1 mM KCl, 1.5 mM MgCl2, 0.92 mM KH2PO4, 25 mM NaHCO3, 20 mM Hepes (pH 7.4), 0.1 mM sodium pyruvate, 21.6 mM sodium lactate, 10 IU/ml penicillin, 1 mg/ml BSA, 20 µg/ml heparin, and 2 mM CaCl2. The cells were incubated in this capacitation medium for 4 h at 39°C in 5% CO2. The capacitation state of the sperm was confirmed after the 4-h incubation in mTALP by examining the ability of the sperm to undergo the acrosome reaction [1].

Immunoblot Analysis

Sperm lysates were prepared by the addition of lysis buffer that contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2% SDS, 1% Triton X-100, 1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.2 mM Na3VO4, and the mixture was vortexed vigorously for 10 min at 4°C. Lysates were then centrifuged at 10 000 x g for 10 min at 4°C. Sample buffer (2x) was added to the supernatant and boiled for 5 min. The extracts were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Western blotting was performed (200 mA for 1 h) using a buffer composed of 25 mM Tris (pH 8.2), 192 mM glycine, and 20% methanol. For Western blotting, the nitrocellulose membranes were blocked with 5% BSA in Tris-buffered saline (pH 7.6) that contained 0.1% Tween-20 (TBST), for 30 min at room temperature. Kinases and actin were immunodetected using monoclonal anti-PIK4CB antibody (diluted 1:10 000), monoclonal anti-PIK3R1 antibody (1:2000), polyclonal anti-phospho-PIK3R1 antibody (1:3000), and horseradish peroxidase (HRP)-conjugated anti-ACTB (ß-actin) antibody (Sigma) (1:3000). The membranes were incubated overnight at 4°C with the primary antibodies diluted in 1% BSA in TBST, except for anti-PIK4CB, which was diluted in 5% BSA in TBST. Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific HRP-linked secondary anti-mouse or anti-rabbit antibodies (Bio-Rad, Hercules, CA) diluted 1:10 000 in TBST and 5% BSA. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham).

Fluorescence Staining of Actin Filaments

Sperm cells were spread on microscope slides. After air-drying, sperm were fixed in 2% formaldehyde in TBS for 10 min, placed in 0.2% Triton X-100 in TBS for 30 min, washed three times at 5-min intervals in distilled water, air-dried, and then incubated with FITC-phalloidin (3 µM in TBS) for 40 minutes, washed four times with water at 10-min intervals, and mounted with FluoroGuard Antifade (Bio-Rad).

Determination of Actin Incorporation into the Triton-Insoluble Cytoskeleton of Sperm, and Immunoblotting Analysis

Sperm suspensions were incubated in mTALP (3 x 108/ml) for 4 h, and the cells were lysed as described previously [47]. Briefly, the sperm cells were washed once with TBS, an equal volume of lysis solution that contained 1.5% Triton X-100 was added and vortexed vigorously (10 min at 4°C), and the mixture was centrifuged at 12 000 x g for 5 min. The actin content of the Triton-insoluble cytoskeleton (F-actin) was determined by removing the supernatant fluid (Triton-soluble G-actin) from the pellet, and then 100 µl of sample buffer that contained 6% SDS was added and the mixture was vortexed vigorously for 10 min. The extracted proteins were separated by SDS-PAGE and immunoblotted with HRP-conjugated anti-ACTB antibody (1:3000).

Preparation of Membrane-Permeable Phosphoinositides

Phosphoinositides were dissolved in chloroform, dried under an N2 stream, and suspended in ice-cold NKM buffer (pH 7.4). The phosphoinositide liposomes were prepared by sonication (two 20-sec pulses, power setting 4) or phosphoinositides were preincubated with type III-s histone from the calf thymus (1:1) for 30 min [48, 49]. In both cases, penetration of PIP2(4,5) into the sperm cells was confirmed by visualization of intracellular PIP2(4,5) by immunocytochemistry using cells that were treated with 10 µM WT to inhibit PI4K and reduce endogenous PIP2(4,5). As a negative control, sperm were incubated with untreated (nonpermeable) phosphoinositides suspended in NKM. No intracellular PIP2(4,5) staining was observed in these cells. The experiments described in the present study used phosphoinositide liposomes.

Immunocytochemistry

The anti-PIP2(4,5) and anti-PIP3(3,4,5) antibodies were used to determine the intracellular levels of PIP2(4,5) and PIP3(3,4,5), as described previously [49–54]. For immunocytochemistry, sperm cells were spread on glass slides, air-dried, fixed in formaldehyde (2%) for 10 min, dipped in 0.5% Triton X-100 in TBS for 30 min, and washed three times at 5-min intervals with TBS. When exogenous PIP2(4,5) or PI (10 min) was added, the cells were washed three times before spreading on slides. Nonspecific reactive sites were blocked for 30 min at room temperature with TBS that contained 5% BSA. The cells were then incubated for 2 h at 37°C with monoclonal anti-PIK4CB or anti-PIK3R1 antibodies diluted 1:20, or at 37°C with mouse anti-PIP2(4,5) or anti-PIP3(3,4,5) biotin-conjugated antibodies diluted 1:100. Next, the slides were washed once in TBS-T and twice (for 5 min) in TBS. The bound antibody was detected using rhodamine RED-X-conjugated anti-mouse-IgG (1:200 dilution, 2 h incubation at 37°C) for PIK3R1 and PIK4CB or using Texas Red-conjugated streptavidin for PIP2(4,5) and PIP3(3,4,5) (100 ng/ml, incubation at 37°C for 2 h), followed by washing once with TBS-T and twice with water at 5-min intervals and mounting in FluoroGuard Antifade. Nonspecific staining was determined by preabsorbing the anti-PIP2(4,5) and anti-PIP3(3,4,5) antibodies with PIP2(4,5) and PIP3(3,4,5) (25 µM), respectively, for 1 h at 37°C prior to incubation with the cells or alternatively, by incubating the sperm without primary antibody; in both cases, no staining was detected.

Microscopy

All images were captured on an Olympus AX70 microscope at 400x magnification. This microscope was equipped with an Olympus DP50 digital camera and with the Viewfinder Lite ver. 1 software (Pixera Corp., Los Gatos, CA). All fluorescence determinations were performed under nonsaturated conditions. Each experiment and staining were performed on the same day, and sperm pictures were photographed within 24 h to reduce the loss of fluorescence. All cell preparations from a single experiment were photographed in the same session and for the same exposure period. The fluorescence intensity was quantified using the MetaMorph Image J software (National Institutes of Health) and the background intensity was subtracted. For F-actin, all experiments were carried out in duplicate and at least 100 cells (5–7 pictures) per slide were quantified for fluorescence intensity. For PIP2(4,5) and PIP3(3,4,5), at least 50 cells were quantified for fluorescence intensity.

Determination of PI4K Activity

Washed cells (5 x 108 cells/ml) were homogenized in ice-cold buffer A (20 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10 µg/ml leupeptin) by sonication (three 10-sec pulses, power setting 4) using a Vibra Cell material sonicator (Sonics and Materials Inc., Danbury, CT). The homogenate was centrifuged for 10 min at 10 000 x g at 4°C, the supernatant was removed, and the protein concentration was determined by the Bradford method [55]. The activity of PI4K in the separated fraction were measured as the incorporation of radioactivity from [{gamma}-32P]ATP into the organic solvent-extractable material, as described previously [56, 57]. The standard reaction mixture (50-µl final volume) contained 50 mM Tris-HCl (pH 7.5), 0.4% Triton X-100, 20 mM MgCl2, 1 mM EGTA, 0.5 mM PI, 0.5 mg/ml BSA, 100 µM [{gamma}-32P]ATP, and 3–10 µg of protein. Inhibitors were added as indicated to the assay mixture, and incubated for 5 min at 25°C before the addition of [{gamma}-32P]ATP. The reaction was started by the addition of [{gamma}-32P]ATP, incubated for 20 min, and terminated by the addition of 3 ml of CHCl3:CH3OH:concentrated HCl at 200:100:0.75 (v:v:v). The organic solvent phase was separated from the [{gamma}-32P]ATP by adding 0.6 ml of 0.6M HCl, mixing vigorously, and letting the mixture stand to allow phase separation. The upper phase was discarded, and the lower phase was extracted with 1.5 ml of CHCl3:CH3OH:0.6M HCl at 3:48:47 (v:v:v), followed by mixing and phase separation. The lower phase was then transferred to scintillation vials, 3.5 ml of scintillation fluid was added, and the radioactivity was counted in a TRI-CARB liquid scintillation counter. The assay for PI4K activity included 0.4% Triton X-100, which blocked PI3K and PI5K activity. It has been demonstrated by TLC that more than 98% of the radioactivity is from PI4P production [56]. The production of PI4P was confirmed by separating the enzymatic products on TLC plates (Merck, Darmstadt, Germany) that were pretreated with 60 mM EDTA and 2% sodium potassium tartarate in 50% ethanol (pH 8). The chromatograms were developed with chloroform:methanol:ammonium hydroxide:water (90:90:7:20) and the phospholipids were visualized by autoradiography.

Determination of PI3K Activity

The cell homogenate was prepared as for the PI4K activity assay. The activity of PI3K in the separated fraction was measured as the incorporation of radioactivity from [{gamma}-32P]ATP into organic solvent-extractable material, as described previously [56]. The standard reaction mixture (50-µl final volume) contained 50 mM Tris-HCl (pH 7.4), 20 mM MgCl2, 1 mM EGTA,

0.5 mM PIP2(4,5), 0.5 mg/ml BSA, 100 µM [{gamma}-32P]ATP, and 3–10 µg of protein. The inhibitor was added as indicated to the assay mixture, and incubated for 5 min at 25°C before the addition of [{gamma}-32P]ATP. The reaction was started by the addition of [{gamma}-32P]ATP, incubated for 20 min, and terminated by the addition of 3 ml of CHCl3:CH3OH:concentrated HCl at 200:100:0.75 (v:v:v). The lipids were extracted and separated by TLC as described for PI4K activity. The phospholipids were visualized by autoradiography and quantified in a TRI-CARB liquid scintillation counter.

Statistical Analysis

Data are expressed as mean ± SD of at least three experiments for all determinations. Statistical significance was calculated by ANOVA with the Bonferroni post-hoc comparison test using the SPSS software (SPSS Inc., Chicago, IL).

RESULTS

Detection of PI3K and PI4K in Bovine Sperm

We first wished to demonstrate the presence of PI3K and PI4K in bovine sperm. Western blot analysis revealed the presence of the 110-kDa PIK4CB (PI4K ß type III isoform) and PIK3R1 (p85 {alpha} regulatory subunit of PI3K) in bovine sperm (Fig. 1A). The anti-PIK3R1 antibody recognized four protein bands of approximately 100 kDa, 85 kDa, 55 kDa, and 45 kDa in whole cell lysates of bovine spermatozoa (Fig. 1A). It is known that the PIK3R1 mRNA can be alternatively spliced, giving rise to a 55-kDa or 45-kDa protein, termed p55 or p45, respectively [26, 58], as well as an 85-kDa or 100-kDa protein, termed p85 or p85+8aa, respectively [29] These results are consistent with the three protein bands of p85, p85+8aa, and p55 found in boar spermatozoa [41]. The localizations of PIK4CB and PIK3R1 in bovine sperm were evaluated by immunocytochemistry. PIK4CB was found to be localized at the principal piece, midpiece, and the postacrosomal region, while PIK3R1 was found only in the sperm midpiece and the postacrosomal region (Fig. 1B).


Figure 01
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  		FIG. 1. Detection and localization of PIK4CB and PIK3R1 in bovine sperm. A) Immunoblot analysis of protein extracts from sperm cells, separated by SDS-PAGE and stained with anti-PIK4CB or anti-PIK3R1 antibody, as indicated. B) Immunocytochemistry of sperm cells: for PIK4CB, (a) sperm treated with anti-PIK4CB antibody and with secondary antibody conjugated to RED-X and (b) negative control without the primary antibody; and for PIK3R1, (c) bovine sperm treated with anti-PIK3R1 antibody and secondary antibody conjugated to RED-X and (d) negative control without the primary antibody. Bar = 20 µm.

PI4K is activated by spermine [59–63], which is a known component of the semen, and is inhibited by 10 µM WT, while 10 nM WT inhibits PI3K [39]. In the present study, we used two different assays to differentiate PI4K and PI3K activities. First, we assessed the enzymatic activity by following PI4P and PIP3(3,4,5) synthesis in bovine sperm extracts using [{gamma}-32P]ATP. The activity of PI4K was inhibited by a high concentration of WT (10 µM) but not by a lower concentration (10 nM) (Fig. 2A). Moreover, 10 µM spermine increased PI4K activity almost 3-fold when added to the reaction mixture, and this increase was completely blocked by 10 µM WT but not by 10 nM WT (Fig. 2A). The inhibition of PI4K activity by 10 µM WT suggests that the type III PI4K isoform is active in bovine sperm. The activity of PI3K was inhibited by 10 nM WT (Fig. 2C). Second, we determined quantitatively the intracellular levels of PIP2(4,5) and PIP3(3,4,5) using specific anti-PIP2(4,5) and anti-PIP3(3,4,5) antibodies, respectively, [49–54] by analyzing the fluorescence intensities of the immunostaining using the Image J software [8]. We found a significant increase in intracellular PIP2(4,5) levels in the presence of spermine, while treatment with 10 µM WT completely blocked this effect; however, no effect on PIP2(4,5) was seen in the presence of 10 nM WT (Fig. 2). The intracellular levels of PIP2(4,5) in the control cells were relatively low (Fig. 2). Regarding PIP3(3,4,5) production, there was a significant increase in intracellular PIP3(3,4,5) levels after 30 min of incubation under capacitation conditions and there was complete inhibition of PIP3(3,4,5) production in sperm treated with 10 nM WT (Fig. 2D).


Figure 02
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  		FIG. 2. P14K and PI3K activities and intracellular levels of PIP2(4,5) and PIP3(3,4,5). A) P14K activities (open bars) in sperm extracts were measured by the radioactive assay described in Materials and Methods. Sperm extracts were added to the reaction mixture with or without WT (10 nM or 10 µM) for 5 min, and then with or without spermine (10 µM) for an additional 5 min. The reaction was started by adding [{gamma}-32P]ATP for 20 min. The results are presented as the percentages of total lipid cpm incorporated into PtdI, whereby 100% activity is equivalent to 241 ± 31 pmol min–1 mg–1 protein. Quantitative analysis of intracellular PIP2(4,5) staining (closed bars) using the anti-PIP2(4,5) antibody. Sperm cells were incubated in mTALP for 10 min with or without WT (10 nM or 10 µM), and then treated with or without spermine (10 µM) for an additional 10 min. Sperm cells were stained and images were captured and quantified as described in Materials and Methods. B) Representative images of intracellular PIP2(4,5) staining of sperm cells treated as described above in A. C) P13K activities in sperm extracts were measured by the radioactive assay described in Materials and Methods. Sperm extracts were incubated in the reaction mixture with or without 10 nM WT for 5 min. The reaction was started by adding [{gamma}-32P]ATP for 20 min. The results are presented as the percentages of total lipid cpm incorporated into PtdI; whereby 100% activity is equivalent to 193 ± 38 pmol min–1 mg–1 protein. D) Representative images of intracellular PIP3(3,4,5) staining of sperm cells treated as described above in C. The data in A and C represent the mean ± SD of three experiments. * Significant difference from the corresponding control, P < 0.01. Bars in B and D = 20 µm.

The use of immunohistochemistry for quantifying PtdIs has been shown for other cell types [49–51, 54, 64, 65]. However, this is the first time that this method has been used in sperm. To evaluate the reliability of this quantitative analysis of intracellular PIP2(4,5) levels, we tested the ability to quantitate exogenous PIP2(4,5) associated with the cells. Sperm cells were incubated with 10 µM WT for 10 min to reduce the endogenous PIP2(4,5), and then increasing concentrations of exogenous PIP2(4,5) liposomes were added. The sperm-associated PIP2(4,5) was immunostained and quantified using the specific anti-PIP2(4,5) and the Image J software. Quantitative analysis of the fluorescence intensity (Fig. 3A) and images (Fig. 3B) indicated a gradual increase in the amount of cellular-associated PIP2(4,5) as a function of the increased amount of exogenous PIP2(4,5) added. These results support the use of the immunostaining assay for the quantitation of intracellular PIP2(4,5) levels.


Figure 03
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  		FIG. 3. Standard curve for intracellular PIP2(4,5) staining using increasing concentrations of exogenous PIP2(4,5) liposomes. A) Sperm cells were incubated in mTALP with 10 µM WT for 10 min, and then with exogenous PIP2(4,5) liposomes for an additional 10 min. Sperm cells were stained and images were captured and quantified as described in Materials and Methods. The data in A. represent the mean ± SD of three experiments. B) Representative images of intracellular PIP2(4,5) staining of sperm treated as described above for A. Bar = 20 µm.

Involvement of PI3K and PI4K in Actin Polymerization

We have recently demonstrated that actin polymerization in bovine sperm is dependent upon PLD activity [8]. Our aim in the present study was to demonstrate the relationship between actin polymerization and intracellular levels of PIP2(4,5), which is a known cofactor for PLD activation [9]. Incubation of sperm cells in the presence of exogenous PI or PIP2(4,5) liposomes revealed significant increases in both the intracellular PIP2(4,5) and F-actin levels (Fig. 4). These effects were blocked by 10 µM WT when PI but not PIP2(4,5) was added to the cells, further indicating the specificity of 10 µM WT for PI4K inhibition in our system (Fig. 4). The correlation between the intracellular levels of PIP2(4,5) and F-actin and the inhibition of both by WT suggest a possible relationship between PIP2(4,5) and F-actin synthesis.


Figure 04
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  		FIG. 4. Intracellular PIP2(4,5) increased by exogenous PI or PIP2(4,5) induces actin polymerization. A) Sperm cells were incubated in mTALP with or without 10 µM WT for 10 min, and 10 µM of PI or PIP2(4,5) liposomes were added for an additional 10 min. Sperm cells were stained for intracellular PIP2(4,5) and F-actin and images were captured as described in Materials and Methods. The data shown are the mean ± SD of three experiments. B) Quantitative analysis of intracellular PIP2(4,5) and F-actin staining of sperm treated as described above for A. Bar = 20 µm. * Significant difference compared to the control (F-actin, without treatment), P < 0.01; ** significant difference compared to the control (PIP2(4,5), without treatment), P < 0.01.

The relationship between PI4K activation and actin polymerization was further supported by the finding that 10 µM spermine increased intracellular PIP2(4,5) (Fig. 2) and F-actin formation (Fig. 5) within 10 min of incubation. This increase in F-actin was blocked by 10 µM WT (but not by 10 nM WT) and the activity could be restored by adding back exogenous PIP2(4,5), which suggests the activation of PI4K, but not PI3K, by 10 µM spermine (Fig. 5). While 10 µM spermine enhanced actin polymerization, higher levels (1 mM) of this agent did not similarly induce F-actin formation (Fig. 5). It is known that this polyamine can bind to PIP2(4,5) and block PLC activity [61, 63, 66, 67]. This binding may inhibit PLD binding to PIP2(4,5) and as a result, PLD cannot be activated and actin polymerization does not occur. Thus, the level of free intracellular PIP2(4,5) appears to be the rate-limiting step for actin polymerization.


Figure 05
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  		FIG. 5. Spermine induces PI4K-dependent actin polymerization. Sperm cells were incubated in mTALP with or without WT (10 nM or 10 µM) for 10 min, and then with or without spermine (10 µM or 1 mM) or with 10 µM PIP2(4,5) liposomes (as indicated) for an additional 10 min. Sperm cells were stained with phalloidin-FITC and the images were captured and quantified as described in Materials and Methods. The data represent the mean ± SD of three experiments. * Significant difference compared to the control (untreated samples), P < 0.01.

Involvement of PI3K and PI4K in Actin Polymerization During Sperm Capacitation

We investigated the correlation between PIP2(4,5) production and actin polymerization during sperm capacitation. Sperm cells incubated in capacitation medium showed a time-dependent increase in intracellular PIP2(4,5), as determined by immunofluorescence and F-actin levels, which were determined by phalloidin-FITC staining (Fig. 6A). The F-actin levels increased gradually during the 4 h of capacitation. The kinetics of PIP2(4,5) production was much faster than that of F-actin formation (Fig. 6A), reaching a maximum after 1 h, and was maintained at this relatively high level until the end of the 4-h incubation under capacitating conditions. To confirm our results, we used Western blot analysis for the detection of F-actin and G-actin at the beginning and end of capacitation. This assay is based on separating the Triton-insoluble cytoskeletal fraction (F-actin) from the Triton-soluble fraction (G-actin). We found that the ratio between F-actin and G-actin at the beginning of capacitation was relatively low, while at the end of capacitation, the ratio was relatively high (Fig. 6, B and C), indicating an increase in F-actin formation during sperm capacitation and further confirming the results obtained with phalloidin-FITC (Fig. 6A). The capacitation state of sperm incubated for 4 h in capacitation medium was detected by following the ability of the cells to undergo the acrosome reaction induced by Ca2+-ionophore A23187. The spontaneous acrosome reaction rate was 18 ± 2% and in the presence of the Ca2+-ionophore, this rate was enhanced to 68 ± 4%. In control experiments in which bovine sperm were incubated for 4 h in medium without heparin, we did not observe any increase in actin polymerization or Ca2+-ionophore-induced acrosome reaction [1].


Figure 06
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  		FIG. 6. Intracellular levels of PIP2(4,5) and F-actin during capacitation. A) Sperm cells were incubated in mTALP and at the indicated times, samples were taken for the PIP2(4,5) assay and F-actin staining. Images were captured and quantified as described in Materials and Methods. The results are presented as the percentages of the fluorescence intensity at 4 h, which was defined as 100% (32 and 42 fluorescence intensity units for PIP2(4,5) and F-actin, respectively). The fluorescence intensity units at 0 h were 17 and 21 for PIP2(4,5) and F-actin, respectively. The data represent the mean ± SD of three experiments. B) Sperm cells were incubated in mTALP for 4 h. The F-actin and G-actin contents at the beginning and end of capacitation (i.e., 0 h and 4 h) were determined by Western blotting. C) Densitometric quantification of five independent Western blots analyses. The results are presented as the F:G actin ratios. The data represent the mean ± SD of five experiments. * Significant difference compared to the control at 0 h, P < 0.01.

In order to distinguish between the PI3K and PI4K activities, the inhibitor WT was added to the cells at 10 nM or 10 µM at the beginning of the capacitation period, and the intracellular PIP2(4,5), PIP3(3,4,5), and F-actin levels were evaluated. After 4 h of incubation under capacitation conditions, there was a significant increase in the levels of intracellular PIP2(4,5), PIP3(3,4,5), and F-actin (Fig. 7). F-actin formation and PIP2(4,5) production during capacitation were blocked by 10 µM WT but not by 10 nM WT, whereas the levels of PIP3(3,4,5) were significantly reduced by 10 nM WT. These results further confirm that PI3K is inhibited by 10 nM WT, and that this enzyme is not involved in PIP2(4,5) or F-actin formation during capacitation. Moreover, these results further support the correlation between PIP2(4,5) production and actin polymerization during sperm capacitation.


Figure 07
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  		FIG. 7. The intracellular increases in PIP2(4,5) and F-actin during capacitation are inhibited by 10 µM WT. A) Sperm cells were incubated in mTALP with or without WT (10 nM or 10 µM) for 4 h. Sperm cells were stained with anti-PIP2(4,5), anti-PIP3(3,4,5) or phalloidin-FITC and the images were captured as described in Materials and Methods. B) Quantitative analysis of intracellular PIP2(4,5), PIP3(3,4,5), and F-actin staining of sperm treated as described above for A. The data represent the mean ± SD of three experiments. * Significant difference in intracellular F-actin compared to the control at 0 h, P < 0.01; ** significant difference in intracellular PIP2(4,5) compared to the control at 0 h, P < 0.05; *** significant difference in intracellular PIP3(3,4,5) compared to the control at 0 h, P < 0.05.

Involvement of PKA and PKC in Phosphoinositide-Dependent Actin Polymerization

In our recent study, we have shown that activation of PKA by dibutyryl cAMP (dbcAMP) or activation of PKC by phorbol myristate acetate (PMA) can induce PLD activation and actin polymerization in bovine sperm within 10 min or 30 min, respectively [8]. Therefore, we examined the involvement of PI3K and PI4K in actin polymerization induced by the activation of PKA or PKC. Incubation of sperm with dbcAMP (to activate PKA) stimulated the formation of F-actin, PIP2(4,5), and PIP3(3,4,5) after 10 min (Fig. 8A). These effects were blocked by the PKA inhibitor H-89 or by 10 nM WT, which specifically inhibits PI3K activity, which suggests that PI3K activity is required for PKA-dependent PIP3(3,4,5), PIP2(4,5), and F- actin formation in bovine sperm. Moreover, dbcAMP activated PI4K (Fig. 8B) and the phosphorylation of PIK3R1 (Fig. 8C), and activation of these kinases was blocked by H-89 (Fig. 8, B and C), which further support the involvement of PKA in PI4K and PI3K activation. Activation of PKC using PMA for 30 min revealed significant stimulation of F-actin and PIP2(4,5) formation, which were inhibited by the PKC inhibitor GF, which suggests that PI4K is activated by PKC (Fig. 9A). However, the intracellular levels of PIP3(3,4,5) were reduced by PMA treatment, and this reduction was restored by GF (Fig. 9A), which suggests that PI3K is down-regulated by PKC activation. This conclusion is further supported by the finding that PI4K activity was enhanced by PMA and this effect was inhibited by GF (Fig. 9B), while the phosphorylation of PIK3R1 was inhibited by PMA and the activity could be restored by GF (Fig. 9C). Finally, the stimulatory effect of PMA on actin polymerization and PIP2(4,5) production were completely blocked by 10 µM WT, which inhibits PI4K (Fig. 2A), but not by 10 nM WT (Fig. 9A), which inhibits PI3K activation (Fig. 9C). These data suggest that PI4K but not PI3K is activated when PKC is activated. Taken together, these data suggest that PI4K can be activated by either the PKA or PKC pathway, while PI3K is activated by PKA and down-regulated by the PKC pathway.


Figure 08
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  		FIG. 8. The involvement of PI3K and PI4K in PKA-dependent actin polymerization. Sperm cells were incubated in mTALP with or without 10 nM WT or H89 50µM for 10 min, and then with or without 1 mM dbcAMP for an additional 10 min. A) Sperm cells were stained with anti-PIP2(4,5), anti-PIP3(3,4,5) or phalloidin-FITC and the images were captured and quantified as described in Materials and Methods. The data represent the mean ± SD of three experiments. * Significant difference in intracellular F-actin compared to the control at 0 min, P < 0.01; ** significant difference in intracellular PIP2(4,5) compared to the control at 0 min, P < 0.05; *** significant difference in intracellular PIP3(3,4,5) compared to the control at 0 min, P < 0.05. B) Sperm extracts were assayed for PI4K activity. The reaction was started by adding [{gamma}-32P]ATP for 20 min. The results are presented as the percentages of total lipid cpm incorporated into PtdI in the control sample; whereby 100% activity is equivalent to 222 ± 60 pmol min–1 mg–1 protein. The data represent the mean ± SD of three experiments. * Significant difference compared to the control, P < 0.01. C) Proteins were extracted and separated by SDS-PAGE and stained with anti-phospho-PIK3R1 antibody; the anti-PIK3R1 and anti-ACTB antibodies served as loading controls. The data shown represent the results from one of three experiments.


Figure 09
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  		FIG. 9. The involvement of PI3K and PI4K in PKC-dependent actin polymerization. Sperm cells were incubated in mTALP with or without 10 nM WT, 10 µM WT or 0.1 nM GF for 10 min, and then with or without 100 ng/ml PMA for an additional 30 min. A) Sperm cells were stained with anti-PIP2(4,5), anti-PIP3(3,4,5) or phalloidin-FITC and the images were captured and quantified as described in Materials and Methods. The data represent the mean ± SD of three experiments. * Significant difference in intracellular F-actin compared to the control at 0 min, P < 0.01; ** significant difference in intracellular PIP2(4,5) compared to the control at 0 min, P < 0.05; *** significant difference in intracellular PIP3(3,4,5) compared to the control at 0 min, P < 0.05. B) Sperm were extracted and incubated in the reaction mixture for PI4K activity assay. The reaction was started by adding [{gamma}-32P]ATP for 20 min. The results are presented as the percentages of total lipid cpm incorporated into PtdI in the control sample; whereby 100% activity is equivalent to 306 ± 47 pmol min–1 mg–1 protein. The data represent the mean ± SD from three experiments. * Significant difference compared to the control, P < 0.01. C) Proteins were extracted, separated by SDS-PAGE, and stained with anti-phospho-PIK3R1 antibody; the anti-PIK3R1 and anti-ACTB antibodies served as loading controls. The data shown represent the results from one of three experiments.

DISCUSSION

PI3K and PI4K Involvement in Actin Polymerization During Sperm Capacitation

In a recent study, we showed that PLD-dependent actin polymerization occurs during mammalian sperm capacitation [8]. It is known from other cell types that PIP2(4,5) is a cofactor for PLD activation [9]. In the present study, we demonstrate a role for PI4K type III and PI3K in actin polymerization during bovine sperm capacitation. We suggest that PIP2(4,5), which is the product of PI4K and PI4P5K activities, mediates actin polymerization during sperm capacitation. This conclusion is supported by the following findings: 1) PIK4CB, PIP2(4,5), and F-actin were colocalized mainly in the midpiece and the postacrosomal region (Figs. 1 and 3), indicating a possible relationship between them; 2) comparison of the kinetics of PIP2(4,5) production and actin polymerization during capacitation revealed that PIP2(4,5) production occurs prior to F-actin formation (Fig. 6), which suggests that PIP2(4,5) formation leads to actin polymerization; 3) rapid actin polymerization was induced after 10 min of incubation with exogenous PIP2(4,5), and the degree of polymerization was comparable to the level of F-actin formed after 4 h under capacitating condition (Figs. 4 and 7), which suggests that PIP2(4,5) production is the rate-limiting step for actin polymerization; 4) spermine, which activates PI4K activity (Fig. 2), caused significant enhancement of the intracellular levels of PIP2(4,5) and F-actin after 10 min of incubation, further indicating that PI4K activation and the production of PIP2(4,5) are the rate-limiting steps for actin polymerization; 5) WT at 10 µM, a concentration known to inhibit PI4-kinase type III but not type II, almost completely inhibited the activity of PI4K (Fig. 2), significantly reduced the intracellular levels of PIP2(4,5), and blocked actin polymerization during sperm capacitation (Fig. 7). On the other hand, 10 nM WT, which inhibits PI3K activity and PIP3(3,4,5) production, affected neither PI4K activity (Fig. 2) nor the levels of intracellular PIP2(4,5) or F-actin enhancement that occurred under capacitating condition (Fig. 7). These data suggest that PI4K, but not PI3K, is involved in actin polymerization during sperm capacitation. In fact, we observed an increase in the intracellular levels of PIP3(3,4,5) during capacitation, and this production was blocked by 10 nM WT (Fig. 7); however this PIP3(3,4,5) is probably not important for PIP2(4,5) production and actin polymerization during sperm capacitation. It has been shown elsewhere that 10 nM WT does not block protein tyrosine phosphorylation during human sperm capacitation, which suggests that PI3K is probably not involved in this process [45]. A recent study in porcine sperm also suggests that PI3K is not involved in sperm capacitation [68]. Thus, the increase in PIP3(3,4,5) levels during sperm capacitation may be important for the acrosome reaction, as we suggested previously [69].

The inhibition of actin polymerization and the reduction in intracellular PIP2(4,5) levels by 10 µM WT and the restoration of these activities by exogenous PIP2(4,5) or PI4P but not by PI or PS (Fig. 4) [69] further indicate that PI4K activation is the rate-limiting step in the actin polymerization process. We suggest that PIP2(4,5) production during capacitation is essential for PLD activation leading to actin polymerization. This idea is supported by the similar kinetics of PLD activation [8] and PIP2(4,5) production (Fig. 6A) during sperm capacitation. Moreover, we have shown previously that actin polymerization induced by exogenous PIP2(4,5) is blocked by PLD inhibition [69], which indicates that PIP2(4,5) is a cofactor for PLD activation in bovine sperm.

Relationships Between PKA, PKC, PI3K, and PI4K

The induction of endogenous F-actin, PIP2(4,5), and PIP3(3,4,5) by the addition of exogenous dbcAMP and the inhibition of these effects by H-89 indicate that PKA mediates all three of these processes (Fig. 8A). Under these conditions, inhibition of PI3K by 10 nM WT induced significant reductions in cellular PIP2(4,5), PIP3(3,4,5), and F-actin (Fig. 8A). On the other hand, activation of PKC by PMA induced significant enhancement of the intracellular PIP2(4,5) and F-actin levels, while the level of PIP3(3,4,5) was reduced (Fig. 8B). Moreover, the enhanced effect of PKC activation was completely blocked by 10 µM WT, whereas 10 nM WT had no effect (Fig. 8B), which indicates the involvement of PI4K, but not PI3K, in PKC-dependent PIP2(4,5) production and actin polymerization. This conclusion is further supported by the activation of PI4K activity by PMA and the inhibition of this activation by GF; conversely, we have demonstrated the inhibition of PI3K by PMA and the reversion of this effect by GF (Fig. 9, B and C).

We have shown previously that PKC is involved in PLD activation and F-actin formation in bovine sperm [8, 69, 70], and in the present study, we show that PKC is involved in actin polymerization and PIP2(4,5) production. Taken together, these data suggest that PI4K is involved in PKC- and PLD-dependent F-actin formation. In the presence of PMA, there is no need for PIP2(4,5) hydrolysis in order to activate PKC, so PKC is active when PI4K is inhibited by 10 µM WT. PKC is probably localized upstream of PI4K in the cascade that leads to actin polymerization. This point is further supported by our demonstration of PI4K activation by PMA and the inhibition of this effect by GF (Fig. 9B), as well as by PMA-dependent PIP2(4,5) production and its inhibition by 10 µM WT (Fig. 8B). These results suggest that PKC mediates PI4K activation. However, when PKA is activated by dbcAMP, conditions under which PLC and PKC activities are inhibited [8], F-actin formation was blocked by inhibiting PI3K (Fig. 8A), which suggests that PKC activity is not required under these conditions.

It seems that two alternative pathways lead to PI4K activation in sperm: 1) activation by PKC independent of PI3K; and 2) activation by PKA, which is mediated by PI3K activity. Thus, it is possible that the relative PKA and PKC activities dictate the PI3K activation state. When PKA activity is high and PKC activity is low, PI3K could be activated, whereas decreasing PKA activity and increasing PKC activity would down-regulate PI3K activity. Since PIP3(3,4,5) was produced during in vitro capacitation (Fig. 7), we suggest that PI3K is active in PIP2(4,5) phosphorylation, although this activity is not necessary for PIP2(4,5) production and actin polymerization during the capacitation process.

It has been suggested previously that PIP3(3,4,5) can activate PI4K and PI4P5K via ARF to produce PIP2(4,5), and that PKC can inhibit PI3K activity [20], a model that fits our findings. We have previously shown that the small G-protein, Rho [1], and ARF [69] are involved in actin polymerization in sperm. Thus, when PKC is activated by PMA, PI3K activity is down-regulated (Fig. 9C), and we also observed a reduction in the cellular levels of PIP3(3,4,5) and a reduction in PI3K-dependent actin polymerization (Fig. 8B). However, under conditions in which PKC is down-regulated by the activation of PKA, as we have shown previously [8], we found PI3K-dependent F-actin formation (Fig. 8A), which is probably mediated via the ARF pathway. We found that actin polymerization induced by dbcAMP/PKA is mediated by PLD [8], and in the present study, we showed that treatment with dbcAMP enhanced intracellular PIP2(4,5) levels that were blocked by H-89. Taken together, these results suggest that the activation of PKA indirectly activates PI4K via the activation of PI3K, leading to PIP2(4,5) production, PLD activation, and actin polymerization.

It is still not clear how PI4K is activated during capacitation. We have shown in the present study that PI4K activity and actin polymerization are stimulated by a relatively low concentration of spermine (10 µM), whereas this stimulation was abrogated at a relatively high concentration of spermine (1 mM) (Fig. 5). It is known that at ejaculation, sperm are exposed to a millimolar concentration of spermine [71]. We have shown previously that the bovine sperm acrosome reaction can be induced by 10 µM spermine but not by 1 mM spermine [72]. In that study, we also showed that spermine was taken up very rapidly by the sperm cells and released rapidly when incubated under capacitation conditions. Therefore, we propose that in the millimolar range, spermine acts as a decapacitation factor, and the decrease in its cellular concentration allows the sperm to undergo the capacitation process [72]. This old concept fits the data of the present study, in which micromolar concentrations of spermine activated PI4K, while concentrations in the millimolar range prevented PLD activation during capacitation, probably due to its binding to PIP2(4,5). Thus, spermine appears to be a physiological regulator of PI4K activity during capacitation.

In summary, we suggest a model in which two possible pathways lead to actin polymerization in sperm: 1) when PKA activity is relatively low and PKC activity is high, enhancement of PIP2(4,5) via the activation of PI4K by spermine or PKC leads to PLD activation and actin polymerization. Under these conditions PIP3(3,4,5) is not involved in actin polymerization; and 2) when PKA activity is relatively high and PKC activity is down-regulated, PKA enhances PIP2(4,5) via the activation of PI3K, which activates PI4K and PI4P5K, leading to PLD activation and actin polymerization.

FOOTNOTES

1Supported by the Chief Scientist of the Israeli Ministry of Health and by the Ihel Foundation to H.B. Back

Correspondence: 2FAX: 972 3 5344766; e-mail: breith{at}mail.biu.ac.il

Received: 23 August 2006.

First decision: 10 October 2006.

Accepted: 27 April 2007.

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