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Biology of Reproduction 63, 1271-1277 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Activation of Protein Kinase C{alpha} in the Lysophosphatidic Acid-Induced Bovine Sperm Acrosome Reaction and Phospholipase D1 Regulation1

Meirav Garbia, Sara Rubinsteina, Yehudit Laxa, and Haim Breitbart2,a

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

ABSTRACT

Protein kinase C (PKC) has been implicated in the sperm acrosome reaction. In the present study, we demonstrate induction of the acrosome reaction and activation of sperm PKC{alpha} by lysophosphatidic acid (LPA), which is known to induce signal transduction cascades in many cell types via binding to specific cell-surface receptors. Under conditions by which LPA activates PKC{alpha}, there is significant stimulation of the acrosome reaction, which is inhibited by PKC inhibitors. Protein kinase C{alpha} belongs to the Ca2+-dependent classical PKC family of isoforms, and indeed we show that its activation depends upon the presence of Ca2+ in the incubation medium. Protein kinase C{alpha} is a known regulator of phospholipase D (PLD). We investigated the possible regulatory relationships between PKC{alpha} and PLD1. Using specific antibodies against PLD1, we demonstrate for the first time its presence in bovine sperm. Furthermore, PLD1 coimmunoprecipitates with PKC{alpha} and the PKC{alpha}-PLD1 complex decomposes after treatment of the cells with LPA or 12-O-tetradecanoyl phorbol-13-acetate, resulting in the translocation of PKC{alpha} to the plasma membrane and translocation of PLD1 to the particulate fraction. A possible bilateral regulation of PKC{alpha} and PLD1 activation during the sperm acrosome reaction is suggested.

kinases, signal transducers, sperm, sperm capacitation/acrosome reaction

INTRODUCTION

The protein kinase C (PKC) family of serine/threonine kinases is central to the signal transduction pathway in many cell types [1, 2]. This family comprises at least 11 different isoforms, divided into three major groups, namely, classical (cPKC), novel (nPKC), and atypical (aPKC) PKC. Classical PKCs and nPKCs translocate from the cytosol to membranous sites upon activation with diacylglycerol (DAG) [3]. The cPKCs depend on Ca2+ for activity [4], whereas the other PKCs are Ca2+ independent [2]. Phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (PMA) can substitute for DAG in the activation of PKC [5].

The presence and involvement of PKC in the mammalian sperm acrosome reaction has been previously demonstrated [6, 7]. We have shown direct activation of PKC{alpha} and ßI by the PKC activator, PMA, in bovine spermatozoa [8] and also that PMA induces the sperm acrosome reaction [9–11]. Moreover, the possible involvement of PKC in the acrosome reaction was demonstrated by the physiological inducers zona pellucida or progesterone and was blocked by PKC inhibitors [12, 13]. Several other molecules, present in the female genital tract, have also been implicated in the activation of the acrosome reaction by specific receptors [14]. The bioactive lipid lysophosphatidic acid (LPA) is one component of the follicular fluid [15] and was shown to enhance in vitro blastocyst formation in a mouse in vitro fertilization system [16], suggesting a possible physiological role for LPA in the fertilization process and embryonic development. Lysophosphatidic acid has a growth factor-like effect on many somatic cell types and can initiate a signal transduction cascade upon its interaction with specific cell membrane receptors through activation of PKC [17]. Several reports indicate that LPA induces its activity through activation of PKC [17].

It is known that LPA is generated as a result of phospholipase D (PLD) activity [18]. Phospholipase D is a widely distributed enzyme that is controlled by hormones, neurotransmitters, growth factors, and cytokines in mammalian cells [19] and is known to generate bioactive LPA from pre-existing lysophosphatidylcholine [20]. It is well established that stimulation of acrosome reaction sperm with natural agonists such as progesterone or zona pellucida causes a rise in DAG by the hydrolysis of various phospholipids [21]. Furthermore, there is clear evidence that PLD plays an important role in signaling during the acrosome reaction of sea urchin spermatozoa [22]. However, studies on mammalian spermatozoa suggest that this phospholipase does not contribute to DAG generation presumably due to its little activity [21]. Thus, the specific role of PKC in the mechanism of sperm acrosome reaction and the mechanism of PLD regulation in the mammalian sperm has not been completely established. Nevertheless, there is evidence that PKC plays a major role in control of PLD. This is supported by numerous studies showing that PKC inhibition or its down-regulation inhibit the stimulation of PLD by agonists in many cell types [23]. Moreover, PKC{alpha} and PLD1 can associate as a complex, and this interaction can be promoted by phorbol ester pretreatment of the cells [24, 25].

In the present study, we have investigated the effect of LPA, a bioactive metabolite of the female genital tract, on PKC{alpha} activation and PLD1 regulation in bovine sperm cells. We demonstrate that LPA can induce the acrosome reaction and provide direct evidence for PKC{alpha} activation in the LPA-induced sperm acrosome reaction. Further results suggest that PKC{alpha} may participate in LPA-mediated PLD activation in bovine sperm, leading to acrosomal exocytosis and successful fertilization.

MATERIALS AND METHODS

Sperm Preparation

Ejaculated bovine sperm provided by Israel Breeders Service (Hasherut Breeder Service, Hafetz Haim, Israel) were collected using an artificial vagina and diluted (1:1, v:v) in NKM medium consisting of NaCl (110 mM), KCl (5 mM), and 3-[N-morpholino]propanesulfonic acid (10 mM), pH 7.4. The cells were washed in NKM medium, by three centrifugations at 780 x g for 10 min, and the final pellets were resuspended in NKM, with the sperm concentration adjusted to 1–3 x 109 cells/ml. Only sperm cells with good motility (>70%) were used in the experiments.

Assessment of Sperm Acrosome Reaction

Washed cells (108 cells/ml) were capacitated for 4 h at 39°C in modified Tyrode medium (mTALP medium) supplemented with (20 µg/ml) heparin and CaCl2 (2 mM) [26, 27]. Lysophosphatidic acid or PMA (Sigma, St. Louis, MO) were then added for another 20 min of incubation. The percentage of acrosome-reacted sperm was determined microscopically on air-dried sperm smears using a biotin-conjugated Pisum sativum agglutinin (PSA) procedure according to Mendoza et al. [28]. An aliquot of spermatozoa (106 cells) was smeared on a glass slide and allowed to air dry. The sperm were then permeabilized by methanol for 30 sec at room temperature, incubated for 10 min with a blocking solution of Tris-buffered saline (TBS) containing 1% BSA, and followed by 30 min incubation with biotin-conjugated PSA (50 µg/ml) at room temperature in the dark. The slides were then washed with TBS and incubated with peroxide-conjugated extravidin (Sigma) (1:400 dilution) for 10 min. All incubations were performed in a moist chamber. The substrate (AEC from Histostain-SP kit, Zymed Laboratories, South San Francisco, CA) was then added for 10 min. Hematoxylin was used for counterstaining (3 min). The slides were mounted with GVA mounting solution (Zymed) and were examined microscopically. For each experiment, at least 200 cells per slide were evaluated. Cells with red staining over the acrosomal cap were considered acrosome intact; those with equatorial red staining or no staining at all were considered acrosome reacted.

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.

Subcellular Fractionation

Sperm cells were fractionated to separate cytosol, membranes, and particulate fraction proteins. Cells (1.5 x 109) were resuspended in 20 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 2 mM EGTA, 2 mM EDTA, 1 mM benzamidine, 1 mM Na3VO4, 10% glycerol, 25 µg/ml leupeptin, 4 µg/ml aprotinin, and 1 mM PMSF (homogenization buffer) and then sonicated (3 x 10-sec pulses, power setting 4) with a Vibra cell, Sonics (Sonics & Materials Inc., Danbury, CT) material sonicator. The homogenate was centrifuged for 10 min at 10 000 x g for pelleting the particulate fraction, containing head and tail fragments. The resulting supernatant was centrifuged at 100 000 x g (60 min, 4°C) for recovery of the cytosolic fraction (supernatant) and the membrane fraction (pellet). The cytosolic fraction was concentrated to at least one tenth of the original volume using a microconcentrator 30 (Amicon, Lexington, MA). The membrane fraction was resuspended in the homogenization buffer supplemented with 0.6% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) [29]. The particulate fraction was solubilized with SDS lysis buffer. Protein concentration of the cytosolic and membrane fractions was determined by the Bradford method [30] using Bio-Rad (Richmond, CA) reagents. For immunoblot analysis, the cell lysates were boiled for 5 min in SDS-PAGE sample buffer [31] and separated on a 7.5% SDS-polyacrylamide gel.

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 mA, 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, pH 7.6, containing 0.1% Tween 20 (TBST), for 30 min at room temperature. The PKC{alpha} isoform and PLD were immunodetected using the following antibodies: rabbit polyclonal anti-PKC{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000, and rabbit polyclonal anti-PLD1 (generated against a peptide corresponding to amino acid residues 675–688 of human PLD1 [32], and kindly provided by Prof. M. Liscovitch of The Weizmann Institute of Science) diluted 1:1000. The membranes were incubated overnight at 4°C with the appropriate primary antibody. Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific horseradish peroxidase (HRP)-linked secondary antibody (Jackson Laboratories, West Grove, PA) diluted 1:10 000 in TBST. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham, Little Chalfont, UK). Specificity of the antibodies was determined by preabsorbing the antibodies with 10 µg of their peptide antigens for 1 h before incubating the antibodies with the membrane. Quantitation of the Western blots was performed using a laser densitometer.

Immunocytochemistry

Sperm (106 cells) were spread on glass slides and then fixed and permeabilized with cold methanol (10 min) followed by cold acetone (10 min). Nonspecific reactive sites were blocked with TBS-containing 1% BSA for 10 min at room temperature. Then the cells were incubated for 2 h at room temperature with the appropriate primary antibody. Between antibody incubations, the cells were washed three times (5 min) in TBS. Primary antibodies were detected by fluorescein isothiocynate (FITC)- or rhodamine-conjugated anti-IgG antibodies (30 min incubation) followed by washing in distilled water and mounted in Fluro Guard Antifade Reagent (Bio-Rad). The cells were then examined in the confocal fluorescence microscope (Bio-Rad MRC-1024). Nonspecific staining, determined by incubation without primary antibody or in the presence of specific antigenic peptide (10 µg), was negligible.

Immunoprecipitation

Proteins were extracted from spermatozoa using triple detergent RIPA lysis buffer consisting of 0.5% deoxycholate, 2% Triton-X-100 and 0.2% SDS, 50 mM NaCl, 5 mM Tris-HCl (pH 7.5), 1 mM Na3VO4, 1 mM benzamidine, 25 mM leupeptin, 4 µg/ml aprotinin, and 1 mM PMSF. Equal protein aliquots were incubated with anti-PKC{alpha} antibody (1 µg) for 2 h, 4°C, and with 60 µl of a 50% slurry of protein A-Sepharose for 1 h, 4°C. The immunoprecipitates were collected by centrifugation and washed four times (12 900 x g, 5 min, 4°C) with TBS containing 0.1% Triton-X-100. The final pellet was resuspended in sample buffer and boiled for 5 min before analyzing on SDS-PAGE and by Western blotting as described above.

RESULTS

The bioactive lipid LPA activates signal transduction cascade via binding to a cell-specific receptor [33]. Lysophosphatidic acid is secreted from various cells and is found in the serum [33] and in the follicular fluid [15]. Because LPA is present in the female reproductive tract, as a first step we tested whether it could induce sperm acrosomal exocytosis. Lysophosphatidic acid in different concentrations was added to capacitated bovine spermatozoa for 20 min. A dose-dependent increase in acrosomal exocytosis was found (Fig. 1). The maximal effect of LPA was found at about 300 µM.



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  		FIG. 1. Dose dependence of LPA-induced acrosome reaction. Sperm were incubated for 4 h in mTALP medium in the presence of heparin (20 µg/ml) and then stimulated with the indicated concentrations (30–1000 µM) of LPA for 20 min. The percentage of acrosome-reacted sperm was assessed by HRP-conjugated PSA as described in Materials and Methods. For each sample, 200 cells were counted. Results are mean ± SEM (n = 3)

No effect of LPA on sperm motility was observed. In many cell types, LPA activates PKC [17]. We have previously shown that PKC{alpha} is present on bovine sperm [8] and is activated as part of the signaling pathway leading to the acrosome reaction [9]. To demonstrate further the role of PKC in LPA-mediated acrosome exocytosis, we tested the effect of the PKC inhibitors staurosporine (STP) and bisindolylmaleimide I (GF) that bind to the catalytic site of PKC. Both agents significantly inhibited LPA-induced acrosomal exocytosis by 91% (STP) or 83% (GF) (Fig. 2). Smaller inhibition was found when acrosomal exocytosis was induced by PMA, 65% or 70% inhibition by STP and GF, respectively (Fig. 2). These data suggest that PKC is involved in the mechanism by which LPA induces acrosomal exocytosis.



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  		FIG. 2. Inhibitory effect of STP and GF on LPA- and PMA-induced acrosome reaction. Sperm were incubated for 4 h in mTALP medium in the presence of heparin (20 µg/ml). The PKC inhibitors STP (10 nM) and GF (10 nM) were added for the last 20 min of capacitation. Lysophosphatidic acid (300 µM) or PMA (100 ng/ml) were subsequently added for 20 min. Percentages of PMA- or LPA-stimulated acrosome-reacted cells was determined as described in Materials and Methods. Results shown represent mean ± SEM (n = 3) after subtracting the spontaneous acrosome reaction (20%) determined in the absence of PMA or LPA. Solid bars, control cells without inhibitor; open bars, GF-treated cells; cross-hatched bars, STP-treated cells. a compared with b, P < 0.02; c compared with d, P < 0.02; c compared with e, P < 0.006

Protein kinase C, upon activation, translocates from the cytosol to cell membranes; thus translocation is an indicator of PKC activation [34]. In order to demonstrate the PKC translocation, sperm cells were fractionated into three fractions, cytosol, plasma membranes and particulate fraction as described in Materials and Methods. The amount of PKC{alpha} was determined in each fraction by Western blot analysis, and the results show that PKC{alpha} is present only in the cytosolic fraction (Fig. 3). The same pattern was revealed in capacitated or noncapacitated sperm cells, suggesting that PKC{alpha} is not activated during capacitation. Treatment of capacitated cells by LPA or PMA for a short time (10 min) induced an enhancement of PKC{alpha} in the plasma membranes and a significant concomitant decrease in its amount in the cytosolic fraction (Fig. 3). The densitometric analysis of the Western blot from four separate experiments revealed that in the control cells, 95% of PKC{alpha} is found in the cytosolic fraction and only 5% in the plasma membrane. Treating the cells with PMA or LPA revealed that the amount of PKC{alpha} in the cytosolic fraction was reduced to 45% or 49%, while its amount in the plasma membrane was enhanced to 55% or 51% in the presence of PMA or LPA, respectively. This suggests that PKC{alpha} is translocated from the cytosol to the cell plasma membrane due to LPA or PMA treatment. Lysophosphatidic acid or PMA also induced PKC translocation in noncapacitated cells (not shown), suggesting that capacitation is not required for activation of PKC{alpha}.



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  		FIG. 3. Subcellular redistribution of PKC{alpha} in response to LPA. Sperm cells were incubated alone (C) or in the presence of LPA (300 µM) (L) or PMA (100 ng/ml) (P) for 10 min. Cells were homogenized and fractionated into cytosol, plasma membrane, and particulate fractions as described in Materials and Methods. A) Protein kinase C{alpha}-immunodetection. Proteins were separated on SDS-PAGE. For total protein separation, protein extracts from 1.5 x 107 cells were loaded on each lane, and for cell fractions equal protein aliquots of 15 µg of total protein per lane were used. Proteins were transferred to nitrocellulose, immunoblotted with PKC-specific polyclonal antibody, and visualized as described in Materials and Methods. Molecular weights of prestained high range marker proteins are indicated (x10-3). The blot shown is representative of three separate experiments. B) Densitometric analysis of PKC{alpha}. Bars represent the relative PKC{alpha} levels expressed as mean optical density (OD) units ± SEM (n = 4) compared with b, P < 0.05; a compared with c, P < 0.03; d compared with e, P < 0.001; d compared with f, P < 0.015

Acrosomal exocytosis is a Ca2+-dependent process. Lysophosphatidic acid induced the acrosome reaction in the presence of Ca2+ and failed to do so in its absence in the incubation medium (data not shown). Because PKC{alpha}, which is activated by LPA, is a Ca2+-dependent isoform, we examined whether this activation depends upon the presence of Ca2+ in the incubation medium as well. It is shown in Figure 4 that LPA-mediated induction of the translocation of PKC{alpha} from the cytosol to the plasma membrane is Ca2+ dependent. Furthermore, we also found that the presence of BSA in the incubation medium is necessary for PKC translocation. Bovine serum albumin apparently prevents the formation of LPA micelles that are unable to bind LPA receptors [35]. In the presence of PMA, the addition of Ca2+ is not required to induce translocation of PKC{alpha} from the cytosol to the plasma membrane (Fig. 4). We assume that in the presence of PMA the low concentration of intracellular Ca2+ is sufficient to promote PKC{alpha} translocation.



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  		FIG. 4. Effect of BSA and Ca2+ on subcellular distribution of PKC{alpha} in response to LPA. Protein immunoblots showing the levels of PKC{alpha} in subcellular fractions of the cells. Sperm cells were incubated with LPA (300 µM) or PMA (100 ng/ml) for 10 min, in the presence or absence of BSA (3 mg/ml) and/or Ca2+ (2 mM). Cells were homogenized, fractionated, and immunodetected for PKC{alpha} as described in Materials and Methods. The blot shown is representative of three separate experiments

The specific role of PKC{alpha} in the acrosome reaction is not established yet. We speculated that PKC{alpha} might be involved in the regulation of sperm PLD. It was suggested previously that PKC{alpha} can regulate the activation of PLD1 in various cell types [23]. In sea urchin spermatozoa, PLD participates in signal transduction leading to acrosomal exocytosis [36]. However, the involvement of PLD in mammalian acrosomal exocytosis is yet unclear. To elucidate further the role of PLD1, antibodies directed against PLD1 were used to identify this protein in bovine sperm. By Western blotting, these antibodies specifically recognized a single protein of approximately 115 kDa (Fig. 5). The specificity of the antibodies was demonstrated by their preabsorption with the specific peptide antigens that completely prevented immunodetection of the 115-kDa protein band. The PLD1-specific antibodies were used to examine the cellular localization of PLD1 through immunocytochemical studies. Using immunofluorescent staining, PLD1 was found to be localized in the acrosomal region of the sperm, suggesting possible involvement in acrosomal exocytosis (Fig. 6).



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  		FIG. 5. Expression of bovine sperm PLD1. Protein immunoblots showing PLD1 in bovine sperm. Total protein was extracted with SDS lysis buffer, separated on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with PLD1-specific polyclonal antibody in the absence (-) or presence (+) of a specific PLD peptide (pep), as described in Materials and Methods. The blot shown is representative of three separate experiments



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  		FIG. 6. Indirect immunofluorescence localization of PLD1 in sperm cells. A) Bovine sperm cells were fixed as described in Materials and Methods and stained with anti-PLD1 polyclonal antibody (1:200 dilution), followed by FITC-conjugated donkey anti-rabbit IgG (1:250 dilution). Staining can be observed in the acrosomal region. B) Control experiment, in which sperm were stained with anti-PLD1 antibody preabsorbed with a specific antigenic peptide (10 µg/ml), followed by FITC-conjugated donkey anti-rabbit IgG (1:250 dilution). No staining was visualized. Controls for the antibodies included primary antibody replaced by preimmune rabbit serum or FITC-conjugated anti-rabbit IgG only. In these specimens, no staining was observed (not shown)

Preabsorption of the PLD1-specific antibodies with the immunizing peptides abrogated immunostaining (Fig. 6). As mentioned above, PKC{alpha} can regulate the activation of PLD1 in many cell types. If protein-protein interaction is the major mechanism by which PKC activates PLD, then the two enzymes might form a complex in the cell that can be decomposed after PKC activation, allowing each enzyme to translocate to cell membranes.

Immunoprecipitation of sperm proteins using anti-PKC{alpha} antibody, revealed the coprecipitation of PKC{alpha} and PLD1 (Fig. 7), suggesting that the two enzymes are colocalized in the cell. Cell fractionation revealed that about 40% of PLD1 is localized in the cytosol and 60% in the particulate fraction (Fig. 8). Similar results were obtained for both capacitated or noncapacitated cells. Treatment of sperm cells with LPA (or PMA) to activate PKC{alpha} revealed a significant increase (from 60% to 88%) of PLD1 in the particulate fraction and a concomitant decrease (from 40% to 12%) in the cytosolic fraction (Fig. 8). These data suggest that activation of PKC{alpha} cause the translocation of PLD1 from the cytosol to the particulate fraction suggesting coregulation of PKC{alpha} and PLD1.



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  		FIG. 7. Phospholipase D associates with PKC{alpha}. Sperm cells were treated with LPA (300 µM) or PMA (100 ng/ml) for 10 min. Cells were then lysed with RIPA lysis buffer, and immunoprecipitates (IP) were prepared using anti-PKC{alpha} antibody ({alpha}PKC{alpha}) (1 µg/ml). Immunoprecipitates were analyzed by SDS-PAGE, followed by transfer of proteins and Western blotting (Blot) with anti-PLD1 ({alpha}PLD1) and anti-PKC{alpha} antibodies. The blots shown are representative of three experiments



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  		FIG. 8. Subcellular distribution of PLD1 in response to LPA. Sperm cells were incubated in the absence (C) or presence of LPA (300 µM) (L) or PMA (100 ng/ml) (P) for 10 min. Cells were homogenized and fractionated into cytosol (Cyt), plasma membrane (Mem), and particulate (Part) fractions as described in Materials and Methods. A) Phospholipase D1 immunodetection. Proteins were separated on SDS-PAGE using equal amounts of protein per lane (15 µg of total proteins). Proteins were transferred to nitrocellulose, immunoblotted with PLD1-specific polyclonal antibody, and visualized as described in Materials and Methods. The blot shown is representative of three separate experiments. B) Densitometric analysis of the PLD1 band. Bars represent the relative PLD1 levels expressed as mean OD units ± SEM (n + 4). a compared with b, P < 0.05; a compared with c, P < 0.005; d compared with e, P < 0.05; d compared with f, P < 0.01

DISCUSSION

We recently showed the activation of PKC{alpha} and ßI in bovine sperm treated with the known PKC activator, PMA [8]. Here we demonstrate that activation of PKC{alpha} by a physiological ligand, LPA, leads to acrosomal exocytosis. The involvement of PKC in sperm acrosomal exocytosis is now well documented [7,9], but there is no information regarding the specific PKC isoforms that participate in the process. Lysophosphatidic acid binds to specific cell surface receptors and affects cells in a manner similar to growth factors [17, 37]. It was recently shown that LPA can cause an increase in intracellular Ca2+ by activating inositol triphosphate (IP3)-dependent Ca2+ channels [38–40], and it can induce exocytosis in various cell types [41, 42]. Here we demonstrate that LPA induces Ca2+-dependent sperm acrosomal exocytosis (Fig. 1), which is inhibited by PKC inhibitors (Fig. 2). Under the same conditions, LPA can induce the specific translocation of PKC{alpha} from the cytoplasm to the sperm plasma membrane (Fig. 3), indicating PKC activation. Translocation to the membrane is a necessary step in PKC activation, as was shown recently in many cell types [3, 34] including bovine sperm [8]. It was shown in smooth muscle cells that LPA can activate PKC{alpha}, PKCß, PKC{epsilon}, and PKC{zeta} by increasing their quantity in the particulate fraction of the cells [43].

Lysophosphatidic acid acts via G-protein-coupled receptors, especially those of the Gi group [37, 44]. Interaction of LPA with the Gq/11-coupled receptor is expected to activate phospholipase C (PLC) to produce IP3 and DAG. The IP3 can activate IP3-dependent Ca2+ channels of intracellular membranes leading to elevation of cytosolic Ca2+ concentration, and together with DAG, to activation of Ca2+-dependent cPKC. The Gq/11 as well as IP3 receptors are present in the acrosomal region of various sperm cells [45, 46]. It is possible that LPA binds to a G1/11-coupled receptor resulting in the activation of PLCß which in turn hydrolyzes phosphatidyl inositol bisphosphate to produce IP3 and DAG, which together cause the activation of PKC{alpha}. The translocation of PKC{alpha} from the cytoplasm to the plasma membrane may ensure rapid phosphorylation of specific substrates that are important for acrosomal exocytosis. We have shown that bovine sperm PKC{alpha} and PKCßI are both translocated from the cytoplasm to the plasma membrane resulting in their activation [8]. In mouse epididymal sperm, PKCß is located in the acrosomal region, close to three of its specific substrates [47]. Furthermore, in human sperm, PKC was colocalized with various cytoskeletal and other structural elements such as myosin, vimentin, and actin, suggesting that these proteins are potential substrates for sperm PKC [48].

Our next question concerned the possible regulatory connection between PKC{alpha} and PLD1. It was reported that PKC{alpha} is an important regulator of PLD1 activation [18, 23]. We show here, the presence of 115-kDa PLD1 in bovine sperm (Fig. 5) localized to the cytosolic fraction and to the particulate fraction but not to the plasma membranes (Fig. 8). In addition we show that the enzyme is localized in the acrosomal region of the sperm head (Fig. 6) and coimmunoprecipitates with PKC{alpha} (Fig. 7). Treatment of sperm with the PKC{alpha} activators LPA or PMA causes fast translocation of PLD1 from the cytoplasm to the particulate fraction (Fig. 8). Under these same conditions, LPA or PMA cause the translocation of PKC{alpha} from the cytoplasm to the cell plasma membrane (Fig. 3). Because PKC{alpha} and PLD1 are colocalized in the cells as revealed by their coimmunoprecipitation (Fig. 7), we suggest that the two enzymes form a complex in the cytosol. After activating PKC{alpha} by LPA or PMA, the two enzymes separate, the PKC{alpha} is translocated to the plasma membrane, and PLD1 is translocated to the particulate fraction. The colocalization of the PKC{alpha} and PLD1 in the cytosol, the localization of PLD1 in the acrosomal region, and the translocation of PKC{alpha} to the plasma membrane and PLD1 to the particulate fraction after LPA or PMA treatment suggest that PLD1 is probably translocated to the outer acrosomal membrane, which is part of the particulate fraction. This suggestion is supported by the fact that the localization of PLD1 in the acrosomal region of the head was not changed after removal of the plasma membrane of LPA- or PMA-treated sperm (see Fig. 6A).

It is known that the acrosome is formed from the Golgi system [49], and PLD was shown to induce the formation of secretory vesicles in the Golgi [50]. Thus, binding of PLD1 to the outer acrosomal membrane could cause hydrolysis of phospholipids to produce phosphatidic acid, which is a fusogenic compound that may stimulate the fusion between the outer acrosomal and the plasma membrane leading to acrosomal exocytosis.

In various cell types, it was shown that after its translocation, active PKC is bound to specific anchor proteins in the membrane, collectively called receptors for activated C kinase or RACKs [51]. We have recently shown that RACK is present in the plasma membrane fraction of bovine sperm [8]. The binding of PKC to RACK helps to position correctly the PKC relative to its substrate and enables efficient phosphorylation.

To date, PKC{alpha} has been considered a regulator for PLD1 activation, but it is also possible to consider PLD1 as a regulator of PKC{alpha} activation. As long as PLD1 is bound to PKC{alpha}, the latter cannot be translocated to the plasma membrane and to bind to RACK, meaning that under these conditions PLD1 prevents the premature activation of PKC{alpha}. When the sperm are activated, by LPA for example, the increase in DAG production causes separation of PKC{alpha} from PLD1 resulting in the translocation of PKC{alpha} to the plasma membrane and its activation. Thus, in resting cells, PLD1 might be considered as receptor for inactive C kinase (RICK). It was suggested that in addition to RACKs, cells also contain RICKs, and the two kinds of protein regulate PKC activity in the cell [52].

In conclusion, we suggest here the involvement of PKC{alpha} in LPA-induced sperm acrosomal exocytosis. In addition we propose the bilateral regulation of PKC{alpha} and PLD1 that might participate in the mechanism leading to acrosomal exocytosis. Lysophosphatidic acid is present in the female reproductive tract, suggesting a possible physiological role in the induction of acrosomal exocytosis.

FOOTNOTES

First decision: 10 May 2000.

1 This research was supported by the Israel Science Foundation funded by The Academy of Sciences and Humanities and by Ihel Foundation to H.B. Back

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

Accepted: June 6, 2000.

Received: April 10, 2000.

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