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Gamete Biology |
a Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, People's Republic of China
b Museo Nacional de Ciencias Naturales, CSIC, 28006 Madrid, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, calcium, gamete biology, signal transduction, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies concerning spermatozoa from animal models, domestic species, or humans have uncovered some common themes in molecular events underlying the changes leading to membrane fusion during acrosomal exocytosis. Stimulation of spermatozoa to undergo acrosomal exocytosis results in the activation of phospholipases and the generation of membrane or intracellular messengers. Thus, stimulation with ZP results in activation of phosphoinositidase C and phosphatidylcholine (PC)-specific phospholipase C and the ensuing generation of various molecular species of diacylglycerol (DAG) [2–5]. Stimulation with ZP also leads to internalization of Ca2+ via channels [6]. This elevation of intracellular Ca2+ could be important for the activation of signaling cascades such as that involving the generation of DAG, because no rise in DAG is observed after ZP stimulation if Ca2+ entry is prevented [2].
Action of ZP is thought to be mediated by at least two signal transduction mechanisms: G proteins and tyrosine kinase [7]. The G proteins have been identified in spermatozoa, and stimulation with G protein agonists causes acrosomal exocytosis [8, 9]. Furthermore, stimulation with ZP in the presence of G protein inhibitors, such as pertussis toxin (PTX), results in no acrosomal exocytosis [3, 10].
Limited information is available regarding other signaling mechanisms activated on stimulation of spermatozoa with ZP [7]. An increase in cAMP has been detected after treatment with ZP [11]. This messenger may be important for downstream events, because acrosomal exocytosis induced by ZP does not take place after exposure to an inhibitor of protein kinase A (a target of cAMP action) [12]. Recent work has revealed that the p21 Ras-extracellular regulated kinase (ERK1/ERK2) cascade can be activated in spermatozoa in response to ZP [13], and this pathway may be important for acrosomal exocytosis [13, 14].
Studies using a model in which spermatozoa are induced to undergo acrosomal exocytosis in response to challenge with a Ca2+ ionophore have revealed that other pathways may have important roles in events underlying acrosomal exocytosis triggered by natural agonists. Stimulation with the ionophore A23187 to undergo the acrosome reaction leads to activation of phospholipase A2 (PLA2), as revealed by an elevation of free arachidonic acid and lysoPC and a decrease in PC (and other phospholipids) [15]. These lipid changes precede and are tightly coupled to A23187-induced exocytosis; furthermore, inhibition of such changes by inclusion of a PLA2 antagonist also results in inhibition of exocytosis [16]. Activation of PLA2 requires millimolar levels of Ca2+ [16], and this activation is stimulated by DAG and other diglycerides [17]. Treatment with progesterone, a natural agonist of acrosomal exocytosis, leads to lipid changes indicative of PLA2 activation [18, 19], but to our knowledge, no studies have yet addressed whether PLA2 is activated after stimulation with ZP.
The present study was therefore undertaken to examine if stimulation of spermatozoa with ZP to undergo acrosomal exocytosis also results in activation of PLA2 and, if so, how PLA2 is modulated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[Methyl-14C]choline chloride (55 mCi/mmol) and [1-14C]arachidonic acid (56 mCi/mmol; toluene solution) were obtained from Amersham Pharmacia Biotech U.K. Ltd. (Little Chalfont, Buckinghamshire). Chemicals (reagent grade) and reagents were purchased from Sigma or Shanghai Chemical Reagents Co. (both of Shanghai, People's Republic of China). Percoll was from Pharmacia LKB (Uppsala, Sweden). The La(NO3)3·6H20 was from E. Merck (Darmstadt, Germany). Organic solvents were of reagent grade and were purchased from Shanghai Chemical Reagents. Arachidonic acid (AA), phospholipids, and neutral lipids used as standards were from Sigma.
Medium
The medium used throughout this study was a low-calcium minimal capacitation medium (LCa-MCM), which consisted of 111.76 mM NaCl, 2.7 mM KCl, 0.49 mM MgCl2, 25.07 mM NaHCO3, 5.56 mM glucose, 10 mM sodium lactate, 1.0 mM sodium pyruvate, 50 µg/ml of kanamycin monosulfate, 20 mM Hepes, and 4 mg/ml of bovine serum albumin (fraction V). The LCa-MCM was at pH 7.9 and had a final osmolality of 300–305 mOsm/kg. No Ca2+ was added to this medium, although when measured, the Ca2+ concentration was 23 µM. This low-Ca2+ medium induces capacitation of guinea pig spermatozoa under in vitro conditions but does not support the acrosome reaction [20, 21]. When required, 2 mM CaCl2 was added.
Collection and Preparation of Spermatozoa
White and Black retired male guinea pigs (weight, 750 ± 30 g) were purchased from the Center for Experimental Animals, Zhejiang University, and were housed in environmentally controlled rooms with a 12L:12D photoperiod and maintained at approximately 20°C. Food and water were provided ad libitum. Animals were killed with CO2. The caudae epididymides and vasa deferentia were incised, and their contents were milked into LCa-MCM medium. Spermatozoa (final concentration, 5 x 107 cells/ml) were incubated for 1 h in a capped jar in a shaking water bath at 38.5°C under air. Sperm viability at this stage was 90–95%, as estimated using a trypan blue exclusion test and phase-contrast microscopy.
Capacitation and Labeling of Spermatozoa
Spermatozoa were labeled with 2 µCi/ml of [methyl-14C]choline chloride or 0.5 µCi/ml of [14C]arachidonic acid by incubating them for 5 h at 38.5°C under air. During this labeling period, the viability of spermatozoa remained constant (85–90%), as estimated using the trypan blue exclusion test and phase-contrast microscopy. Spermatozoa were washed through a Percoll gradient (30%, 55%, and 85% Percoll in LCa-MCM medium) by centrifugation for 18 min at 690 x g. After centrifugation, the supernatant was removed, leaving in each tube approximately 0.3 ml of the infranatant (85% Percoll), in which the spermatozoa were loosely pelleted. The pellet was diluted 1:10 with LCa-MCM and centrifuged again at 440 x g for 8 min. After centrifugation, the supernatant was removed, and the spermatozoa were diluted in the medium to be tested (final concentration, 3–5 x 107 cells/ml). At this stage, 
85% viable cells were found.
Isolation and Preparation of ZP
Female guinea pigs (age, 21–22 days) of the White with Flower Spots strain were killed with CO2, and the ovaries were removed. The ZP were isolated as described previously [22] with some modifications. Briefly, ovaries of 20 guinea pigs were homogenized in 2 ml of ice-cold buffer (120 mM NaCl, 10 mM NaH2PO4, 10 mM trisodium citrate [dihydrate], 1 mM p-aminobenzamidine, 1 mM EGTA, and 0.2% [w/v] polyvinyl alcohol [PVA]). The ovaries were placed in a glass homogenizer and subjected to 20 strokes of the pestle. The homogenate (
2 ml) was gently loaded on the top of a two-step gradient of 1.5 ml of 10% and 20% Percoll/buffer with 0.2% (w/v) PVA in seal-cap plastic tubes. Percoll solutions were prepared using the buffer mentioned above. The tubes were centrifuged for 18 min at 4°C and 6000 x g. The ZP were isolated by examining samples of various layers under a phase-contrast microscope. The band containing the majority of ZP was located in the interphase between the homogenate and the 10% Percoll/buffer. The typical yield of this isolation protocol was 250–300 ZP/ovary. The ZP were stored in the buffer with 0.2% (w/v) PVA at -20°C. On the day of the experiment, they were thawed, and the concentration was adjusted to 20 ZP/µl. They were then solubilized by incubation at 60°C for 1 h. The preparation was centrifuged at 13 000 x g for 8 min at 4°C to remove particulate debris, and the supernatant was used for experiments.
Experimental Design
Stock solutions of PTX (250 µg/ml), aristolochic acid (ATA; sodium salt, 10 mM), La3+ (15 mM), and EGTA (200 mM) were made up weekly in tridistilled water and were kept at -20°C. Stocks of the DAG kinase inhibitor R59022 (200 µM) were prepared in dimethyl sulfoxide (DMSO), kept at -20°C, and used within 1 mo. For use, further dilutions were made in DMSO. Final concentrations of DMSO were 1% or lower, and they did not affect motility or acrosomal integrity.
For experiments, samples (0.5 ml each) of capacitated sperm suspensions (final concentration, 3–5 x 107 cells/ml) in LCa-MCM medium with or without 2 mM Ca2+ were incubated with test reagents (or their solvents as controls; see Results) at 38.5°C under air for 5–15 min. Spermatozoa were then treated with ZP (1–5 ZP/µl) and incubated for another 15 min under similar conditions, and then their lipids were extracted and analyzed. To test effects on the acrosome reaction, unlabelled spermatozoa were first exposed to reagents (or their solvents as controls) and then treated with ZP (or left untreated as controls) and incubated further. Subsamples were then taken to assess the occurrence of exocytosis using phase-contrast microscopy.
Lipid Analyses in Labeled Spermatozoa
For quantification of arachidonic acid, incubations of spermatozoa prelabeled with [14C]arachidonic acid were terminated by the addition of chilled chloroform:methanol (1:2, v/v), and lipids were extracted and analyzed as described previously [19] with some modifications. After the incubations were stopped (and the lipids extracted), phospholipids were separated by thin-layer chromatography (TLC) on silica-gel 60 F254 precoated glass plates (20 x 20 cm, 0.25-mm thickness; E. Merck), pretreated by spraying with 1% (w/v) potassium oxalate, activated by heating at 110°C for 60 min, and developed twice in the solvent n-hexane:diethyl-ether:acetic acid (70:30:10, v/v/v). The plates were air-dried well between the first and the second run. Lipid spots were identified by comparison with authentic standards (arachidonic acid, mono-oleoylglycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoylglycerol) run in the same plate by staining with iodine vapors or detected by autoradiography using Fuji RX film (Tokyo, Japan) and exposing it for 4 wk at room temperature (25°C). The individual spots were scraped off, and the radioactivity in each was determined by liquid scintillation counting (LKB 1209 counter; Denmark Wallac Inc., Turku, Finland).
To measure changes in lysoPC and PC, incubations of spermatozoa prelabeled with [methyl-14C]choline chloride were terminated and lipids extracted as described above. Phospholipids were separated using a one-dimensional TLC system. Plates were pretreated by spraying with 1% (w/v) potassium oxalate, activated by heating at 110°C for 60 min, and developed first in the solvent chloroform:methanol:water:concentrated ammonia (28:10:0.5:1.5, v/v/v/v) and then developed in the solvent chloroform:methanol:water:acetic acid (32:4:1:0.4, v/v/v/v). Lipid spots were revealed by iodine staining and identified by comparison with authentic standards of PC and lysoPC run in the same plate. Spots were scraped off, and the radioactivity in each was determined by liquid scintillation counting.
Statistical Analyses
Results are the mean ± SEM. For statistical analyses, data were transformed [log10 for lipid levels and arcsine 
(% of acrosome-reacted cells ÷ 100) for exocytosis]. Multiple group comparison was performed by one-way ANOVA, followed by Bonferroni multiple-comparison corrections. Values of P < 0.05 were regarded as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation of guinea pig spermatozoa in Ca2+-deficient medium (LCa-MCM) for 6 h and subsequent exposure to millimolar Ca2+ (2 mM) resulted in low numbers (15%) of spermatozoa exhibiting acrosome reactions. Treatment of similarly preincubated spermatozoa with increasing concentrations of ZP (0.5–5 ZP/µl) in the presence of 2 mM Ca2+ led to a concentration-dependent stimulation of acrosomal exocytosis. Stimulation of exocytosis was maximal (60% acrosome reactions) when sperm cells were stimulated with 4 ZP/µl (Fig. 1). Treatment of spermatozoa with up to 5 ZP/µl did not affect sperm motility or viability.
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Lipid Changes Following Stimulation with ZP
Incubation of guinea pig spermatozoa with either [14C]arachidonic acid or [14C]choline chloride resulted in good and consistent labeling of sperm lipids. When spermatozoa were prelabeled during capacitation with either one or the other radioactive precursor, washed to remove excess radioactive precursor, stimulated with ZP, and lipids examined at various times after stimulation, the changes in AA or lysoPC, respectively, could be followed. In parallel, unlabelled capacitated spermatozoa were stimulated with ZP for similar periods of time and then examined for the occurrence of exocytosis.
Stimulation of capacitated spermatozoa with 1 ZP/µl led to a time-dependent release of arachidonic acid, which was maximal 15–20 min after stimulation; thereafter, levels of arachidonic acid decreased (Fig. 2A). Similarly stimulated spermatozoa revealed a time-dependent rise in lysoPC levels that peaked at 15 min and decreased thereafter (Fig. 2B). Acrosomal exocytosis followed a similar pattern, except that levels remained high after 15 min. Therefore, good agreement exists between the time-courses of arachidonic acid and lysoPC releases and that of acrosomal exocytosis (Fig. 2, A and B).
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Ca2+ Requirement for PLA2 Activation and Subsequent Exocytosis
To assess if arachidonic acid and lysoPC releases require the presence of extracellular Ca2+ or Ca2+ internalization, capacitated spermatozoa labeled with radioactive precursors were exposed to media with or without added Ca2+ or were stimulated with ZP in Ca2+-containing medium with or without EGTA (a Ca2+ chelator) or La3+ (a Ca2+ channel inhibitor).
When spermatozoa were precapacitated in LCa-MCM, washed, and resuspended in medium with 2 mM Ca2+, a rise was observed in both arachidonic acid (Fig. 3A) and lysoPC (Fig. 3B), and a decrease was observed in PC (Fig. 3C), compared to the levels of these lipids as seen when spermatozoa were resuspended in medium without added Ca2+. The percentage of cells without acrosomes increased 15 min after resuspension in medium with 2 mM Ca2+ in comparison to levels seen after resuspension in medium without added Ca2+ (Fig. 3D).
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If spermatozoa were stimulated with ZP in the presence of Ca2+, an increase in arachidonic acid (Fig. 3A) and lysoPC (Fig. 3B) levels and a decrease in PC level (Fig. 3C) followed. However, if spermatozoa were treated with ZP after chelation of extracellular Ca2+ by the addition of EGTA, levels of these three lipids were not different from those seen in control spermatozoa (2 mM Ca2+, no ZP treatment). Acrosomal exocytosis did not take place in spermatozoa treated with ZP in the presence of EGTA (Fig. 3D). Levels of free Ca2+ in medium with 2 mM Ca2+ and 2 mM EGTA were 320 µM.
When the Ca2+ channel inhibitor La3+ was used instead of EGTA, the results agreed. Inclusion of La3+ when spermatozoa were treated with ZP led to no increase in lysoPC, no decrease in PC, and no acrosome reactions (Fig. 4).
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Taken together, these results indicate that Ca2+ internalization in precapacitated spermatozoa, on resuspension in medium containing high Ca2+ and after ZP treatment, results in PLA2 activation and exocytosis.
Signaling Mechanisms Modulating PLA2 Activation and Exocytosis
We tested if ZP-induced releases of arachidonic acid and lysoPC (indicative of PLA2 activation) are modulated by G proteins and whether DAG plays a role in the sequence leading to PLA2 activation. To this end, capacitated spermatozoa were stimulated with ZP in the absence or presence of PTX, to test for G protein involvement, or the compound R59022, a DAG kinase inhibitor. We predicted that if G proteins (of the Gi class) were involved, inclusion of PTX when spermatozoa were stimulated with ZP should inhibit responses related to PLA2 activation and exocytosis. On the other hand, we hypothesized that if DAG was an important metabolite in events leading to PLA2 activation, inclusion of a DAG kinase inhibitor (which would result in a rise in DAG levels) together with ZP stimulation should lead to an increase in arachidonic acid/lysoPC release and exocytosis.
In the first series of experiments, capacitated guinea pig spermatozoa were incubated with various concentrations of PTX for 5 min and then stimulated with 1 ZP/µl for 15 min. Examination of spermatozoa for occurrence of the acrosome reaction revealed that PTX caused a concentration-dependent inhibition of ZP-stimulated exocytosis, with maximal effects seen with 1.5–2.0 µg/ml of PTX (Fig. 5). These concentrations of PTX did not affect sperm motility or integrity. As a negative control, we also tested the effect of PTX B oligomer (an enzymatically inactive component of PTX) [23]. The PTX B oligomer was resuspended in the same solvent used for PTX and added to the sperm suspensions 5 min before addition of ZP (as done for PTX). Inclusion of PTX B oligomer did not inhibit ZP-induced acrosomal exocytosis. The following results were obtained (n = 3; mean acrosome reactions ± SEM): control (2 mM Ca2+), 15 ± 1%; 1 ZP/µl plus Ca2+, 35 ± 1%; 1.5 µg/ml of PTX for 5 min and then 1 ZP/µl plus Ca2+, 10 ± 1%; and 1.5 µg/ml of PTX B oligomer for 5 min and then 1 ZP/µl plus Ca2+, 35 ± 1%.
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In another series of experiments, spermatozoa were capacitated and labeled with lipid precursors in low-Ca2+ medium, washed, resuspended in Ca2+-containing medium, incubated for 5 min with or without PTX (1.6 µg/ml), treated with ZP (1 ZP/µl) for 15 min, and then had their lipids extracted and analyzed. As in experiments described above, stimulation with ZP led to a rise in arachidonic acid (Fig. 6A) and lysoPC levels (Fig. 6B) and a decrease in PC level (Fig. 6C). In parallel, unlabeled samples, this treatment led to acrosomal exocytosis (Fig. 6D). Inclusion of PTX before stimulation with ZP abrogated all these responses (Fig. 6).
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To examine the effects of the DAG kinase inhibitor R59022, guinea pig spermatozoa were capacitated and labeled with a lipid precursor in a low-Ca2+ medium, washed, resuspended in Ca2+-containing medium, incubated for 5 min without or with 2 µM R59022 (a concentration known to inhibit DAG kinase in various cell systems and to result in a rise in DAG levels [24]), and then stimulated with ZP (1 ZP/µl). After 15 min of further incubation, sperm lipids were extracted and analyzed. As can be seen in Figure 7A, spermatozoa pre-exposed to the compound R59022 and then treated with ZP experienced a higher increase in lysoPC levels than those not pre-exposed to R59022. In spermatozoa preincubated with R59022 in the presence of Ca2+ but not stimulated with ZP, levels of lysoPC were higher than those seen in controls (spermatozoa exposed only to Ca2+) and not different from those found after ZP stimulation (Fig. 7A). In parallel experiments, spermatozoa were similarly treated but were examined after ZP stimulation for the occurrence of acrosomal exocytosis (Fig. 7B). Spermatozoa pre-exposed to the compound R59022 before ZP stimulation experienced much higher levels of exocytosis than spermatozoa not pre-exposed to this compound. Spermatozoa exposed solely to the DAG kinase inhibitor R59022, in the presence of Ca2+, experienced some acrosome reactions, with levels (20%) similar to those seen after stimulation with ZP (Fig. 7B). The latter, together with the increase in lysoPC after exposure to the DAG kinase inhibitor alone, suggests that under these conditions, a slight rise in endogenous DAG levels could lead to acrosome reactions in some spermatozoa.
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ATA Inhibits ZP-Induced PLA2 Activation and Exocytosis
To test further if ZP-stimulated acrosome reaction involved PLA2 activation, capacitated guinea pig spermatozoa were pre-exposed to ATA, an effective inhibitor of PLA2 in human neutrophils [25, 26] and ram spermatozoa [27, 28], before stimulation with ZP. Use of increasing concentrations of ATA inhibited the ZP-induced exocytosis in a concentration-dependent fashion (Fig. 8). This compound, when used alone, did not affect sperm motility or integrity.
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We sought confirmation that the effect of ATA was, indeed, on PLA2 activity. Thus, guinea pig spermatozoa were capacitated and labeled with lipid precursors in low-Ca2+ medium, washed, resuspended in Ca2+-containing medium, exposed to ATA for 5 min, and then stimulated with ZP. After 15 min of further incubation, lipids were extracted and analyzed. Results are shown in Figure 9. When spermatozoa were exposed to ATA before ZP treatment, levels of arachidonic acid were significantly lower than those seen after treatment with ZP (i.e., no ATA pre-exposure) and resembled the levels seen in control samples (Fig. 9A). Similar results were obtained when lysoPC was quantified: exposure to ATA before ZP treatment led to an inhibition of the ZP-induced rise in lysoPC levels (Fig. 9B). In agreement with these results, levels of PC, which decreased after ZP treatment, remained high (and similar to those seen in controls) when spermatozoa were pretreated with ATA (Fig. 9C). In parallel experiments, spermatozoa were exposed to ATA (or just to its solvent as controls), stimulated with ZP, and then examined for occurrence of the acrosome reaction. It was found that spermatozoa pre-exposed to ATA did not experience acrosomal exocytosis in response to ZP stimulation (Fig. 9D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of precapacitated guinea pig spermatozoa with solubilized ZP resulted in a high proportion of cells undergoing exocytosis; the effect was concentration dependent, with maximal response at 4 ZP/µl. This result is in agreement with earlier findings demonstrating that the ZP stimulates the acrosome reaction in spermatozoa of various species [29, 30], including the guinea pig [31].
In spermatozoa precapacitated in low-Ca2+ medium and simultaneously incubated with radioactive precursors ([14C]choline chloride and [14C]arachidonic acid) to label sperm phospholipids, stimulation with ZP in the presence of millimolar Ca2+ led to a time-dependent increase in free arachidonic acid and formation of lysoPC, and this was accompanied by a decrease in PC levels. These lipid changes are indicative of PLA2 activity. Previous work has shown that stimulation of ram [15] and boar [32] spermatozoa with the Ca2+ ionophore A23187 results in a time-dependent release of free arachidonic acid, formation of lysoPC, and hydrolysis of phospholipids, with PC being the main source of arachidonic acid. Similarly, stimulation of human sperm with A23187 results in release of free arachidonic acid [18]. Furthermore, stimulation of capacitated spermatozoa with progesterone has revealed lipid changes indicative of PLA2 activation in humans [18] and boars [19]. The present work shows, to our knowledge for the first time, evidence indicating that ZP also triggers lipid changes related to PLA2 activation. Thus, stimulation of spermatozoa from various mammalian species with molecular probes (e.g., Ca2+ ionophore) or natural agonists of exocytosis (e.g., progesterone, ZP) leads to activation of PLA2 and generation of arachidonic acid (and, possibly, other free fatty acids) and lysoPC (and, perhaps, other lysolipids).
Evidence for the participation of PLA2 in events underlying the mammalian sperm acrosome reaction has been accumulating for years. However, such evidence has been mainly circumstantial (for reviews, see [17, 33]), because none of these studies has actually quantified PLA2 activity (e.g., by labeling cells and measuring fatty acid release on stimulation) and, therefore, has not provided direct evidence of PLA2 participation in exocytosis. The participation of PLA2 in events leading to membrane fusion during acrosomal exocytosis has been recently explored using other strategies. Various PLA2 inhibitors shown to inhibit sperm PLA2 during in vitro assays [34–37] were used to demonstrate inhibition of exocytosis triggered by molecular probes such as the Ca2+ ionophore A23187 [15]. Inhibition was reversed by inclusion of PLA2-generated metabolites [15]. Quantification of metabolites generated by PLA2 in labeled sperm cells has confirmed that the effect of the inhibitor was, indeed, on PLA2, because no PLA2-derived metabolites were generated in the presence of PLA2 inhibitors after stimulation with A23187 [15]. The latter work has been carried out using molecular probes (e.g., the Ca2+ ionophore), and to our knowledge, no previous study has dealt with the relationship between PLA2 activation/inhibition and ensuing exocytosis on stimulation with a natural agonist (e.g., progesterone, ZP). The present results are, thus, the first to identify that PLA2 activation after ZP stimulation may be essential for naturally triggered exocytosis. We found that inhibition of PLA2 activity (as confirmed by lack of arachidonic acid release and lysoPC formation) in the presence of the PLA2 inhibitor ATA was accompanied by lack of exocytosis. This tight coupling between lipid changes and exocytosis induced by ZP was further confirmed by examining the Ca2+-dependency of lipid changes and exocytosis and by exploring the possible participation of G proteins and DAG in pathways leading to PLA2 activation (see below). All conditions or treatments resulting in release of arachidonic acid and lysoPC formation were accompanied by the occurrence of exocytosis, whereas all conditions/treatments that caused the inhibition of ZP-induced changes in these lipids were paralleled by lack of exocytosis.
The ZP-induced activation of PLA2 required the presence of extracellular Ca2+ and, presumably, internalization of Ca2+. Stimulation of capacitated spermatozoa with ZP induces Ca2+ influx [6, 38, 39], and PLA2 activation requires Ca2+ [17, 33]. Studies assaying PLA2 activity either in sperm extracts in vitro [16] or in labeled spermatozoa stimulated with A23187 [15] or progesterone [19] have revealed that the enzyme requires millimolar levels of the cation. In the present study, resuspension of guinea pig spermatozoa precapacitated in medium with micromolar Ca2+ in medium with 2 mM Ca2+ (but without stimulation with ZP) resulted in a slight increase in arachidonic acid and lysoPC levels, which suggests that even without ZP stimulation, an exposure to high Ca2+ levels leads to some internalization of the cation and a limited activation of PLA2. Stimulation with ZP in medium with millimolar Ca2+ resulted in considerable increases in arachidonic acid and lysoPC levels, indicating full activation of PLA2. That this effect is caused by Ca2+ internalization is suggested by the observations that inclusion of EGTA (to chelate the extracellular Ca2+) or La3+ (to block Ca2+ entry via channels) before stimulation with ZP prevented increases in AA and lysoPC.
Regulation of PLA2 may involve other membrane and intracellular signaling mechanisms, as seen in spermatozoa [17, 33] or somatic cells [40, 41]. In somatic cells, evidence has been gathered in favor of these other mechanisms of PLA2 regulation: 1) G protein-mediated PLA2 activation, 2) activation via phosphorylation by mitogen-activated protein (MAP) kinase, and 3) modulation of PLA2 activity by DAG. In some cells, DAG may stimulate protein kinase C (PKC); in turn, this would result in phosphorylation of MAP kinase and subsequent activation of PLA2. In other cells, however, DAG could stimulate PLA2 directly, without participation of the PKC pathway [42–45]. In spermatozoa, little is known about the mechanisms that regulate the activation of PLA2.
One possible mechanism regulating PLA2 in spermatozoa is that involving G proteins [17]. Clearly, Gi proteins transduce ZP-initiated signals during sperm acrosomal exocytosis [3, 46, 47]. Our results indicate that activation of a G protein (at least of the Gi class) is involved (directly or indirectly) in the pathway leading to PLA2 activation, because inclusion of PTX before ZP stimulation blocks arachidonic acid and lysoPC generation and exocytosis. Future studies should address the question of which pathway links G protein activation and PLA2. It is not clear whether G proteins are directly related to PLA2 activity or, more likely, whether G proteins activate a signaling pathway, such as Ca2+ influx [38, 48], cAMP formation [11], or phosphoinositidase C-mediated DAG generation [3], that would eventually result in PLA2 activation.
Another possible pathway modulating sperm PLA2 is the DAG-PKC-MAP kinase cascade [49]. The direct activation of PLA2 by PKC has been postulated [50], but to our knowledge, no evidence has been reported for this interaction in vivo [51, 52]. Alternatively, DAG might act directly on PLA2 [53, 54].
In the present study, we explored if DAG would modulate activation of PLA2 by manipulating the endogenous levels of DAG. Enhancement of endogenous DAG levels by inhibition of the DAG kinase (an enzyme that metabolizes DAG) has been demonstrated by use of the compound R59022 [24], and we have used this strategy in the present study. We anticipated that an increase in endogenous DAG (when sperm cells were stimulated in the presence of R59022) would result in higher PLA2 activity and exocytosis if DAG has a role in activation of PLA2. This was the case: Spermatozoa preincubated with the DAG kinase inhibitor R59022 and stimulated with ZP revealed higher levels of lysoPC and much higher levels of exocytosis than sperm cells not exposed to the DAG kinase inhibitor.
Our results, therefore, indicate that DAG activates PLA2, although we cannot at this stage discriminate whether such activation occurs directly or indirectly, via other downstream events. In the case of an indirect action, it is possible that DAG could act via PKC and MAP kinase, because current information indicates that these signaling elements are present in spermatozoa (for PKC, see [55]; for MAP kinase cascade, see [13, 14]).
In conclusion, our findings indicate that PLA2 activation is an important and essential event in acrosomal exocytosis stimulated by ZP. Activation of PLA2 results in the release of arachidonic acid and formation of lysoPC, two metabolites important in the events regulating membrane fusion. Activation of PLA2 by ZP requires internalization of Ca2+, and the enzyme's activity appears to be regulated by G protein- and DAG-modulated pathways.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Qi-xian Shi, Zhejiang Academy of Medical Sciences, 182 Tian Mu Shan Road, Hangzhou, Zhejiang 310013, People's Republic of China. FAX: 0571 8886 9045; e-mail: qxshi{at}mail.hz.zj.cn
Received: 31 March 2002.
First decision: 23 April 2002.
Accepted: 20 September 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 for the zona pellucida-induced acrosome reaction. Science 2001 292:920-923This article has been cited by other articles:
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  W. Y. Chen, W. M. Xu, Z. H. Chen, Y. Ni, Y. Y. Yuan, S. C. Zhou, W. W. Zhou, L. L. Tsang, Y. W. Chung, P. Hoglund, et al. Cl- Is Required for HCO3- Entry Necessary for Sperm Capacitation in Guinea Pig: Involvement of a Cl-/HCO3- Exchanger (SLC26A3) and CFTR Biol Reprod, January 1, 2009; 80(1): 115 - 123. [Abstract] [Full Text] [PDF]
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 A. A. Moura, H. Koc, D. A. Chapman, and G. J. Killian Identification of Proteins in the Accessory Sex Gland Fluid Associated With Fertility Indexes of Dairy Bulls: A Proteomic Approach J Androl, March 1, 2006; 27(2): 201 - 211. [Abstract] [Full Text] [PDF]
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A. Furimsky, N. Vuong, H. Xu, P. Kumarathasan, M. Xu, W. Weerachatyanukul, M. Bou Khalil, M. Kates, and N. Tanphaichitr Percoll Gradient-Centrifuged Capacitated Mouse Sperm Have Increased Fertilizing Ability and Higher Contents of Sulfogalactosylglycerolipid and Docosahexaenoic Acid-Containing Phosphatidylcholine Compared to Washed Capacitated Mouse Sperm Biol Reprod, March 1, 2005; 72(3): 574 - 583. [Abstract] [Full Text] [PDF]
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 S. Bao, D. J. Miller, Z. Ma, M. Wohltmann, G. Eng, S. Ramanadham, K. Moley, and J. Turk Male Mice That Do Not Express Group VIA Phospholipase A2 Produce Spermatozoa with Impaired Motility and Have Greatly Reduced Fertility J. Biol. Chem., September 10, 2004; 279(37): 38194 - 38200. [Abstract] [Full Text] [PDF]
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