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Fertilization-Induced Activation of Phospholipase C in the Sea Urchin Egg
Developmental Biology 215, 147–154 (1999) Article ID dbio.1999.9472, available online at http://www.idealibrary.com on
Fertilization-Induced Activation of Phospholipase C in the Sea Urchin Egg 1 Brenda J. Rongish, Wenjun Wu, and William H. Kinsey 2 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160
Fertilization results in the biphasic activation of polyphosphoinositide-specific phospholipase C (PLC) activity with an initial increase in activity coincident with the sperm-induced calcium transient, followed by a more sustained increase prior to mitosis. Immunoprecipitation studies demonstrated that the g isoform of PLC is present in both the unfertilized and the fertilized egg and contributes to the initial phase of PLC activation. Fertilization also resulted in translocation of a significant fraction of PLC-g from the cytosol to the membrane compartment of the egg. © 1999 Academic Press Key Words: fertilization; phospholipase C; calcium; sea urchin.
INTRODUCTION Fertilization results in the activation of a series of preprogrammed biochemical events which are required to prevent polyspermy, activate cell metabolism, and release the egg from cell cycle arrest. A key step is the transient release of calcium from internal stores, the sperm-induced “calcium transient,” which triggers the cortical reaction and activates all of the essential components of the egg activation pathway (Whitaker and Steinhardt, 1982). Two mechanisms of calcium mobilization have been identified in the sea urchin egg: the InsP 33 receptor (Parys et al., 1994) and the ryanodine receptor (McPherson et al., 1992). While both types of channels are expressed in eggs, the InsP 3 receptor has proven to be the dominant mechanism for initiation of the calcium release in most species of eggs (Lee and Shen, 1998; Miyazaki et al., 1992; Nuccitelli et al., 1993) and the ryanodine receptor probably plays a redundant role (Galione et al., 1993) or contributes to the ampli1
This investigation was funded by Grant NIH-HD 14846. To whom correspondence should be addressed at Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3902 Rainbow Boulevard, Kansas City, Kansas 66160. Fax: (913) 588-2710. E-mail: [email protected]. 3 Abbreviations used: EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N,N-tetraacetic acid; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; PtdIns, phosphatidylinositol; InsP 3, inositol 1,4,5-P 3; SBTI, soybean trypsin inhibitor; BSA, bovine serum albumin; P-Tyr, phospho-L-tyrosine; Pipes, piperazine-N,N-bis(2ethanesulfonic acid); PI-3 kinase, phosphatidylinositol 3-kinase. 2
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tude of the calcium transient once initiated by InsP 3 (Shearer et al., 1999). The mechanism by which InsP 3 levels are controlled in the fertilized egg remains an important question. Several studies in the sea urchin system have documented fertilization-induced changes in polyphosphoinositide metabolism at fertilization (Turner, et al., 1984; Kamel et al., 1985; Swann and Whitaker, 1990; Ciapa et al., 1992; Jones et al., 1998) and a recent study demonstrated the biphasic accumulation of InsP 3 in the fertilized sea urchin egg (Lee and Shen, 1998). Several approaches have been used to study the isoforms of phospholipase C (PLC) present in the egg and the mechanism by which they are regulated after fertilization. The isoforms of PLC that have been identified in the mammalian egg include b 1, b 3, g 1, and g 2 (Dupont et al., 1996; Mehlmann et al., 1998), while only PLC-g has been identified in the sea urchin egg to date (De Nadai et al., 1998). Functional studies using chemical inhibitors of PLC (Lee and Shen, 1998) as well as fusion proteins which act as competitive inhibitors of PLC-g (Carroll et al., 1997, 1999; Shearer,, et al., 1999) have demonstrated that PLC activity (specifically PLC-g activity) is required for the fertilizationinduced calcium transient in marine invertebrate eggs. Similar studies in mammalian eggs have provided conflicting evidence for a role of PLC-g (Mehlmann et al., 1998; Sette et al., 1998), in initiation of the initial calcium transient or for the later cyclic calcium transients resulting from fertilization. Interestingly, very thorough studies on the role of G protein-activated PLC-b (Williams et al., 1998) have indicated no clear role for this isozyme in the calcium
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transient of mammalian eggs. The mammalian egg may be able to use both forms of PLC or may make use of another isoform to generate InsP 3 at fertilization. Despite the clear role of PLC in regulating InsP 3 levels in the fertilized egg, the actual effect of fertilization on the activity of PLC isoforms has received only limited attention (De Nadai et al., 1998). The purpose of the present study was to quantitate the effect of fertilization on overall PLC activity and specifically on PLC-g activity in the sea urchin egg. The results demonstrate that even unfertilized eggs contain substantial polyphosphoinositide-specific PLC activity which can become active when [Ca 21] i reaches permissive levels. Fertilization induces an early phase of activation of PLC activity coincident with the calcium transient followed by a second, more prolonged activation that occurs well after the S phase of the cell cycle and continues through mitosis. The activity of PLC-g also increases coincident with the calcium transient, but does not increase during the later phase of PLC activation. Fertilization also resulted in the rapid translocation of PLC-g activity from the cytosol to the membrane compartment of the egg, suggesting that PLC-g is under complex regulatory control at fertilization.
MATERIALS AND METHODS Buffers. PKME consisted of 425 mM KCl, 50 mM Pipes, 10 mM MgCl 2, 10 mM EGTA, 10 mM Na 3VO 4, 0.01 mg/ml SBTI (Sigma, St. Louis, MO), and 10 mg/ml aprotinin (Sigma), pH 6.8. PLC buffer consisted of 50 mM NaH 2PO 4,100 mM KCl, 1 mM EGTA, pH 6.8. Solubilization buffer was PLC buffer with 1% n-octyl b-Dglucopyranoside, 100 mM Na 3VO 4, pH 6.8. Eggs and embryos. Eggs were collected from the sea urchin Lytechinus variegatus and dejellied by passage through Nytex (200 mm) cloth, washed, and suspended in artificial sea water (Marine Enterprises Int., Baltimore, MD). Cytosolic or detergent extracts were prepared from egg suspensions (2% v/v) that had been fertilized by addition of “dry” sperm and incubated at 20°C for various periods of time. The egg suspension was diluted by addition of 4 vol of ice-cold PKME and quickly pelleted in a hand centrifuge. The eggs were then resuspended in 1ml of PLC buffer (cytosol preparation) or PLC solubilization buffer (detergent extracts) and homogenized in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 100,000g for 5 min in a SW 50.1 rotor (Beckman Instruments) and the soluble material was assayed for enzyme activity immediately. Immunoprecipitation. Immunoprecipitation of PLC-g was done by incubating the detergent-solubilized egg extract (30 mg protein) with 0.1 mg of a mixed monoclonal antibody preparation specific for PLC-g 1 (Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C. The immune complexes were collected by adsorption to 5 ml of protein A–Sepharose (Pharmacia, Piscataway, NJ), which had been blocked with casein (Sigma), and then washed twice with PLC solubilization buffer and once with PLC buffer. PLC assay. PLC activity was measured in 25 ml of PLC buffer containing 4 nmol of a mixture of PtdIns 4,5-P 2 (Sigma) and [ 3H]PtdIns 4,5-P 2 (Amersham) (2.5 mCi/mmol) in a mixed micelle formed by 0.25% octylglucoside 1 0.1% deoxycholate (Wahl et al., 1992). CaCl 2 was added to a final concentration of 850 mM to give
FIG. 1. Calcium dependence of PLC activity in the sea urchin egg. PLC activity of cytosolic extracts prepared from unfertilized eggs was measured under calcium–EGTA buffering conditions designed to provide defined levels of free calcium. Buffer components were KCl (100 mM), NaH 2PO 4 (50 mM), EGTA (1.36 mM), Mg 21 (340 mM), Ca 21 (42 mM–1.042 mM). Data represent the average of two experiments, each performed in duplicate 6 SE.
a free calcium concentration of 1.8mM. Free calcium levels were calculated using the WINMAXC program (C. Patton, Stanford University). The reaction was carried out at 25°C for 60 min and then stopped by addition of 1 mg BSA followed by 200 ml of 10% TCA. The TCA-soluble material was then counted in a scintillation counter.
RESULTS Quantitation of total PLC activity in the egg. PtdIns 4,5-P 2-specific PLC activity was measured using the substrate Ptd[ 3H]Ins 4,5-P 2 presented in a mixed micelle containing ocytl-b-D-glucopyranoside:deoxycholate (4.5:1). The reaction at 25°C was linear for up to 3 hours. HPLC analysis of the TCA-soluble products of this reaction confirmed that [ 3H]inositol 1,4,5-P 3 was the predominant reaction product and that lysophospholipids resulting from phospholipase A activity were not a significant contaminant of this reaction (not shown). As in other systems (Wahl et al., 1992), the PLC activity in the egg was absolutely dependent on the concentration of free Ca 21 with very little activity detected with less than 2.5 3 10 27 M free Ca 21 and half-maximal activity requiring 1 mM free Ca 21 (Fig. 1). Similar calcium dependence was seen in extracts prepared from unfertilized
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FIG. 2. Effect of fertilization on total PLC activity in the egg. PLC activity was measured in detergent extracts prepared before and at different times after fertilization. The timing of the cortical reaction (30 s postinsemination) and of mitosis (90 –105 min postinsemination) was monitored in parallel samples incubated under identical conditions. Values represent the average of three experiments, each involving duplicate samples, 6 SE.
eggs and zygotes collected during the first few minutes after fertilization. All subsequent assays were done at 1.8 mM free Ca 21. The data presented in Fig. 1 also demonstrate that the unfertilized egg contains abundant PLC activity which could become active if [Ca 21] i were elevated to 1 mM or above. Since the level of [Ca 21] i in the unfertilized egg is reported to be in the range of 100 –150 nM (reviewed in Shen, 1995), it is likely that the population of PLC enzymes would be essentially inactive in the unfertilized egg. To determine whether fertilization had any effect on overall PLC activity, enzyme assays were performed on detergent extracts prepared at different times after fertilization. The concentration of sperm used in these experiments was sufficient to achieve partial elevation of the fertilization envelope in at least 80% of the eggs within 30 s after addition of the sperm. The Ptd[ 3H]Ins 4,5-P 2-specific PLC activity in the unfertilized L. variegatus egg was between 45 and 60 pmol/min/mg. Analysis of L. variegatus sperm revealed a level of PLC activity of 1400 pmol/min/mg which is nearly identical to that reported for human sperm (Ribbes, 1987) and is substantially higher than that of the egg. In the present experiments, the excess sperm were removed by washing with PKME before homogenization. Therefore, about 6 ng of sperm protein would remain in the egg sample and could contribute 8.4 fmol/min of activity or about 1.3% of the activity in the eggs. The effect of fertilization on PLC activity is seen in Fig. 2. Fertilization resulted in a biphasic increase in PLC activity with an initial phase occurring during the first 2 min and a later, more prolonged phase continuing through the first mitotic division. The initial increase in PLC activity was
detected as early as 30 s postinsemination and increased by approximately twofold before returning to baseline levels. This initial increase in PLC activity corresponds temporally with the propagation of the calcium transient in the egg (Mohri and Chambers, 1995) and with the reported phase 1 accumulation of InsP 3 (Lee and Shen, 1998). As the zygote assembled the mitotic spindle (60 –75 min) and began cytokinesis (90 –105min) the PLC activity increased to a level approximately fourfold higher than in the unfertilized egg. This elevated level of PLC activity was maintained at the two-cell stage (105 min). In summary, while PLC activity is ultimately dependent on the concentration of free calcium, the activation state of PLC does change in a biphasic manner with an early activation event occurring between 0.5 and 2 min postinsemination, followed by a later increase in activity at 60 to 75 min that persisted through the first cleavage. Identification of phospholipase C-g in the sea urchin egg. As a first step in identification of the isoforms of PLC that participate in the response to fertilization, an antibody to human PLC-g was used to detect this protein in the sea urchin egg. Combined immunoprecipitation and Western blot analysis revealed the presence of a 143-kDa protein in detergent extracts of sea urchin eggs (Fig. 3). This mixed monoclonal anti-human-PLC-g antibody preparation (Upstate Biotechnology Inc.) was first used in the sea urchin system by De Nadai et al. (1998) and is the only antibody of several that we have tried that will crossreact with the sea urchin protein. The anti-PLC-g immunoprecipitates were found to be highly enriched in phospholipase C activity, with 2300 pmol/min of activity typically immunoprecipitated from 1 mg of unfertilized egg protein. This specific
FIG. 3. Detection of PLC-g in the sea urchin egg. Detergent extracts of unfertilized eggs (50 mg protein) were incubated with 0.1 mg of either the mixed monoclonal anti-PLC-g antibody (A) or two unrelated monoclonal antibodies (B, C) at a 1:500 dilution. The immune complexes were adsorbed to protein A–Sepharose which had been preblocked with purified casein (1 mg/ml) and resolved on a 10% SDS–polyacrylamide gel. The gel was transferred to a nylon membrane, blocked, and incubated with the mixed anti-PLC-g monoclonal antibody at a dilution of 1:1000. Secondary antibody incubation was with rabbit anti-mouse IgG–peroxidase, and the bound peroxidase was detected by chemiluminescence (ECL, Amersham).
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FIG. 4. Calcium dependence of PLC-g immunoprecipitated from sea urchin eggs. PLC-g immunoprecipitated from detergent extracts prepared before and 1 min after fertilization were assayed for PLC activity under calcium–EGTA buffering conditions designed to provide defined levels of free calcium. Control immunoprecipitates (anti-Grb2 antibody) were assayed simultaneously and the PLC-g-specific activity was calculated by subtracting the activity in control samples from that of the PLC-g immunoprecipitates. Values represent the average of two experiments, each performed in duplicate 6 SE.
activity is substantially higher than the 50 pmol/min/mg measured for total PLC activity in egg extracts (Fig. 2) suggesting that inhibitors of PLC-g may be present in the egg cytosol or that endogenous lipids interfere with the measurement of PLC activity in crude detergent extracts (see Discussion). The observed specific activity for PLC-g of 2300 pmol/min/mg is similar to the value of 5500 pmol/ min/mg reported for the mammalian enzyme (Wahl et al., 1992). Calcium dependence of PLC-g. The effect of calcium on the PLC-g activity immunoprecipitated from unfertilized eggs and zygotes extracted at 1 min postfertilization is presented in Fig. 4. In both unfertilized and fertilized egg samples, half-maximal activity was detected at 750 nm free calcium. This value is somewhat higher than that reported for PLC-g immunoprecipitated from EGF-stimulated human A-431 cells where half-maximal activity occurred at 316 nM free calcium (Wahl et al., 1992) and may reflect species differences. Further attempts to detect possible changes in calcium dependence following fertilization were carried out by using lower substrate/detergent ratios and by replacing deoxycholate with various amounts of Triton X-100 in the assay. The presence of Triton X-100 in the
substrate micelle has been demonstrated to enhance detection of changes in calcium dependence of PLC-g in growth factor-stimulated cells (Nishibe et al., 1990); however, we were not able to detect fertilization-dependent changes in calcium dependence in immunoprecipitates from unfertilized and fertilized eggs. Effect of fertilization on PLC-g activity. The ability to detect PLC activity in the PLC-g immunoprecipitates made it possible for us to quantitate the effect of fertilization on PLC-g activity as a subset of total PLC activity. As seen in Fig. 5, PLC-g activity increased slightly within 30 to 45 s after fertilization. This change was found to be statistically significant (P 5 0.05), although the maximal level of PLC-g activity was not reached until 60 –90 s postinsemination. Activity declined slightly after 2 min, but remained higher than that immunoprecipitated from unfertilized eggs until 60 min, at which time PLC activity returned to the unfertilized level. It is interesting to note that PLC-g activity was not elevated between 60 and 110 min postfertilization as was observed for the total PLC activity of the zygote. One possible explanation might be association of PLC-g with the detergent-insoluble cytoskeleton; however, Western blot analysis of duplicate immunoprecipitates did not reveal any significant changes in the immunoprecipitation recovery of PLC-g which might cause artifactual changes in PLC-g activity during this period of development. We interpret the above results, therefore, to indicate that the g isoform of PLC participates in the fertilization-induced activation of PLC during the first 60 s postinsemination. This enzyme is also active during the late phase of PLC activation but does not exhibit increased activity in this type of immunoprecipitation assay.
FIG. 5. Effect of fertilization on PLC-g activity. PLC-g-specific activity was measured after immunoprecipitation from detergent extracts prepared from unfertilized eggs and from zygotes at different times after fertilization. Values represent the activity in the anti-PLC-g immunoprecipitates less (2) the activity in control immunoprecipitates and are the average of three experiments 6 SE.
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FIG. 6. Subcellular distribution of PLC-g activity. Samples (1 ml) of eggs were homogenized in 5 ml of PKME buffer before (E) and 1 min after (F) fertilization. The homogenates were then centrifuged through sucrose gradients consisting of sucrose in PKME (60% (4 ml), 45% (5 ml), 45–20% linear gradient (22 ml)) overnight in an SW28 rotor (Beckman) at 25,000 rpm and fractions were collected. Aliquots (50 ml) of each fraction were solubilized in 0.5 ml of PLC solubilization buffer and PLC-g-specific activity was immunoprecipitated and assayed as described under Materials and Methods. The top of the gradient is at fraction 1.
Subcellular localization. In order to determine whether the subcellular distribution of PLC-g changed in response to fertilization, we used a sucrose density gradient method (Ribot et al., 1983) to fractionate unfertilized and fertilized eggs and then measured PLC-g activity immunoprecipitated from the different fractions (Fig. 6). PLC-g activity from unfertilized eggs remained, for the most part, at the top of the gradient where the cytosolic proteins and pigment-rich yolk fractions are distributed. However, preparations from zygotes made 60 s postinsemination exhibited a more complex distribution with over one-half of the PLC activity associated with membrane bands, including a band eluting at a buoyant density of 1.23 g/ml (fraction 25) which we have previously shown to be highly enriched in plasma membrane (Ribot et al., 1983). The results of this experiment demonstrate that fertilization involves rapid and significant changes in the distribution of the PLC-g enzyme, with the result that a large fraction of the activity becomes membrane associated. Phosphorylation of PLC-g. Tyrosine phosphorylation of PLC-g is a well-established control mechanism utilized in a variety of cell types and a growing body of evidence suggests that PLC-g interacts with PTKs in the egg (see Discussion). In an attempt to detect possible changes in the P-Tyr content of PLC-g at fertilization, we performed Western blot analysis of PLC-g immunoprecipitates prepared from unfertilized eggs and early zygotes using a monoclonal antibody to P-Tyr (Fig. 7). The PLC-g band shown in the top panel is recognized by the anti-P-Tyr antibody, and antibody binding is significantly reduced but not completely
eliminated by the presence of 5 mM P-Tyr (control). The monoclonal P-Tyr antibody used in this experiment (Zymed Laboratories Inc., San Francisco, CA) has a very high affinity for P-Tyr and competition with free P-Tyr is only partially successful in our experience. The relative amount of P-Tyr detected in PLC-g did not change significantly after fertilization (Fig. 7, lanes B–D). Similar results were obtained in over seven experiments using the Zymed antibody as well as the 4G10 anti-P-Tyr monoclonal from Upstate Biotechnology Inc. Additional efforts to control degradation by phosphatases included the use of sodium pyrophosphate, sodium fluoride, phenylarsine oxide, and peroxyvanadate, with no change in results. The data show that PLC-g is phosphorylated on tyrosine in the unfertilized egg and that any changes in phosphorylation state which might occur at fertilization probably involve a small subset of the total PLC-g pool.
DISCUSSION Localized release of calcium from internal stores is required for several aspects of the egg activation process, including the slow block to polyspermy, activation of egg metabolism (Epel, 1990), and reentry into the cell cycle (Ciapa et al., 1994; Becchetti and Whitaker, 1997). Studies in both vertebrate and invertebrate systems have demonstrated the importance of polyphosphoinositide hydrolysis in the regulation of free calcium in response to fertilization (Oberdorf et al., 1989; Miyazaki et al., 1992). Three different isoforms of PLC have been detected in mammalian eggs (Dupont et al., 1996; Mehlmann et al., 1996), and while the limited crossreactivity of PLC antibodies among different species has not allowed similar analysis in invertebrate eggs, it is likely that several PLC isoforms are present in all species. Given this array of PLC enzymes, the details of the
FIG. 7. Detection of phosphotyrosine in PLC-g immunoprecipitated from sea urchin eggs. L. variegatus eggs collected before (A) and at 15 s (B), 30 s (C), and 60 s (D) after fertilization were solubilized in PLC solubilization buffer containing 100 mM Na 3VO 4 and 10 mM phenylarsine oxide as phosphatase inhibitors. PLC-g was immunoprecipitated and blotted to a nylon membrane and blocked in 1% BSA as described under Materials and Methods and then P-Tyr was detected using a monoclonal anti-P-Tyr antibody (Zymed Laboratories, Inc., San Francisco, CA) at a 1:1000 dilution, followed by goat anti-mouse IgG–peroxidase and chemiluminescent detection. Control blots were incubated with the anti-P-Tyr antibody 1 5mM phosphotyrosine as a competitive inhibitor (Con).
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calcium regulation mechanisms at each stage of egg activation could be quite complex. The objective of the present study was to begin biochemical characterization of the PLC activities in the sea urchin egg with special emphasis on the PLC-g isoform. The total PLC activity of the sea urchin egg as measured with [H 3]PtdIns 4,5-P 2 exhibits a near absolute dependence on Ca 21 as reported in many other systems (Rhee and Bae, 1996). This activity probably represents the sum of several PLC isoforms, but it is clear that prior to fertilization, very little PtdIns-P 2 hydrolysis can occur at the low resting calcium levels which are typically reported to be between 100 and 150 nM (Shen, 1995). Measurement of total PLC activity at different points during the egg activation process revealed that the PLC isoforms in the egg are under complex regulatory control. When assayed at optimal calcium concentration, the PLC activity in the egg appears to be regulated by additional mechanisms with the result that a biphasic increase in activity occurs during the period between fertilization and the first cell division. The initial increase in PLC activity begins as early as 30 s postinsemination and remains elevated for approximately 5 min. This burst of activity corresponds to the sperm-induced calcium transient and would provide InsP 3 for generation of the calcium transient as well as during the sperm incorporation step which has been associated with InsP 3 accumulation as well (Lee and Shen, 1998). The second phase of PLC activation occurred between 45 and 60 min postinsemination and persisted through the first cell division at 110 min. The maximal PLC activity during this late phase of activation was more than three times that achievable in the newly fertilized egg. This pool of activated PLC would provide enhanced capacity to achieve calcium signaling during the later stages of egg activation which involve mitotic spindle activity and cytokinesis, or it may reflect an increase in the catabolic activity as the zygote prepares to utilize stored yolk components for development. The complex changes in PLC activity described above highlight the importance of identifying the specific isoforms of PLC that are active in the egg. Recent studies demonstrated a 145-kDa PLC activity that is recognized by two different antibodies to mammalian PLC-g in eggs of the sea urchin Paracentrotus lividus (De Nadai et al., 1998). This made possible the study of the PLC-g isoform during the egg activation process. We found that the PLC-g enzyme from L. variegatus is quite active in extracts from both unfertilized and fertilized eggs. The specific activity in PLC-g immunoprecipitates was much higher than in samples of total egg extract. This apparent stimulation may indicate the presence of PLC inhibitors in the egg cytoplasm which would be removed by immunoprecipitation of the enzyme. Another potential explanation is that endogenous phospholipids such as phosphatidylinositol and the polyphosphoinositides present in crude cell extracts could compete with the [ 3H]phosphatidylinositol 4,5-bisphosphate used as substrate in the assay. The PLC-g enzyme exhibits a strong calcium depen-
dence, with less than 10% of maximal activity detected at 200 nM [Ca 21] i and half-maximal activity requiring 700 – 800 nM [Ca 21] i. No significant change in the calcium dependence of PLC-g activity was detected in immunoprecipitates from fertilized eggs, although the maximal level of activity did change (see below). As mentioned above, the resting levels of [Ca 21] i in the unfertilized egg could support only minimal PLC-g activity, but our data indicate that as intracellular [Ca 21] I levels approach 600 nM, the enzyme would exhibit large changes in activity. The peak [Ca 21] i levels observed during the sperm-induced calcium transient (2–2.5 mM) (Shen, 1995) would support maximal activity of the egg PLC-g enzyme. In addition to the potential for regulation of PLC-g activity by calcium, the sea urchin egg also exhibited the capacity for activation of the total PLC-g activity in response to fertilization. Immunoprecipitates prepared from eggs as early as 30 s postinsemination exhibited greater maximal PLC activity than that of the unfertilized egg. Elevated PLC-g activity was found to persist for up to 20 min, after which it returned to the unfertilized level. This result demonstrates that the PLC-g contributes to the initial phase of PLC activation described above, although participation of other PLC isoforms is not ruled out. This result extends the findings of De Nadia et al. (1998) and supports the conclusion of PLC-g SH2 domain microinjection studies in the marine invertebrate egg (Carroll et al., 1997, 1999; Shearer et al., 1999). Interestingly, PLC-g activity was not elevated during the second phase of PLC activation that occurred between 60 and 110 min postinsemination. This surprising result might indicate that other PLC isozymes are responsible for the second phase of PLC activation that continues through the first mitosis. An alternative explanation would be that the second phase of PLC activation requires cytosolic factors which would be removed by immunoprecipitation. Examples of such factors could include phosphatidic acid or arachidonic acid, both of which might act to stimulate PLC-g (Hwang et al., 1996; Jones and Carpenter, 1993; Sekiya et al., 1999). Further characterization of the second phase of PLC activation is now under way as part of a separate study. The possibility that PLC-g undergoes changes in interactions with other protein or lipid components is reinforced by our finding that fertilization did result in the translocation of a significant percentage of the enzyme to membrane fractions including a plasma membrane-enriched fraction. This phenomenon has been observed in a wide variety of systems and can result from changes in PI-3 kinase activity (Gupta et al., 1999), associations with profilin (Goldschmidt-Clermont et al., 1991), or possibly other membrane-associated proteins. The membrane association of PLC-g is thought to greatly enhance its effectiveness by providing better access to the substrate polyphosphoinositide. It is clear that simple measurement of PLC-g activity can reveal only part of the changes which affect the ability of PLC-g to hydrolyze PtdIns 4,5-P 2 in the intact egg. This it particularly important as it relates to the production of
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InsP 3 in small regions of cytoplasm such as those in the immediate vicinity of the fertilizing sperm, where the sperm-induced calcium transient begins. Our data indicate that PLC-g would be active at very low levels at the [Ca 21] i present in the unfertilized egg. In order for PLC-g to initiate a calcium transient in response to a fertilizing sperm, it is likely that some subset of the PLC-g pool must either increase its sensitivity to Ca 21 or be activated by another mechanism. Changes in the Ca 21 sensitivity of PLC-g have been demonstrated to occur as a response to tyrosine phosphorylation of PLC-g by the activated EGF receptor (Wahl et al., 1992), and it is possible that tyrosine phosphorylation of a small, highly localized subset of the egg PLC-g pool could trigger sufficient InsP 3 production in the vicinity of the fertilizing sperm to initiate Ca 21 release (see below). As Ca 21 levels increase in the vicinity of the fertilizing sperm, additional pools of PLC-g would become active, amplifying the rate of InsP 3 accumulation in the cytoplasm. A critical question remaining at this time is whether the activation of PLC-g following fertilization occurs as a result of tyrosine phosphorylation as occurs, for example, in the response of certain cells to growth factors (Wahl et al., 1992; Shiroo et al., 1992; Kuruvilla et al., 1994). Artificial activation of exogenous (Shilling et al., 1994) or endogenous PTK activity in the egg (Shen et al., 1999) can induce the calcium transient with subsequent cortical reaction. This indicates that increased tyrosine phosphorylation of PLC-g could initiate the calcium transient. However, studies using chemical and peptide inhibitors of PTKs have provided conflicting evidence for a direct role of tyrosine phosphorylation in activation of the sperm-induced calcium transient (Kinsey, 1997; Sato et al., 1998; Glahn et al., 1999) during normal fertilization. While a recent study has demonstrated that a Src-family kinase activity in the egg can associate with exogenous PLC-g domain-specific fusion proteins (Gusti et al., 1999), it is not known whether this association actually occurs in the egg or whether this enzyme phosphorylates PLC-g at fertilization. De Nadai and co-workers (1998) detected fertilization-dependent changes in the ability of anti-P-Tyr antibodies to immunoprecipitate PLC-g. However, this approach suffers from the inability to distinguish between tyrosine phosphorylation of PLC-g and the association of PLC-g with P-Tyrcontaining docking proteins. In the present study, Western blot analysis allowed us to detect some level of phosphotyrosine in PLC-g immunoprecipitated from the sea urchin egg, but we were not able to detect any consistent increase in P-Tyr content after fertilization. One possible explanation is that the PLC-g is maintained in the phosphorylated state prior to fertilization, but requires additional factors or protein–protein associations to become activated sufficiently to initiate the calcium transient. This model would explain the poor effectiveness of chemical PTK inhibitors in blocking the calcium transient, as well as the successful inhibition of the calcium transient by SH2 domaincontaining fusion proteins which act by competing for protein–protein interactions involving P-Tyr. A second pos-
sibility is that the fertilizing sperm may trigger tyrosine phosphorylation of only a small, highly localized subset of the total PLC-g pool which is not detectable in immunoprecipitates of total cellular PLC-g. Further studies using more advanced approaches will be necessary to answer this question.
REFERENCES Becchetti, A. and Whitaker, M. (1997). Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo-inositol. Development 124, 1099 –1107. Carroll, D. J., Albay, D. T., Terasaki, M., Jaffe, L. A., and Foltz, K. R. (1999). Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs. Dev. Biol. 206, 232–247. Carroll, D. J., Ramarao, C. S., Mehlmann, L. M., Roche, S., Terasaki, M., and Jaffe, L. A. (1997). Calcium release at fertilization in starfish eggs is mediated by phospholipase Cgamma. J. Cell Biol. 138, 1303–1311. Ciapa, B., Borg, B., and Whitaker, M.. (1992). Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs. Development 115, 187–195. Ciapa, B., Pesando, D., Wilding, M., and Whitaker, M. (1994). Cell-cycle calcium transients driven by cyclic changes in inositol trisphosphate levels. Nature 368, 875– 878. De Nadai, C., Cailliau, K., Epel, D., and Ciapa, B. (1998). Detection of phospholipase Cg in sea urchin eggs. Dev. Growth. Differ. 40, 669 – 676. Dupont, G., McGuinness, O. M., Johnson, M. H., Berridge, M. J., and Borgese, F. (1996). Phospholipase C in mouse oocytes: Characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca 21 spiking. Biochem. J. 316, 583–591. Epel, D. (1990). The initiation of development at fertilization. Cell. Differ. Dev. 29, 1–12. Galione, A., McDougall, A., Busa, W. B., Willmott, N., Gillot, I., and Whitaker, M. (1993). Redundant mechanisms of calciuminduced calcium release underlying calcium waves during fertilization of sea urchin eggs. Science 261, 348 –352. Glahn, D., Mark, S. P., Behr, R. K., and Nuccitelli, R. (1999). Tyrosine kinase inhibitors block sperm-induced egg activation in Xenopus laevis. Dev. Biol. 205, 171–180. Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, L. M., Rhee, S. G., and Pollard, T. (1991). Regulation of phospholipase C-gamma-1 by profilin and tyrosine phosphorylation. Science 251, 1231–1233. Gupta, N., Scharenberg, A. M., Fruman, D. A., Cantley, L. C., Kinet, J. P., and Long, E. O. (1999). The SH2 domain-containing inositol 59-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling. J. Biol. Chem. 274, 7489 –7494. Gusti, A. F., Carroll. D. J., Abassi, Y. A., and Foltz, K. R. (1999). Evidence that a starfish egg Src family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization. Dev. Biol. 208, 189 –199. Hwang, S. C., Jhon, D. Y., Bae, Y. S., Kim, J. H., and Rhee, S. G. (1996). Activation of phospholipase C-gamma by the concerted action of tau proteins and arachidonic acid. J. Biol. Chem. 271, 18342–18349.
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Jaffe, L. A., Turner, P. R., Kline, D., Kado, R. T., and Shilling, F. (1988). G-proteins and egg activation. Cell. Differ. Dev. Suppl., 15–18. Jones, G. A., and Carpenter, G. (1993). The regulation of phospholipase C-gamma 1 by phosphatidic acid. Assessment of kinetic parameters. J. –Biol. Chem. 268, 20845–20850. Jones, K. T., Cruttwell, C., Parrington, J., and Swann, K. (1998). A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca 21 release in sea urchin egg homogenates. FEBS Lett. 437, 297–300. Kamel, L. C., Bailey, J., Schoenbaum, L., and Kinsey, W. (1985). Phosphatidylinositol metabolism during fertilization in the sea urchin egg. Lipids 20, 350 –356. Kinsey, W. H. (1997). Tyrosine kinase signalling at fertilization. Biochem. Biophys. Res. Commun. 240, 519 –522. Kuruvilla, A., Pielop, C., and Shearer, W. T. (1994). Plateletactivating factor induces the tyrosine phosphorylation and activation of phospholipase C-gamma 1, Fyn and Lyn kinases, and phosphatidylinositol 3-kinase in a human B cell line. J. Immunol. 153, 5433–5442. Lee, S. J., and Shen, S. S. (1998). The calcium transient in sea urchin eggs during fertilization requires the production of inositol 1,4,5-trisphosphate. Dev. Biol. 193, 195–208. McPherson, S. M., McPherson, P. S., Mathews, L., Campbell, K. P., and Longo, F. J. (1992). Cortical localization of a calcium release channel in sea urchin eggs. J. Cell Biol. 116, 1111–1121. Mehlmann, L. M., Carpenter, G., Rhee, S. G., and Jaffe, L. A. (1998). SH2 domain-mediated activation of phospholipase Cgamma is not required to initiate Ca 21 release at fertilization of mouse eggs. Dev. Biol. 203, 221–232. Mehlmann, L. M., Mikoshiba, K., and Kline, D. (1996). Redistribution and increase in cortical inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse oocyte. Dev. Biol. 180, 489 – 498. Miyazaki, S., Yuzaki, J., Nakada, K., Shirakawa, H., Nakanishi, S., Nakade, S., and Mikoshiba, K. (1992). Block of Ca 21 wave and Ca 21 oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs. Science 257, 251–255. [published erratum appears in Science, 1992, Oct 9;258(5080):following 203] Nishibe, S., Wahl, M., Hernandez-Sotomayor, S., Tonks, N., Rhee, S., and Carpenter, G. (1990). Increase of the catalytic activity of phospholipase C-g1 by tyrosine phosphorylation. Science 25, 1253–1255. Nuccitelli, R., Yim, D. L., and Smart, T. (1993). The sperm-induced Ca 21 wave following fertilization of the Xenopus egg requires the production of Ins(1, 4, 5)P3. Dev. Biol. 158, 200 –212. Oberdorf, J., Vilar, R. C., and Epel, D. (1989). The localization of PI and PIP kinase activities in the sea urchin egg and their modulation following fertilization. Dev. Biol. 131, 236 –242. Parys, J. B., McPherson, S. M., Mathews, L., Campbell, K. P., and Longo, F. J. (1994). Presence of inositol 1,4,5-trisphosphate receptor, calreticulin, and calsequestrin in eggs of sea urchins and Xenopus laevis. Dev. Biol. 161, 466 – 476. Rhee, S. G., and Bae, Y. S. (1997). Regulation of phosphoinositidespecific phospholipase C isozymes. J. Biol. Chem. 272, 15045– 15048.
Ribbes, H., Plantavid, M., Bennet, P. J., Chap, H., and Douste-Blazy, L. (1987). Phospholipase C from human sperm specific for phosphoinositides. Biochim. Biophys. Acta 919, 245–254. Ribot, H., Decker, S. J., and Kinsey, W. H. (1983). Preparation of plasma membranes from fertilized sea urchin eggs. Dev. Biol. 97, 494 – 499. Sato, K., Iwasaki, T., Tamaki, I., Aoto, M., Tokmakov, A., and Fukami, Y (1998). Involvement of protein-tyrosine phosphorylation and dephosphorylation in sperm-induced Xenopus egg activation. FEBS Lett. 424, 113–118. Sekiya, F., Bae, Y. S., andRhee, S. G. (1999). Regulation of phospholipase C isozymes: Activation of phospholipase C-gamma in the absence of tyrosine phosphorylation. Chem. Physiol. Lipids 1–2, 2–11. Sette, C., Bevilacqua, A., Geremia, R., and Rossi, P. (1998). Involvement of phospholipase C gamma1 in mouse egg activation induced by a truncated form of C-kit tyrosine kinase present in spermatozoa. J. Cell Biol. 142, 1063–1074. Shearer, J., De Nadai, C., Emily-Fenouil, F., Gache, C., Whitaker, M., and Ciapa, B.(1999). Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos. Development 126, 2273–2284. Shen, S. S. (1995). Mechanisms of calcium regulation in sea urchin eggs and their activities during fertilization. Curr. Top. Dev. Biol. 30, 63–101. Shen, S. S., Kinsey, W., and Lee, S. J. (1999). Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg. Devel. Growth Differ. 41, 345–355. Shilling, F. M., Carroll, D. J., Muslin, A. J., Escobedo, J. A., Williams, L. T., and Jaffe, L. A. (1994). Evidence for both tyrosine kinase and G-protein-coupled pathways leading to starfish egg activation. Dev. Biol. 162, 590 –599. Shiroo, M., Goff, L., Biffen, J., Shivnan, E., and Alexander, D. (1992). CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx. EMBO J. 11, 4887– 4897. Swann, K., and Whitaker, M. J. (1990). Second messengers at fertilization in sea-urchin eggs. J. Reprod. Fertil. Suppl. 42, 141–153. Turner, P. R., Sheetz, M. P., and Jaffe, L. A. (1984). Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature 310, 414 – 415. Wahl, M. I., Jones, G. A., Nishibe, S., Rhee, S. G., and Carpenter, G. (1992). Growth factor stimulation of phospholipase C-gamma 1 activity: Comparative properties of control and activated enzymes. J. Biol. Chem. 267, 10447–10456. Whitaker, M. J., and Steinhardt, R. A. (1982). Ionic regulation of egg activation. Q. Rev. Biophys. 15, 593– 666. Williams, C. J., Mehlmann, L. M., Jaffe, L. A., Kopf, G. S., and Schultz, R. M. (1998). Evidence that Gq family G proteins do not function in mouse egg activation at fertilization. Dev. Biol. 198, 116 –127. Received for publication July 29, 1999 Revised August 24, 1999 Accepted August 27, 1999
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Fertilization-Induced Activation of Phospholipase C in the Sea Urchin Egg 1 Brenda J. Rongish, Wenjun Wu, and William H. Kinsey 2 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160
Fertilization results in the biphasic activation of polyphosphoinositide-specific phospholipase C (PLC) activity with an initial increase in activity coincident with the sperm-induced calcium transient, followed by a more sustained increase prior to mitosis. Immunoprecipitation studies demonstrated that the g isoform of PLC is present in both the unfertilized and the fertilized egg and contributes to the initial phase of PLC activation. Fertilization also resulted in translocation of a significant fraction of PLC-g from the cytosol to the membrane compartment of the egg. © 1999 Academic Press Key Words: fertilization; phospholipase C; calcium; sea urchin.
INTRODUCTION Fertilization results in the activation of a series of preprogrammed biochemical events which are required to prevent polyspermy, activate cell metabolism, and release the egg from cell cycle arrest. A key step is the transient release of calcium from internal stores, the sperm-induced “calcium transient,” which triggers the cortical reaction and activates all of the essential components of the egg activation pathway (Whitaker and Steinhardt, 1982). Two mechanisms of calcium mobilization have been identified in the sea urchin egg: the InsP 33 receptor (Parys et al., 1994) and the ryanodine receptor (McPherson et al., 1992). While both types of channels are expressed in eggs, the InsP 3 receptor has proven to be the dominant mechanism for initiation of the calcium release in most species of eggs (Lee and Shen, 1998; Miyazaki et al., 1992; Nuccitelli et al., 1993) and the ryanodine receptor probably plays a redundant role (Galione et al., 1993) or contributes to the ampli1
This investigation was funded by Grant NIH-HD 14846. To whom correspondence should be addressed at Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3902 Rainbow Boulevard, Kansas City, Kansas 66160. Fax: (913) 588-2710. E-mail: [email protected]. 3 Abbreviations used: EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N,N-tetraacetic acid; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; PtdIns, phosphatidylinositol; InsP 3, inositol 1,4,5-P 3; SBTI, soybean trypsin inhibitor; BSA, bovine serum albumin; P-Tyr, phospho-L-tyrosine; Pipes, piperazine-N,N-bis(2ethanesulfonic acid); PI-3 kinase, phosphatidylinositol 3-kinase. 2
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tude of the calcium transient once initiated by InsP 3 (Shearer et al., 1999). The mechanism by which InsP 3 levels are controlled in the fertilized egg remains an important question. Several studies in the sea urchin system have documented fertilization-induced changes in polyphosphoinositide metabolism at fertilization (Turner, et al., 1984; Kamel et al., 1985; Swann and Whitaker, 1990; Ciapa et al., 1992; Jones et al., 1998) and a recent study demonstrated the biphasic accumulation of InsP 3 in the fertilized sea urchin egg (Lee and Shen, 1998). Several approaches have been used to study the isoforms of phospholipase C (PLC) present in the egg and the mechanism by which they are regulated after fertilization. The isoforms of PLC that have been identified in the mammalian egg include b 1, b 3, g 1, and g 2 (Dupont et al., 1996; Mehlmann et al., 1998), while only PLC-g has been identified in the sea urchin egg to date (De Nadai et al., 1998). Functional studies using chemical inhibitors of PLC (Lee and Shen, 1998) as well as fusion proteins which act as competitive inhibitors of PLC-g (Carroll et al., 1997, 1999; Shearer,, et al., 1999) have demonstrated that PLC activity (specifically PLC-g activity) is required for the fertilizationinduced calcium transient in marine invertebrate eggs. Similar studies in mammalian eggs have provided conflicting evidence for a role of PLC-g (Mehlmann et al., 1998; Sette et al., 1998), in initiation of the initial calcium transient or for the later cyclic calcium transients resulting from fertilization. Interestingly, very thorough studies on the role of G protein-activated PLC-b (Williams et al., 1998) have indicated no clear role for this isozyme in the calcium
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transient of mammalian eggs. The mammalian egg may be able to use both forms of PLC or may make use of another isoform to generate InsP 3 at fertilization. Despite the clear role of PLC in regulating InsP 3 levels in the fertilized egg, the actual effect of fertilization on the activity of PLC isoforms has received only limited attention (De Nadai et al., 1998). The purpose of the present study was to quantitate the effect of fertilization on overall PLC activity and specifically on PLC-g activity in the sea urchin egg. The results demonstrate that even unfertilized eggs contain substantial polyphosphoinositide-specific PLC activity which can become active when [Ca 21] i reaches permissive levels. Fertilization induces an early phase of activation of PLC activity coincident with the calcium transient followed by a second, more prolonged activation that occurs well after the S phase of the cell cycle and continues through mitosis. The activity of PLC-g also increases coincident with the calcium transient, but does not increase during the later phase of PLC activation. Fertilization also resulted in the rapid translocation of PLC-g activity from the cytosol to the membrane compartment of the egg, suggesting that PLC-g is under complex regulatory control at fertilization.
MATERIALS AND METHODS Buffers. PKME consisted of 425 mM KCl, 50 mM Pipes, 10 mM MgCl 2, 10 mM EGTA, 10 mM Na 3VO 4, 0.01 mg/ml SBTI (Sigma, St. Louis, MO), and 10 mg/ml aprotinin (Sigma), pH 6.8. PLC buffer consisted of 50 mM NaH 2PO 4,100 mM KCl, 1 mM EGTA, pH 6.8. Solubilization buffer was PLC buffer with 1% n-octyl b-Dglucopyranoside, 100 mM Na 3VO 4, pH 6.8. Eggs and embryos. Eggs were collected from the sea urchin Lytechinus variegatus and dejellied by passage through Nytex (200 mm) cloth, washed, and suspended in artificial sea water (Marine Enterprises Int., Baltimore, MD). Cytosolic or detergent extracts were prepared from egg suspensions (2% v/v) that had been fertilized by addition of “dry” sperm and incubated at 20°C for various periods of time. The egg suspension was diluted by addition of 4 vol of ice-cold PKME and quickly pelleted in a hand centrifuge. The eggs were then resuspended in 1ml of PLC buffer (cytosol preparation) or PLC solubilization buffer (detergent extracts) and homogenized in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 100,000g for 5 min in a SW 50.1 rotor (Beckman Instruments) and the soluble material was assayed for enzyme activity immediately. Immunoprecipitation. Immunoprecipitation of PLC-g was done by incubating the detergent-solubilized egg extract (30 mg protein) with 0.1 mg of a mixed monoclonal antibody preparation specific for PLC-g 1 (Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C. The immune complexes were collected by adsorption to 5 ml of protein A–Sepharose (Pharmacia, Piscataway, NJ), which had been blocked with casein (Sigma), and then washed twice with PLC solubilization buffer and once with PLC buffer. PLC assay. PLC activity was measured in 25 ml of PLC buffer containing 4 nmol of a mixture of PtdIns 4,5-P 2 (Sigma) and [ 3H]PtdIns 4,5-P 2 (Amersham) (2.5 mCi/mmol) in a mixed micelle formed by 0.25% octylglucoside 1 0.1% deoxycholate (Wahl et al., 1992). CaCl 2 was added to a final concentration of 850 mM to give
FIG. 1. Calcium dependence of PLC activity in the sea urchin egg. PLC activity of cytosolic extracts prepared from unfertilized eggs was measured under calcium–EGTA buffering conditions designed to provide defined levels of free calcium. Buffer components were KCl (100 mM), NaH 2PO 4 (50 mM), EGTA (1.36 mM), Mg 21 (340 mM), Ca 21 (42 mM–1.042 mM). Data represent the average of two experiments, each performed in duplicate 6 SE.
a free calcium concentration of 1.8mM. Free calcium levels were calculated using the WINMAXC program (C. Patton, Stanford University). The reaction was carried out at 25°C for 60 min and then stopped by addition of 1 mg BSA followed by 200 ml of 10% TCA. The TCA-soluble material was then counted in a scintillation counter.
RESULTS Quantitation of total PLC activity in the egg. PtdIns 4,5-P 2-specific PLC activity was measured using the substrate Ptd[ 3H]Ins 4,5-P 2 presented in a mixed micelle containing ocytl-b-D-glucopyranoside:deoxycholate (4.5:1). The reaction at 25°C was linear for up to 3 hours. HPLC analysis of the TCA-soluble products of this reaction confirmed that [ 3H]inositol 1,4,5-P 3 was the predominant reaction product and that lysophospholipids resulting from phospholipase A activity were not a significant contaminant of this reaction (not shown). As in other systems (Wahl et al., 1992), the PLC activity in the egg was absolutely dependent on the concentration of free Ca 21 with very little activity detected with less than 2.5 3 10 27 M free Ca 21 and half-maximal activity requiring 1 mM free Ca 21 (Fig. 1). Similar calcium dependence was seen in extracts prepared from unfertilized
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FIG. 2. Effect of fertilization on total PLC activity in the egg. PLC activity was measured in detergent extracts prepared before and at different times after fertilization. The timing of the cortical reaction (30 s postinsemination) and of mitosis (90 –105 min postinsemination) was monitored in parallel samples incubated under identical conditions. Values represent the average of three experiments, each involving duplicate samples, 6 SE.
eggs and zygotes collected during the first few minutes after fertilization. All subsequent assays were done at 1.8 mM free Ca 21. The data presented in Fig. 1 also demonstrate that the unfertilized egg contains abundant PLC activity which could become active if [Ca 21] i were elevated to 1 mM or above. Since the level of [Ca 21] i in the unfertilized egg is reported to be in the range of 100 –150 nM (reviewed in Shen, 1995), it is likely that the population of PLC enzymes would be essentially inactive in the unfertilized egg. To determine whether fertilization had any effect on overall PLC activity, enzyme assays were performed on detergent extracts prepared at different times after fertilization. The concentration of sperm used in these experiments was sufficient to achieve partial elevation of the fertilization envelope in at least 80% of the eggs within 30 s after addition of the sperm. The Ptd[ 3H]Ins 4,5-P 2-specific PLC activity in the unfertilized L. variegatus egg was between 45 and 60 pmol/min/mg. Analysis of L. variegatus sperm revealed a level of PLC activity of 1400 pmol/min/mg which is nearly identical to that reported for human sperm (Ribbes, 1987) and is substantially higher than that of the egg. In the present experiments, the excess sperm were removed by washing with PKME before homogenization. Therefore, about 6 ng of sperm protein would remain in the egg sample and could contribute 8.4 fmol/min of activity or about 1.3% of the activity in the eggs. The effect of fertilization on PLC activity is seen in Fig. 2. Fertilization resulted in a biphasic increase in PLC activity with an initial phase occurring during the first 2 min and a later, more prolonged phase continuing through the first mitotic division. The initial increase in PLC activity was
detected as early as 30 s postinsemination and increased by approximately twofold before returning to baseline levels. This initial increase in PLC activity corresponds temporally with the propagation of the calcium transient in the egg (Mohri and Chambers, 1995) and with the reported phase 1 accumulation of InsP 3 (Lee and Shen, 1998). As the zygote assembled the mitotic spindle (60 –75 min) and began cytokinesis (90 –105min) the PLC activity increased to a level approximately fourfold higher than in the unfertilized egg. This elevated level of PLC activity was maintained at the two-cell stage (105 min). In summary, while PLC activity is ultimately dependent on the concentration of free calcium, the activation state of PLC does change in a biphasic manner with an early activation event occurring between 0.5 and 2 min postinsemination, followed by a later increase in activity at 60 to 75 min that persisted through the first cleavage. Identification of phospholipase C-g in the sea urchin egg. As a first step in identification of the isoforms of PLC that participate in the response to fertilization, an antibody to human PLC-g was used to detect this protein in the sea urchin egg. Combined immunoprecipitation and Western blot analysis revealed the presence of a 143-kDa protein in detergent extracts of sea urchin eggs (Fig. 3). This mixed monoclonal anti-human-PLC-g antibody preparation (Upstate Biotechnology Inc.) was first used in the sea urchin system by De Nadai et al. (1998) and is the only antibody of several that we have tried that will crossreact with the sea urchin protein. The anti-PLC-g immunoprecipitates were found to be highly enriched in phospholipase C activity, with 2300 pmol/min of activity typically immunoprecipitated from 1 mg of unfertilized egg protein. This specific
FIG. 3. Detection of PLC-g in the sea urchin egg. Detergent extracts of unfertilized eggs (50 mg protein) were incubated with 0.1 mg of either the mixed monoclonal anti-PLC-g antibody (A) or two unrelated monoclonal antibodies (B, C) at a 1:500 dilution. The immune complexes were adsorbed to protein A–Sepharose which had been preblocked with purified casein (1 mg/ml) and resolved on a 10% SDS–polyacrylamide gel. The gel was transferred to a nylon membrane, blocked, and incubated with the mixed anti-PLC-g monoclonal antibody at a dilution of 1:1000. Secondary antibody incubation was with rabbit anti-mouse IgG–peroxidase, and the bound peroxidase was detected by chemiluminescence (ECL, Amersham).
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FIG. 4. Calcium dependence of PLC-g immunoprecipitated from sea urchin eggs. PLC-g immunoprecipitated from detergent extracts prepared before and 1 min after fertilization were assayed for PLC activity under calcium–EGTA buffering conditions designed to provide defined levels of free calcium. Control immunoprecipitates (anti-Grb2 antibody) were assayed simultaneously and the PLC-g-specific activity was calculated by subtracting the activity in control samples from that of the PLC-g immunoprecipitates. Values represent the average of two experiments, each performed in duplicate 6 SE.
activity is substantially higher than the 50 pmol/min/mg measured for total PLC activity in egg extracts (Fig. 2) suggesting that inhibitors of PLC-g may be present in the egg cytosol or that endogenous lipids interfere with the measurement of PLC activity in crude detergent extracts (see Discussion). The observed specific activity for PLC-g of 2300 pmol/min/mg is similar to the value of 5500 pmol/ min/mg reported for the mammalian enzyme (Wahl et al., 1992). Calcium dependence of PLC-g. The effect of calcium on the PLC-g activity immunoprecipitated from unfertilized eggs and zygotes extracted at 1 min postfertilization is presented in Fig. 4. In both unfertilized and fertilized egg samples, half-maximal activity was detected at 750 nm free calcium. This value is somewhat higher than that reported for PLC-g immunoprecipitated from EGF-stimulated human A-431 cells where half-maximal activity occurred at 316 nM free calcium (Wahl et al., 1992) and may reflect species differences. Further attempts to detect possible changes in calcium dependence following fertilization were carried out by using lower substrate/detergent ratios and by replacing deoxycholate with various amounts of Triton X-100 in the assay. The presence of Triton X-100 in the
substrate micelle has been demonstrated to enhance detection of changes in calcium dependence of PLC-g in growth factor-stimulated cells (Nishibe et al., 1990); however, we were not able to detect fertilization-dependent changes in calcium dependence in immunoprecipitates from unfertilized and fertilized eggs. Effect of fertilization on PLC-g activity. The ability to detect PLC activity in the PLC-g immunoprecipitates made it possible for us to quantitate the effect of fertilization on PLC-g activity as a subset of total PLC activity. As seen in Fig. 5, PLC-g activity increased slightly within 30 to 45 s after fertilization. This change was found to be statistically significant (P 5 0.05), although the maximal level of PLC-g activity was not reached until 60 –90 s postinsemination. Activity declined slightly after 2 min, but remained higher than that immunoprecipitated from unfertilized eggs until 60 min, at which time PLC activity returned to the unfertilized level. It is interesting to note that PLC-g activity was not elevated between 60 and 110 min postfertilization as was observed for the total PLC activity of the zygote. One possible explanation might be association of PLC-g with the detergent-insoluble cytoskeleton; however, Western blot analysis of duplicate immunoprecipitates did not reveal any significant changes in the immunoprecipitation recovery of PLC-g which might cause artifactual changes in PLC-g activity during this period of development. We interpret the above results, therefore, to indicate that the g isoform of PLC participates in the fertilization-induced activation of PLC during the first 60 s postinsemination. This enzyme is also active during the late phase of PLC activation but does not exhibit increased activity in this type of immunoprecipitation assay.
FIG. 5. Effect of fertilization on PLC-g activity. PLC-g-specific activity was measured after immunoprecipitation from detergent extracts prepared from unfertilized eggs and from zygotes at different times after fertilization. Values represent the activity in the anti-PLC-g immunoprecipitates less (2) the activity in control immunoprecipitates and are the average of three experiments 6 SE.
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FIG. 6. Subcellular distribution of PLC-g activity. Samples (1 ml) of eggs were homogenized in 5 ml of PKME buffer before (E) and 1 min after (F) fertilization. The homogenates were then centrifuged through sucrose gradients consisting of sucrose in PKME (60% (4 ml), 45% (5 ml), 45–20% linear gradient (22 ml)) overnight in an SW28 rotor (Beckman) at 25,000 rpm and fractions were collected. Aliquots (50 ml) of each fraction were solubilized in 0.5 ml of PLC solubilization buffer and PLC-g-specific activity was immunoprecipitated and assayed as described under Materials and Methods. The top of the gradient is at fraction 1.
Subcellular localization. In order to determine whether the subcellular distribution of PLC-g changed in response to fertilization, we used a sucrose density gradient method (Ribot et al., 1983) to fractionate unfertilized and fertilized eggs and then measured PLC-g activity immunoprecipitated from the different fractions (Fig. 6). PLC-g activity from unfertilized eggs remained, for the most part, at the top of the gradient where the cytosolic proteins and pigment-rich yolk fractions are distributed. However, preparations from zygotes made 60 s postinsemination exhibited a more complex distribution with over one-half of the PLC activity associated with membrane bands, including a band eluting at a buoyant density of 1.23 g/ml (fraction 25) which we have previously shown to be highly enriched in plasma membrane (Ribot et al., 1983). The results of this experiment demonstrate that fertilization involves rapid and significant changes in the distribution of the PLC-g enzyme, with the result that a large fraction of the activity becomes membrane associated. Phosphorylation of PLC-g. Tyrosine phosphorylation of PLC-g is a well-established control mechanism utilized in a variety of cell types and a growing body of evidence suggests that PLC-g interacts with PTKs in the egg (see Discussion). In an attempt to detect possible changes in the P-Tyr content of PLC-g at fertilization, we performed Western blot analysis of PLC-g immunoprecipitates prepared from unfertilized eggs and early zygotes using a monoclonal antibody to P-Tyr (Fig. 7). The PLC-g band shown in the top panel is recognized by the anti-P-Tyr antibody, and antibody binding is significantly reduced but not completely
eliminated by the presence of 5 mM P-Tyr (control). The monoclonal P-Tyr antibody used in this experiment (Zymed Laboratories Inc., San Francisco, CA) has a very high affinity for P-Tyr and competition with free P-Tyr is only partially successful in our experience. The relative amount of P-Tyr detected in PLC-g did not change significantly after fertilization (Fig. 7, lanes B–D). Similar results were obtained in over seven experiments using the Zymed antibody as well as the 4G10 anti-P-Tyr monoclonal from Upstate Biotechnology Inc. Additional efforts to control degradation by phosphatases included the use of sodium pyrophosphate, sodium fluoride, phenylarsine oxide, and peroxyvanadate, with no change in results. The data show that PLC-g is phosphorylated on tyrosine in the unfertilized egg and that any changes in phosphorylation state which might occur at fertilization probably involve a small subset of the total PLC-g pool.
DISCUSSION Localized release of calcium from internal stores is required for several aspects of the egg activation process, including the slow block to polyspermy, activation of egg metabolism (Epel, 1990), and reentry into the cell cycle (Ciapa et al., 1994; Becchetti and Whitaker, 1997). Studies in both vertebrate and invertebrate systems have demonstrated the importance of polyphosphoinositide hydrolysis in the regulation of free calcium in response to fertilization (Oberdorf et al., 1989; Miyazaki et al., 1992). Three different isoforms of PLC have been detected in mammalian eggs (Dupont et al., 1996; Mehlmann et al., 1996), and while the limited crossreactivity of PLC antibodies among different species has not allowed similar analysis in invertebrate eggs, it is likely that several PLC isoforms are present in all species. Given this array of PLC enzymes, the details of the
FIG. 7. Detection of phosphotyrosine in PLC-g immunoprecipitated from sea urchin eggs. L. variegatus eggs collected before (A) and at 15 s (B), 30 s (C), and 60 s (D) after fertilization were solubilized in PLC solubilization buffer containing 100 mM Na 3VO 4 and 10 mM phenylarsine oxide as phosphatase inhibitors. PLC-g was immunoprecipitated and blotted to a nylon membrane and blocked in 1% BSA as described under Materials and Methods and then P-Tyr was detected using a monoclonal anti-P-Tyr antibody (Zymed Laboratories, Inc., San Francisco, CA) at a 1:1000 dilution, followed by goat anti-mouse IgG–peroxidase and chemiluminescent detection. Control blots were incubated with the anti-P-Tyr antibody 1 5mM phosphotyrosine as a competitive inhibitor (Con).
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calcium regulation mechanisms at each stage of egg activation could be quite complex. The objective of the present study was to begin biochemical characterization of the PLC activities in the sea urchin egg with special emphasis on the PLC-g isoform. The total PLC activity of the sea urchin egg as measured with [H 3]PtdIns 4,5-P 2 exhibits a near absolute dependence on Ca 21 as reported in many other systems (Rhee and Bae, 1996). This activity probably represents the sum of several PLC isoforms, but it is clear that prior to fertilization, very little PtdIns-P 2 hydrolysis can occur at the low resting calcium levels which are typically reported to be between 100 and 150 nM (Shen, 1995). Measurement of total PLC activity at different points during the egg activation process revealed that the PLC isoforms in the egg are under complex regulatory control. When assayed at optimal calcium concentration, the PLC activity in the egg appears to be regulated by additional mechanisms with the result that a biphasic increase in activity occurs during the period between fertilization and the first cell division. The initial increase in PLC activity begins as early as 30 s postinsemination and remains elevated for approximately 5 min. This burst of activity corresponds to the sperm-induced calcium transient and would provide InsP 3 for generation of the calcium transient as well as during the sperm incorporation step which has been associated with InsP 3 accumulation as well (Lee and Shen, 1998). The second phase of PLC activation occurred between 45 and 60 min postinsemination and persisted through the first cell division at 110 min. The maximal PLC activity during this late phase of activation was more than three times that achievable in the newly fertilized egg. This pool of activated PLC would provide enhanced capacity to achieve calcium signaling during the later stages of egg activation which involve mitotic spindle activity and cytokinesis, or it may reflect an increase in the catabolic activity as the zygote prepares to utilize stored yolk components for development. The complex changes in PLC activity described above highlight the importance of identifying the specific isoforms of PLC that are active in the egg. Recent studies demonstrated a 145-kDa PLC activity that is recognized by two different antibodies to mammalian PLC-g in eggs of the sea urchin Paracentrotus lividus (De Nadai et al., 1998). This made possible the study of the PLC-g isoform during the egg activation process. We found that the PLC-g enzyme from L. variegatus is quite active in extracts from both unfertilized and fertilized eggs. The specific activity in PLC-g immunoprecipitates was much higher than in samples of total egg extract. This apparent stimulation may indicate the presence of PLC inhibitors in the egg cytoplasm which would be removed by immunoprecipitation of the enzyme. Another potential explanation is that endogenous phospholipids such as phosphatidylinositol and the polyphosphoinositides present in crude cell extracts could compete with the [ 3H]phosphatidylinositol 4,5-bisphosphate used as substrate in the assay. The PLC-g enzyme exhibits a strong calcium depen-
dence, with less than 10% of maximal activity detected at 200 nM [Ca 21] i and half-maximal activity requiring 700 – 800 nM [Ca 21] i. No significant change in the calcium dependence of PLC-g activity was detected in immunoprecipitates from fertilized eggs, although the maximal level of activity did change (see below). As mentioned above, the resting levels of [Ca 21] i in the unfertilized egg could support only minimal PLC-g activity, but our data indicate that as intracellular [Ca 21] I levels approach 600 nM, the enzyme would exhibit large changes in activity. The peak [Ca 21] i levels observed during the sperm-induced calcium transient (2–2.5 mM) (Shen, 1995) would support maximal activity of the egg PLC-g enzyme. In addition to the potential for regulation of PLC-g activity by calcium, the sea urchin egg also exhibited the capacity for activation of the total PLC-g activity in response to fertilization. Immunoprecipitates prepared from eggs as early as 30 s postinsemination exhibited greater maximal PLC activity than that of the unfertilized egg. Elevated PLC-g activity was found to persist for up to 20 min, after which it returned to the unfertilized level. This result demonstrates that the PLC-g contributes to the initial phase of PLC activation described above, although participation of other PLC isoforms is not ruled out. This result extends the findings of De Nadia et al. (1998) and supports the conclusion of PLC-g SH2 domain microinjection studies in the marine invertebrate egg (Carroll et al., 1997, 1999; Shearer et al., 1999). Interestingly, PLC-g activity was not elevated during the second phase of PLC activation that occurred between 60 and 110 min postinsemination. This surprising result might indicate that other PLC isozymes are responsible for the second phase of PLC activation that continues through the first mitosis. An alternative explanation would be that the second phase of PLC activation requires cytosolic factors which would be removed by immunoprecipitation. Examples of such factors could include phosphatidic acid or arachidonic acid, both of which might act to stimulate PLC-g (Hwang et al., 1996; Jones and Carpenter, 1993; Sekiya et al., 1999). Further characterization of the second phase of PLC activation is now under way as part of a separate study. The possibility that PLC-g undergoes changes in interactions with other protein or lipid components is reinforced by our finding that fertilization did result in the translocation of a significant percentage of the enzyme to membrane fractions including a plasma membrane-enriched fraction. This phenomenon has been observed in a wide variety of systems and can result from changes in PI-3 kinase activity (Gupta et al., 1999), associations with profilin (Goldschmidt-Clermont et al., 1991), or possibly other membrane-associated proteins. The membrane association of PLC-g is thought to greatly enhance its effectiveness by providing better access to the substrate polyphosphoinositide. It is clear that simple measurement of PLC-g activity can reveal only part of the changes which affect the ability of PLC-g to hydrolyze PtdIns 4,5-P 2 in the intact egg. This it particularly important as it relates to the production of
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InsP 3 in small regions of cytoplasm such as those in the immediate vicinity of the fertilizing sperm, where the sperm-induced calcium transient begins. Our data indicate that PLC-g would be active at very low levels at the [Ca 21] i present in the unfertilized egg. In order for PLC-g to initiate a calcium transient in response to a fertilizing sperm, it is likely that some subset of the PLC-g pool must either increase its sensitivity to Ca 21 or be activated by another mechanism. Changes in the Ca 21 sensitivity of PLC-g have been demonstrated to occur as a response to tyrosine phosphorylation of PLC-g by the activated EGF receptor (Wahl et al., 1992), and it is possible that tyrosine phosphorylation of a small, highly localized subset of the egg PLC-g pool could trigger sufficient InsP 3 production in the vicinity of the fertilizing sperm to initiate Ca 21 release (see below). As Ca 21 levels increase in the vicinity of the fertilizing sperm, additional pools of PLC-g would become active, amplifying the rate of InsP 3 accumulation in the cytoplasm. A critical question remaining at this time is whether the activation of PLC-g following fertilization occurs as a result of tyrosine phosphorylation as occurs, for example, in the response of certain cells to growth factors (Wahl et al., 1992; Shiroo et al., 1992; Kuruvilla et al., 1994). Artificial activation of exogenous (Shilling et al., 1994) or endogenous PTK activity in the egg (Shen et al., 1999) can induce the calcium transient with subsequent cortical reaction. This indicates that increased tyrosine phosphorylation of PLC-g could initiate the calcium transient. However, studies using chemical and peptide inhibitors of PTKs have provided conflicting evidence for a direct role of tyrosine phosphorylation in activation of the sperm-induced calcium transient (Kinsey, 1997; Sato et al., 1998; Glahn et al., 1999) during normal fertilization. While a recent study has demonstrated that a Src-family kinase activity in the egg can associate with exogenous PLC-g domain-specific fusion proteins (Gusti et al., 1999), it is not known whether this association actually occurs in the egg or whether this enzyme phosphorylates PLC-g at fertilization. De Nadai and co-workers (1998) detected fertilization-dependent changes in the ability of anti-P-Tyr antibodies to immunoprecipitate PLC-g. However, this approach suffers from the inability to distinguish between tyrosine phosphorylation of PLC-g and the association of PLC-g with P-Tyrcontaining docking proteins. In the present study, Western blot analysis allowed us to detect some level of phosphotyrosine in PLC-g immunoprecipitated from the sea urchin egg, but we were not able to detect any consistent increase in P-Tyr content after fertilization. One possible explanation is that the PLC-g is maintained in the phosphorylated state prior to fertilization, but requires additional factors or protein–protein associations to become activated sufficiently to initiate the calcium transient. This model would explain the poor effectiveness of chemical PTK inhibitors in blocking the calcium transient, as well as the successful inhibition of the calcium transient by SH2 domaincontaining fusion proteins which act by competing for protein–protein interactions involving P-Tyr. A second pos-
sibility is that the fertilizing sperm may trigger tyrosine phosphorylation of only a small, highly localized subset of the total PLC-g pool which is not detectable in immunoprecipitates of total cellular PLC-g. Further studies using more advanced approaches will be necessary to answer this question.
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