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RESEARCH |
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel
Correspondence should be addressed to R Shalgi; Email: shalgir{at}post.tau.ac.il
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Many biological systems in which Ca2+serves as a second messenger are also regulated by protein kinase C (PKC) through its effect, either positive or negative, on the cellular signal transduction processes (Nishizuka 1988). Various studies have demonstrated expression and even possible involvement of PKC in the egg activation process (Gangeswaran & Jones 1997, Johnson & Capco 1997, Raz et al. 1998b, Pauken & Capco 2000, Luria et al. 2000, Eliyahu et al. 2001). There is evidence that activation of PKC induces CGE, but there is probably no direct linkage between PKC activation and the exit from metaphase II (MII; Jones 1998, Raz et al. 1998a). Studies performed in our laboratory revealed that conventional PKC (cPKC) isoenzymes were activated at fertilization and upon parthenogenetic activation either by 12-O-tetradecanoyl phorbol-13-acetate (TPA) or by 1-oleoyl-2-acetylglycerol (OAG), but not by the calcium ionophore ionomycin (Eliyahu & Shalgi 2002). Furthermore, we were able to inhibit CGE by a myristoylated PKC pseudosubstrate (myrPKC
), which is a specific PKC inhibitor, thus suggesting that exocytosis can be triggered independently either by [Ca2+]i rise or by PKC. In a recent study, it was demonstrated that cPKCs translocate to the egg plasma membrane following a pattern that is shaped by the amplitude, duration and frequency of Ca2+transients (Halet et al. 2004).
PKC is known to be associated with a wide range of cytoskeletal components and/or to phosphorylate them (Inagaki et al. 1987). The actin cytoskeleton has long been proposed as the regulatory site of exocytosis. Evidence from several cell types suggests that a membrane-associated filamentous actin (F-actin) acts as a barrier to exocytosis by excluding secretory vesicles from the plasma membrane (Burgoyne et al. 1989, Vitale et al. 1991, Trifaro et al. 1992) and that its depolymerization is a primary step in the translocation of secretory vesicles to the plasma membrane (Lelkes et al. 1986, Sontag et al. 1988, Muallem et al. 1995). However, other studies have demonstrated polymerization, rather than depolymerization, of actin in some somatic cells undergoing exocytosis (Sha’afi & Molski 1987, Apgar 1991). The presence of a subplasmalemal, actin-based cytoskeleton in eggs is well known (Spudich et al. 1988, Bonder et al. 1989, Roeder & Gard 1994, Becker & Hart 1996). The microfilaments of this cytoskeleton play a role in many dynamic processes during mammalian egg maturation and fertilization (Sutovsky et al. 1997, Connors et al. 1998, Terada et al. 2000). Processes such as sperm incorporation, CGE, spindle movement and polar body II (PBII) extrusion (Capco et al. 1992, Gallicano et al. 1992, 1995, Wang et al. 2000) are accompanied by reorganization of the actin cytoskeleton (Terada et al. 2000).
Various studies investigating the effect of actin-perturbing drugs on CGE have produced inconclusive results. CGE could be inhibited in the eggs of only some vertebrate species and only after treatment with some of the actin-perturbing drugs (Tahara et al. 1996, DiMaggio et al. 1997, Terada et al. 2000, McAvery et al. 2002), while other studies showed no inhibition of CGE (Boyle et al. 2001, Sun et al. 2001). Results regarding the effect of PKC activators on actin filaments are also ambiguous. Activation of PKC by TPA or by diacylglycerol (DAG) caused polymerization of actin, while PKC antagonist inhibited the formation of actin storage (Takashi 1997). However, in other studies, stimulation of PKC by phorbol esters was accompanied by a focal and transient disruption of the cortical F-actin network (Vitale et al. 1991, Tchakarov et al. 1998, Trifaro et al. 2002). These contradictory results reflect the lack of clarity regarding the role of PKC, actin and actin-associated proteins in the regulation of secretory events (Muallem et al. 1995).
Evidence from several cell types suggests that F-actin is associated with myristoylated alanin-rich C kinase substrate (MARCKS; Rosen et al. 1990, Aderem 1992, Swierczynski & Blackshear 1995, Rossi et al. 1999). MARCKS cross-links actin filaments and anchors the actin network to the plasma membrane (Rossi et al. 1999). In the present study we investigated the possible interaction between actin and PKC, both of which are known to participate in exocytosis. We examined the hypothesis that the cortical actin filaments serve as a barrier that excludes cortical granules (CGs) from the membrane of MII rat eggs. Changes in the distribution of actin filaments occur as a result of PKC activation during egg activation and enable CGE. Our results demonstrated that CGE cannot be triggered by a simple polymerization/depolymerization of actin filaments but rather PKC activation is required as well.
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Materials and Methods |
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In vivo fertilized eggs
PMSG–hCG-primed immature female rats were caged overnight with fertile males. Rats were killed 15 h after hCG administration. Egg isolation and cumulus cell removal were performed as described above for MII eggs. The eggs were classified according to the various stages of fertilization (Eliyahu & Shalgi 2002): sperm binding (SB), fertilization cone (FC) and PBII stages (0–15, 15–60 and 60–180 min after sperm attachment respectively).
Parthenogenetic activation
MII-ovulated eggs were parthenogenetically activated by two different activators that are capable of inducing full CGE in rat eggs (Raz et al. 1998a, Eliyahu & Shalgi 2002). The first activator was TPA (30–100 ng/ml; Sigma); a 5-min incubation in the presence of TPA followed by an additional 5- or 10-min incubation in fresh medium lacking the activator. A stock solution of 1 mg/ml TPA was prepared in dimethylsulfoxide (DMSO) and stored at –20°C. The second activator was calcium ionophore (2 µM; ionomycin 407950, Calbiochem, San Diego, CA, USA); a 3-min incubation in the presence of ionomycin followed by an additional 17-min incubation in fresh medium lacking the activator. A stock solution of 10 mM ionomycin was prepared in DMSO and stored at 4 °C.
Actin polymerization and depolymerization
MII eggs were incubated for 30 min with the actin-depolymerizing drug cytochalasin D (CD; 10 µg/ml, Sigma), or with the polymerizing drug jasplakinolide/jaspamide (JAS; 1 x 10–7 nM, Molecular Probes, Eugene, OR, USA).
Antibodies and staining agents
Primary antibodies: anti-actin rabbit polyclonal IgG (A-5060, Sigma); anti-cPKC alpha mouse monoclonal IgG (P-16520, Transduction Laboratories, Lexington, KY, USA); anti-MARKCS goat polyclonal IgG (sc-6454, Santa Cruz, CA, USA). Peroxidase-conjugated secondary antibodies: goat anti-rabbit IgG (sc-2004, Santa Cruz); goat anti-mouse IgG (sc-2005, Santa Cruz); donkey anti-goat IgG (sc-2020, Santa Cruz). Cy-conjugated secondary antibody: donkey anti-goat IgG (Jackson Immunoresearch Laboratories, PA, USA). Staining agents: Texas Red phalloidin (T-7471, Molecular Probes); Biotinylated lens culinaris agglutinin (LCA)–biotin (B-1045, Vector, Burlingame, CA, USA); Texas Red streptavidin (SA-5006; Vector).
Immunoprecipitation and immunoblotting
An antibody against anti-actin was chemically coupled to protein A sepharose in order to prepare an immobilized antibody affinity reagent for immunoprecipitation (IP buffer), according to Talmor et al.(1998). Batches of 1000 eggs, that either had or had not been subjected to activating agents, were lysed in 100 µl IP buffer and kept at –70 °C. Upon thawing, the egg lysates were incubated overnight at 4 °C with 10 µl immobilized antibody (25% suspension) and then washed by centrifugation with IP buffer and separated by 10% SDS–PAGE under non-reducing conditions. Proteins were transferred onto a nitrocellulose membrane (Amersham) using a wet blotting apparatus (Hoeffer, San Francisco, CA, USA). For immunoblot analysis, blots were blocked with Tris-buffered saline with 5% dry milk and incubated in the presence of various specific primary antibodies. Bound antibodies were recognized by secondary antibodies conjugated to horseradish peroxidase. Detection was performed by an ECL detection system (Pierce, Rockford, IL, USA). Approximate molecular masses were determined by comparison with the migration of prestained protein standards (Amersham).
Immunofluorescence staining and laser-scanning confocal microscopy
Eggs at various developmental stages were fixed for 10 min at room temperature in Dulbecco’s phosphate-buffered saline (DPBS) with 3% paraformaldehyde and 0.01% glutaraldehyde. Eggs were then washed in a solution of 3% foetal calf serum (FCS; Biological Industries, Beit-Haemek, Israel) in DPBS (DPBS/FCS). ZPs were removed post-fixation by 0.25% pronase (Sigma) in DPBS/FCS. The ZP-free eggs were washed in DPBS/FCS. For detection of CGE, fixed eggs were labeled by LCA and Texas Red streptavidin (Eliyahu & Shalgi 2002).
For labeling egg proteins, the plasma membrane of ZP-free eggs was permeabilized during 10-min incubation in 0.05% NP-40 in DPBS/FCS solution followed by several rinses in 0.005% NP-40 in DPBS/FCS.
For actin labeling eggs were incubated for 2 h in the presence of Texas Red phalloidin (1.1 nM) or with anti-MARCKS (1:100) followed by an incubation in the presence of a fluorescence-labeled Cy secondary antibody (1:300). Chromatin labeling and the fertilization stage of the eggs were assessed by incubating the eggs for 10 min in the presence of 1 µg/ml Hoechst (Sigma). The progression of eggs past the MII stage was analyzed by monitoring the separation of the chromosomal dyads. The various stages of fertilization were determined by following the sperm and egg chromatin. The labeling of CG exudates, actin, MARCKS and DNA was visualized and photographed by a Zeiss confocal laser-scanning microscope (CLSM; Oberkochen, Germany). The Zeiss LSM 410 is equipped with a 25 mW krypton–argon laser, a 10 mW helium–neon laser (488, 543 and 633 maximum lines) and an u.v. laser (Coherent Inc. Laser Group, Santa Clara, CA, USA). A x 40 numerical aperture/1.2 planapochromat water immersion lens (Axiovert 135 M, Zeiss) was used for all imaging. Eggs were scanned using the CLSM through the Z-axis to perform a section at the equatorial plane of the egg. The stain intensity was measured using the correct mean density values obtained by the LSM software. The fluorescence intensity was expressed relative to values obtained for MII eggs, which were set to 1.0 (arbitrary units).
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Expression of actin in the egg
We used Western blot analysis to evaluate the total amount of actin in non-activated MII eggs and in TPA-activated eggs. Egg proteins, before or after parthenogenetic activation of the eggs by TPA, were separated on SDS–PAGE. As shown in Fig. 1
, the amount of actin, a 46 kDa protein, was similar in both non-activated MII eggs and in TPA-activated eggs.
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). As expected, a single 84 kDa PKC alpha band was detected in non-activated MII eggs whereas TPA-activated eggs exhibited two bands, at 84 and 88 kDa, that may represent the unphosphorylated and the phosphorylated forms of PKC alpha respectively. Several treatments were used as control groups: IP buffer containing only the immune complex; incubation of the nitrocellulose membrane with any IgG antibody (polyclonal anti-Src antibody); incubation of the egg lysate with any antibody (polyclonal anti-Src antibody) conjugated to protein A sepharose. None of the control groups exhibited either an 84 kDa or an 88 kDa band on the nitrocellulose membrane (data not shown).
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) with a marked concentration at the cortex and at the plasma membrane, especially above the metaphase plate (Fig. 4A and E). A decrease in the fluorescence signal of F-actin was detected upon fertilization, at the SB stage, as compared with MII eggs (Fig. 3B and E), the decrease was more pronounced at the FC/PBII stages (Fig. 3C and F). In TPA-activated eggs, we observed a time-dependent decrease of the fluorescence signal (Fig. 4F–H), as compared with untreated MII eggs (Fig. 4E). As expected, eggs treated with TPA remained at the MII stage (Fig. 4B–D). The temporal decrease observed in the fluorescence intensity of TPA-activated eggs might reflect the depolymerization/reorganization of F-actin.
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). CD caused aggregation of F-actin within the cytoplasm and a decreased fluorescence intensity, reflecting depolymerization of actin (Fig. 5E). JAS, on the other hand, caused an increase in the fluorescence intensity of F-actin throughout the egg, reflecting polymerization of F-actin (Fig. 5F). Eggs treated with either CD or JAS did not progress beyond the MII stage (Fig. 5A–C).
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). MII eggs were incubated in the presence of either CD or JAS and then activated by 30 ng/ml TPA in the presence of either drug. Untreated MII eggs served as a negative control for CGE (Fig. 6A), whereas, eggs exposed only to TPA served as a positive control (Fig. 6D). MII eggs, as well as eggs exposed to either JAS or CD, did not undergo CGE (Fig. 6A–C), nor did they overcome the second metaphase arrest (not shown). As expected, eggs treated with 30 ng/ml TPA, a concentration that triggers only partial CGE (Raz et al. 1998a), exhibited moderate fluorescence intensity (Fig. 6D). Addition of CD, but not of JAS, caused enhancement of the CGE fluorescence intensity, indicating that the eggs had undergone complete CGE (JAS, Fig. 6E; CD, Fig. 6F). There were no significant differences in the ability of eggs to undergo complete CGE between MII-arrested eggs and eggs treated with CD or JAS alone (Fig. 6G, P >0.05, two-way ANOVA test). However, exposing eggs to CD followed by exposure to 30 ng/ml TPA, doubled the percentage of eggs undergoing complete CGE as compared with eggs incubated with TPA alone (88.6 and 46.4% respectively, P < 0.005, two-way ANOVA test).
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, the egg lysates exhibited a protein band with an apparent molecular mass of 80 kDa, which is consistent with the expected molecular mass for MARCKS protein (Wu et al. 1982, Aderem 1992). The protein band of non-activated MII eggs was much stronger than that of TPA- or of ionomycin-activated eggs (Fig. 7). Control eggs were incubated in the presence of anti-MARCKS primary antibody that had previously been exposed for 1 h to a specific MARCKS peptide (2 µg/ml). No band was observed in this lane (data not shown).
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). MARCKS was highly concentrated at the membrane of MII eggs (Fig. 8B). A distinct colocalization between MARCKS and actin was noted at the plasma membrane (Fig. 8D). Our results demonstrate, for the first time, expression and localization of MARCKS protein and its colocalization with actin in the rat egg.
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Discussion |
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The amount of actin in both non-activated MII eggs and in TPA-activated eggs was similar. This finding is not surprising as most cells have large actin storage ready for an immediate response to intracellular events (Welch et al. 1997). A coimmunoprecipitation assay was used to identify a possible, direct or indirect, interaction between PKC and actin. A band of PKC alpha, an isoenzyme known to participate in exocytosis in other cell types (Benussi et al. 1998) was detected in lysates of non-activated MII eggs. Lysates of activated eggs exhibited two bands of PKC alpha, which probably represent the unphosphorylated and the phosphorylated forms of the isoenzyme as described by Bornancin & Parker (1997). cPKC isoenzymes are known to undergo autophosphorylation during activation. The interaction observed between actin and the phosphorylated and unphosphorylated forms of PKC implies an involvement of both proteins in the egg activation process.
Non-activated MII eggs demonstrated a homogenous distribution of F-actin throughout the cytosol as well as a specific localization at the egg membrane, mainly above the meiotic spindle. Activation of the eggs by sperm or by TPA caused depolymerization/reorganization of the F-actin, as manifested by a decrease in the intensity of the fluorescence signal. The most significant changes observed took place in the cytosol and not at the plasma membrane. We attribute it to the strong actin signal detected at the plasma membrane of non-activated MII eggs, which can mask minor fluorescence changes. Another possibility is that the actin- binding proteins that anchor the F-actin to the plasma membrane render the membranous actin more stable than the cytosolic actin. However, since the total amount of actin appeared similar in Western blots of both activated and non-activated eggs, we can safely assume that there is a change in the ratio of G-actin to F-actin.
TPA induced polymerization of actin in Tubifex eggs while PKC antagonists inhibited the polymerization (Takashi 1997). Depolymerization of actin by CD enhances CGE in zebrafish eggs, while polymerization of actin by phalloidin inhibits CGE (Becker & Hart 1999). They conclude that F-actin acts as a mechanical barrier to exocytosis by excluding CG vesicles from the plasma membrane and that this barrier has to be removed prior to membrane fusion. To study the affect of PKC activation on actin reorganization eggs were treated with actin-polymerizing and -depolymerizing drugs. As expected, CD caused depolymerization of actin while JAS caused polymerization. In an attempt to follow the interplay between F-actin reorganization, PKC activation and CGE, eggs were treated with either CD or JAS with or without a further exposure to TPA. Although CD or JAS alone did not induce CGE, CD but not JAS increased the intensity of the CGE fluorescence signal caused by TPA. The mechanism by which CGs are anchored to the egg cortex is unclear. The observation observation that CD did not cause spontaneous CGE indicates that the reorganization of actin is not adequate to allow CGE, thus implying that the egg cytoskeletal cortex is not a simple barrier that blocks constitutive exocytosis, as suggested for other cells. A more likely possibility is that actin-associated proteins and/or PKC need to be activated in order to cause CGE. We suggest that these proteins are activated in response to parthenogenetic activation by TPA, and they, in turn, could cause the remodeling of the cortical microfilamentous actin that is ultimately required for CGE.
Evidence from several cell types suggests that MARCKS cross-links F-actin and anchors the actin network to the plasma membrane (Rosen et al. 1990, Aderem 1992, Swierczynski & Blackshear 1995, Rossi et al. 1999). An important initial step towards understanding the role of MARCKS during egg activation was the study of its expression, localization and colocalization with actin. We have shown that MARCKS is present in abundance in MII eggs but that levels decrease upon egg activation, as depicted by the decreased band intensity in Western blot analysis. We should bear in mind that the decrease in band intensity could also reflect a decrease in the affinity of the antibody to the protein, caused by phosphorylation of MARCKS. Immunohistochemistry and confocal scanning microscopy enabled us to demonstrate MARCKS localization at the plasma membrane of non-activated MII eggs and its colocalization with F-actin. In view of the aforementioned data, we assume that MARCKS is associated with F-actin and we suggest the hypothesis that, for CGE to occur, PKC has to phosphorylate an actin-bound protein such as MARCKS.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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K Haim, I Ben-Aharon, and R Shalgi Expression and immunolocalization of the calpain-calpastatin system during parthenogenetic activation and fertilization in the rat egg Reproduction, January 1, 2006; 131(1): 35 - 43. [Abstract] [Full Text] [PDF]
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80 kDa as calculated from the migration of protein standards with known molecular masses. At least three independent experiments were performed.