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Reproduction (2008) 135 613-624
DOI: 10.1530/REP-07-0554
Copyright © 2008 Society for Reproduction and Fertility
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RESEARCH

Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis

Lina Tsaadon, Ruth Kaplan-Kraicer and Ruth Shalgi

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


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Sperm–egg fusion induces cortical granules exocytosis (CGE), a process that ensures the block to polyspermy. CGE can be induced independently by either a rise in intracellular calcium concentration or protein kinase C (PKC) activation. We have previously shown that myristoylated alanine-rich C kinase substrate (MARCKS) cross-links filamentous actin (F-actin) and regulates its reorganization. This activity is reduced either by PKC-induced MARCKS phosphorylation (PKC pathway) or by its direct binding to calmodulin (CaM; CaM pathway), both inducing MARCKS translocation, F-actin reorganization, and CGE. Currently, we examine the involvement of Ca2+/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca2+/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active {alpha}CaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt {alpha}CaMKII. Microinjecting eggs with mutant-unphosphorylatable MARCKS reduced the intensity of 12-O-tetradecanoylphorbol 13-acetate or ionomycin-induced CGE by 50%, indicating that phosphorylation of MARCKS by novel and/or conventional PKCs (n/cPKCs) is a pivotal event associated with CGE. Moreover, we were able to demonstrate cPKCs involvement in ionomycin-induced MARCKS translocation and CGE. These results led us to propose that MARCKS, rather than CaMKII, as a key mediator of CGE.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Mammalian oocytes are ovulated while being arrested at the metaphase of the second meiotic division (MII). The fertilizing spermatozoon initiates within the egg a series of biochemical events that lead to rapid mitotic divisions, giving rise to the developing embryo. The transition from MII egg to a developing embryo is brought about by a series of events, generally referred to as ‘egg activation’ (reviewed by Runft et al. 2002, Talmor-Cohen et al. 2002, Jones 2005), but conceptually divided into ‘early’ and ‘late’ events (reviewed by Ducibella et al. 2006). The early events of egg activation commence with a transient rise in intracellular calcium concentration ([Ca2+]i) followed by Ca2+ oscillations. Shortly thereafter, the activated egg undergoes cortical granules exocytosis (CGE), followed by cell cycle progression. The cortical granules (CGs), residing just beneath the plasma membrane of MII-arrested eggs, secrete their content into the perivitelline space causing alteration of the zona pellucida (ZP) and the plasma membrane, thus establishing a block to polyspermy and ensuring successful egg activation and embryo development (Wassarman et al. 2001, Sun 2003, Gardner & Evans 2006).

It is accepted that the initial rise in [Ca2+]i is both necessary and sufficient for triggering the consecutive events of egg activation (reviewed by Runft et al. 2002). Extensive effort was invested in research directed toward answering the paradigm of how the calcium signal is translated into stimuli that evoke biological processes such as CGE and resumption of the second meiotic division (RMII). Sperm–egg interaction triggers the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2; Jones et al. 2000) to inositol 1, 4, 5-trisphosphate (IP3) that induces elevation of [Ca2+]i and formation of diacylglycerol (DAG) that activates various members of protein kinase C (PKC). The dichotomous effect of Ca2+ during egg activation was supported by the observation that CGE and RMII require a different number of Ca2+ transients to commence (Ducibella et al. 2002) and a relatively low rise in [Ca2+]i was sufficient for inducing partial CGE, whereas a ‘full scale’ of [Ca2+]i rise was required for the completion of CGE and RMII (Raz et al. 1998a). The difference can be attributed to different Ca2+ requirements or to different downstream effectors participating in each process.

Various studies suggest diverse roles for PKC within the egg (reviewed by Halet 2004): regulation of oocyte maturation (Viveiros et al. 2001), induction of CGE (Eliyahu & Shalgi 2002), modification of the egg actin cytoskeleton (Eliyahu et al. 2005, 2006), and participation in early embryonic development (reviewed by Capco 2001). Both Ca2+-dependent and Ca2+-independent PKC isoforms were identified in rodent eggs (Gangeswaran & Jones 1997, Raz et al. 1998b). The change in the intracellular distribution of various PKC isoforms, as manifested by its translocation from the cytoplasm to the plasma membrane of the egg, was followed during fertilization (Eliyahu & Shalgi 2002, Halet et al. 2004) as well as during parthenogenetic activation of eggs (Gallicano et al. 1995, Eliyahu & Shalgi 2002). However, it should be noted that activation of PKC by 12-O-tetradecanoylphorbol 13-acetate (TPA) triggered only CGE but had no effect on either RMII or [Ca2+]i rise (Raz et al. 1998a), and that Ca2+ chelators had almost no effect on PKC activation at fertilization (Tatone et al. 2003). RMII can be triggered only by [Ca2+]i rise, whereas CGE can be triggered either by [Ca2+]i rise or by PKC activation (Gangeswaran & Jones 1997, Johnson & Capco 1997, Raz et al. 1998b, Pauken & Capco 2000, Eliyahu & Shalgi 2002).

Calmodulin (CaM), one of the most common Ca2+ transducers, is a regulator of both CGE and RMII (reviewed by Abbott & Ducibella 2001). CaM is present in abundant quantities within eggs and has numerous substrates, of which Ca2+/CaM-dependent protein kinase II (CaMKII) is the most important. CaMKII activity oscillates in synchrony with Ca2+ oscillations (Markoulaki et al. 2003, 2004). A Ca2+-mediated stimulation of egg CaMKII activity is required for activation of the anaphase-promoting complex (APC), which in turn is responsible for cyclin B degradation and sister chromatids separation (Madgwich et al. 2005, reviewed by Ducibella et al. 2006). It was hypothesized that CaMKII is required also for CGE and for establishing the block to polyspermy (reviewed by Abbott & Ducibella 2001). Although some reports indicate that CaMKII, like CaM, is localized to sites of exocytosis (Shen et al. 1998, Easom 1999) and is involved in CGE (Tatone et al. 1999, 2002, Markoulaki et al. 2003); a recent report indicated an abnormal CGE in mouse eggs injected with a constitutively active form of CaMKII (CA-CaMKII; Knott et al. 2006).

We have previously suggested that the filamentous actin (F-actin) at the egg cortex is a dynamic network that can be maneuvered toward allowing CGE by activated PKC and its downstream proteins, such as myristoylated alanine-rich C kinase substrate (MARCKS). We and others have demonstrated that MARCKS, a protein known to cross-link F-actin in other cell types and a major PKC substrate, is expressed in rat and mouse eggs (Eliyahu et al. 2005, Michaut et al. 2005 respectively) and is colocalized with actin in non-activated MII eggs (Tsaadon et al. 2006). MARCKS translocates to the egg cortex and dissociates from F-actin in ionomycin-activated eggs, a process that can lead to the breakdown of the actin network, thus allowing CGE to occur. In an earlier study (Eliyahu et al. 2006), we emphasized the link between PKC, actin, CaM, and MARCKS in mediating CGE. We proposed that, as in secretory cells (Danks et al. 1999, Vaaraniemi et al. 1999, Wohnsland et al. 2000, Arbuzova et al. 2002), phosphorylation of the egg's MARCKS by PKC or interaction of MARCKS with CaM can cause translocation of MARCKS from the plasma membrane to the egg's cortex and disassembly from the F-actin, thus allowing the CGs to fuse with the plasma membrane and induce CGE. PKC activation promoted MARCKS phosphorylation, whereas a PKC inhibitor (myrPKC{psi}) decreased both MARCKS translocation and CGE. We were able to cause translocation of MARCKS and showed an increase in the amount of CaM bound to MARCKS by activating the eggs with ionomycin. MARCKS translocation and CGE were inhibited by the CaM pharmacological inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7; Eliyahu et al. 2006). Although biochemical approaches have provided evidence for a link between MARCKS translocation and CGE, and pharmacological inhibitors aided in analyzing the role of MARCKS in the process, one should be wary of data misinterpretation tainted only by pharmacological effects on other cellular targets.

In order to suggest a role of potential mediators in the process of CGE, we employed, in the present study, molecular tools for focusing specifically and directly on CaMKII and MARCKS. We microinjected rat eggs with a complementary RNA (cRNA) of the rat CA-{alpha}CaMKII or with an unphosphorylatable mutant form of MARCKS and examined the ability of the injected eggs to undergo CGE and RMII. The results led us to propose that MARCKS, but not CaMKII, is a key mediator in CGE.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Effect of W7 on MARCKS translocation, CGE and RMII in eggs parthenogenetically activated by OAG
We have already suggested that CGE is triggered by PKC activation or CaM activation, both pathways acting by causing translocation of the MARCKS protein (Eliyahu et al. 2006). In an attempt to figure out whether these two pathways compensate for one another, we activated, in the present study, both pathways by 1-oleoyl-2-acetylglycerol (OAG), a phorbol ester that causes PKC activation as well as Ca2+ elevation (Raz et al. 1998a), in the presence of W7, a CaM inhibitor, and followed translocation of MARCKS, CGE and RMII.

OAG-induced translocation of MARCKS from the plasma membrane (Fig. 1a, A') to the cortex (Fig. 1a, B') of the egg and triggered both CGE and RMII (Fig. 1b, B'). W7 alone induced neither MARCKS translocation (Fig. 1a, C'), nor CGE or RMII (Fig. 1b, C'). W7 was able to block OAG-induced RMII (Fig. 1b, D'), but was unable to inhibit either OAG-induced MARCKS translocation (Fig. 1a, D') or CGE (Fig. 1b, D'). CGE was not affected by blocking of the CaM activation pathway, indicating a compensatory pathway, most probably the PKC activation pathway. Further examination of each pathway was performed by employing molecular techniques.


Figure 1
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  		Figure 1 Effect of W7 on MARCKS translocation, CGE and RMII in OAG-activated eggs. Confocal images of eggs fixed: at MII stage (A and A'), after a 5-min activation by 50 µg/ml OAG followed by an additional 5-min incubation in fresh medium lacking the activator (B and B'), after a 30-min incubation in the presence of 20 µM W7 (C and C'), after a 30-min incubation in the presence of 20 µM W7 followed by 5-min activation by OAG (50 µg/ml) in the presence of 20 µM W7, followed by 5-min incubation in TH medium containing 20 µM W7, but lacking OAG (D and D'). Light microscopy (A–D), confocal microscopy (A'–D'). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed (three to four eggs were measured for each group in each experimental day). Bar=10 µm. (a) Fixed eggs were labeled with anti-MARCKS goat polyclonal IgG (1:50) recognized by Cy-3 secondary antibody (1:250) and chromosomes were labeled by Hoechst 33342 (1 µg/ml). MARCKS staining was imaged using CLSM. MARCKS, red; chromosomes, blue. (b) Fixed eggs were labeled by LCA–biotin (1:100). LCA–biotin was detected by Texas Red Streptavidin (1:1000) and chromosomes were labeled by Hoechst 33342 (1 µg/ml). CGE was imaged using CLSM. CGE labeling, red; chromosomes, blue.

 
Involvement of {alpha}CaMKII in egg activation
Accumulating evidence from our previous study implies a direct role for CaM in the process of CGE (Eliyahu et al. 2006). We postulated that CaM, beyond being CaMKII activator, is an active participating link in the pathway leading to CGE by directly binding to MARCKS, thus causing MARCKS translocation. We have hypothesized that CaM, rather than CaMKII, is involved in CGE. In order to contradict the involvement of CaMKII in promoting CGE, we microinjected eggs with a CA-{alpha}CaMKII mutant and followed the ability of the eggs to undergo CGE and RMII.

Expression of {alpha}CaMKII isoform in rat eggs
The {alpha}CaMKII isoform was undetected in mouse eggs, whereas the βCaMKII isoform was predominantly expressed (Abbott et al. 2001). Since we were provided with a rat cDNA encoding the {alpha}CaMKII wt isoform, our initial step was to determine whether the {alpha}CaMKII isoform is expressed in rat eggs using Western blot analysis. Proteins of either non-activated MII eggs or eggs activated for 20 min by ionomycin (2 µM) were separated on SDS-PAGE (Fig. 2). A single band was detected at ~50 kDa, consistent with the expected molecular mass (MW) of the {alpha}CaMKII isoform. The 50 kDa band of ionomycin-activated eggs appeared to be slightly stronger (30%) than the one of non-activated MII eggs (Fig. 2a), maybe due to a better recognition between the antibody and the activated form of the protein. Anti-cdc2 p34 antibody served as a control for standardizing the amount of protein loaded on each lane. Staining intensity of bands indicated a similar amount of proteins in the lysates of non-activated and activated eggs (Fig. 2b). The experiment was repeated three times with similar results.


Figure 2
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  		Figure 2 Expression of {alpha}CaMKII in rat eggs. Eggs before or after parthenogenetic activation by ionomycin (2 µM; 20 min) were pooled, lysed and the proteins were separated on SDS-PAGE (300 eggs per lane). The proteins were immunoblotted with either anti-{alpha}CaMKII goat polyclonal IgG (a; 1:250) or with anti-cdc2 p34 rabbit polyclonal IgG (b; 1:1000). Secondary antibodies, peroxidase-conjugated donkey anti-goat IgG (1:2500) or goat anti-rabbit IgG (1:5000) respectively, were detected by an ECL detection system. Anti-cdc2 p34 antibody was used as a control for standardizing the amount of protein loaded on each lane. The arrow in (a) points at {alpha}CaMKII (50 kDa) and the arrow in (b) points at cdc2 p34 (34 kDa), as calculated from the migration of protein standards with known kDa. The 50 kDa band of ionomycin-activated eggs (Iono 20') appeared stronger than that of non-activated MII eggs (MII). At least three independent experiments were performed.

 
Microinjection of {alpha}CaMKII protein and cRNA into the eggs
Once the expression of the {alpha}CaMKII isoform in rat eggs was confirmed, the ability of CA-{alpha}CaMKII to induce CGE and RMII was examined by microinjecting MII eggs with an {alpha}CaMKII-overexpressed protein (0.56 mg/ml) or with rat CA-{alpha}CaMKII cRNA (0.5 mg/ml). We monitored the in vitro activity of the CA-overexpressed protein kinase by SignaTECT CaMKII assay system (Promega) and [{gamma}-32P]ATP (data not shown) as well as the ex vivo translation and distribution of the CA-{alpha}CaMKII cRNA, 3 h after microinjecting the eggs, in order to ensure that mutated proteins maintain their original intracellular localization (Hatch & Capco 2001). Control, non-injected MII eggs exhibited a very pale staining (Fig. 3B'), reflecting non-specific binding of the anti-FLAG antibody. wt (Fig. 3C') and CA (Fig. 3D') proteins were expressed throughout the egg cytoplasm with a marked concentration at the cortex. The developmental stage of the eggs had no effect on the subcellular distribution of the kinase (Fig. 3C' and D'). No fluorescence was observed in eggs incubated only in the presence of the second antibody (Fig. 3A').


Figure 3
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  		Figure 3 Ex vivo translation of the microinjected wt and CA cRNAs of {alpha}CaMKII. Eggs were microinjected with wt or CA cRNAs of {alpha}CaMKII and incubated for 3 h prior to fixation: control secondary antibody (A and A'), non-injected MII eggs (B and B'), eggs microinjected with wt {alpha}CaMKII cRNA (C and C'), and eggs microinjected with CA-{alpha}CaMKII cRNA (D and D'). Expression of injected {alpha}CaMKII was detected using anti-FLAG antibody (1:1000) recognized by Cy-3 secondary antibody (1:1000) and chromosomes were labeled by Hoechst 33342 (1 µg/ml). Anti-FLAG staining was imaged using CLSM. Anti-FLAG, red; chromosomes, blue. Light microscopy (A–D), confocal microscopy (A'–D'). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed (three to four eggs were measured for each group in each experimental day). Bar=10 µm.

 
The effect of CA-{alpha}CaMKII on CGE and RMII
We injected MII eggs with wt or CA-overexpressed {alpha}CaMKII proteins (0.56 mg/ml) and incubated the eggs for 1.5 h to allow recovery before monitoring RMII (Fig. 4a) and CGE intensity (Table 1). We also injected eggs with {alpha}CaMKII cRNAs (0.5 mg/ml), either wt or CA forms, allowed for a 3-h translation period and monitored RMII (Fig. 4b) and the CGE intensity (Table 1). Since CaMKII is known to induce RMII in eggs, the rate of RMII in eggs injected with CA-{alpha}CaMKII served as a positive control. We were able to induce RMII in 57% or 69% of the eggs by injecting the overexpressed CA-{alpha}CaMKII protein (Fig. 4a) or its cRNA (Fig. 4b) respectively. Injection of either wt {alpha}CaMKII protein or wt {alpha}CaMKII cRNA into the eggs induced significantly lower rates of RMII (35% and 27% respectively; P<0.01). Ionomycin or SrCl2 served as additional controls, inducing 86% or 83% RMII respectively (Fig. 4b).


Figure 4
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  		Figure 4 RMII induction by microinjected {alpha}CaMKII-overexpressed protein or cRNA. Eggs were microinjected with: overexpressed wt or CA-{alpha}CaMKII proteins (a), wt or CA-{alpha}CaMKII cRNAs or PBS (b). Eggs were fixed 1.5- or 3-h post-injection respectively, and chromosomes were labeled by Hoechst 33342 (1 µg/ml) to examine RMII occurrence. Eggs activated by ionomycin or SrCl2 served as positive controls. Each bar represents results from at least three experiments with 30 eggs at least in each treatment group. One-way ANOVA and Fisher's exact tests were performed to determine the significance of differences between treatment groups and the wt proteins or cRNA-injected eggs (*P<0.01, }RMII).

 

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  		Table 1 Cortical granules exocytosis (CGE) fluorescence intensity after microinjection with overexpressed calmodulin-dependent protein kinase II ({alpha}CaMKII) proteins or cRNAs.

 
We arbitrarily assigned the value of 1.00 to the CGE intensity of eggs injected with wt protein or wt cRNA. The CGE intensity of treated eggs was calculated relatively by calculating the ratio between the CGE intensity of each experimental group and that of wt-injected eggs. CGE in eggs subjected to ionomycin, SrCl2, or fertilization in vivo served as positive controls (1.92±0.04, 2.53±0.6, 2.57±0.2 respectively; Table 1). Untreated MII eggs exhibited a minimal degree of CGE (0.25±0.12, 0.27±0.11; Table 1). Eggs injected with either PBS, CA-overexpressed protein, or cRNA had CGE intensities (1.05±0.12, 0.93±0.13, or 1.08±0.09 respectively; Table 1) similar to that of wt-injected eggs (1.00; P>0.01), implying that CaMKII is not involved in CGE. Typical representative micrographs of CGE intensity for each treatment group are depicted in Fig. 5: non-injected MII egg (Fig. 5A and A'); wt-, CA-, or PBS-injected eggs (Fig. 5B, B', C, C', D, D' respectively), SrCl2-activated eggs (Fig. 5E and E') and in vivo-fertilized eggs (Fig. 5F and F'). We subjected eggs that had already been injected with wt or CA-{alpha}CaMKII cRNAs to 2 mM SrCl2 in order to find out whether the eggs had exhausted their CGE potential. We found (data not shown) an additive effect of SrCl2, on top of {alpha}CaMKII cRNA injection, regarding CGE. As mentioned earlier, CGE can be induced by activated PKC or by its downstream substrates, such as MARCKS. Next, we attempted to examine whether direct interfering with MARCKS will disrupt the ability of the eggs to undergo CGE and RMII.


Figure 5
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  		Figure 5 Representative micrographs of CGE intensity of the different treatment groups. Non-injected MII eggs (A and A'); wt, CA-{alpha}CaMKII cRNAs, or PBS-injected eggs (B and B', C and C', D and D' respectively); SrCl2-activated eggs (E and E') and in vivo-fertilized eggs (F and F'). Fixed eggs were labeled by LCA–biotin (1:100). LCA–biotin was detected by Texas Red Streptavidin (1:1000). CGE was imaged using CLSM. Light microscopy (A–F), confocal microscopy (A'–F'). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed. Bar=10 µm.

 
Involvement of MARCKS in egg activation
In a previous study, we have used biochemical approaches to provide evidence linking MARCKS translocation to CGE (Eliyahu et al. 2006). In all experiments conducted until now, no direct attempt to mutate or neutralize MARCKS was performed. An accepted method of demonstrating involvement of CaM in CGE via its direct binding with MARCKS would be to try and affect the occurrence of CGE by eliminating the CaM-binding properties of MARCKS. Since the CaM-binding sites are embedded within MARCKS effector domain, performing a mutation designated to hinder the CaM-MARCKS binding is a hard task. As a result, we chose to examine the effect of another direct mutation to the MARCKS protein (m3) on the ability of the eggs to undergo CGE. The three serine residues at the PKC phosphorylation site of MARCKS were replaced at the m3 mutant with alanine, thus preventing phosphorylation of MARCKS by PKC.

Microinjection of MARCKS cRNA into the eggs
We injected MII eggs with missense (ms), wt, or m3 MARCKS cRNAs, all carrying FLAG tag. Injected eggs were cultured for 3 h to allow protein translation before being monitored for CGE intensity and RMII. We monitored the ex vivo translation of the microinjected wt and m3 MARCKS cRNAs and the distribution of the translated protein and compared it with the intracellular localization of the endogenous protein (Eliyahu et al. 2006). Both wt- and m3-translated proteins were localized mainly on the plasma membrane as well as throughout the cytoplasm (Fig. 6C' and D'). Control, non-injected MII eggs and ms cRNA-injected eggs exhibited a pale fluorescence staining, reflecting non-specific binding of the anti-FLAG antibody (Figs 6A' and 7B' respectively).


Figure 6
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  		Figure 6 Ex vivo translation of the microinjected ms, wt, and m3 MARCKS cRNAs. Eggs microinjected with ms, wt, or m3 MARCKS cRNAs were fixed after 3 h: non-injected MII eggs (A and A'), eggs microinjected with ms cRNA (B and B'), with wt cRNA (C and C') and with m3 cRNA (D and D'). Expression of injected MARCKS was detected using anti-FLAG antibody (1:1000) recognized by Cy-3 secondary antibody (1:1000). Anti-FLAG staining was imaged using CLSM. Light microscopy (A–D), confocal microscopy (A'–D'). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed (three to four eggs were measured for each group in each experimental day). Bar=10 µm.

 

Figure 7
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  		Figure 7 The effect of m3 MARCKS cRNA on CGE intensity induced by TPA. Non-injected MII eggs (A and A'), non-injected TPA-activated eggs (B and B'), TPA-activated eggs microinjected with ms, wt, or m3 MARCKS cRNAs (C and C', D and D', E and E' respectively). Fixed eggs were labeled by LCA–biotin (1:100). LCA–biotin was detected by Texas Red Streptavidin (1:1000). CGE was imaged using CLSM. Light microscopy (A–E), confocal microscopy (A'–E'). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed. Results from representative experiment are presented.

 
The effect of m3 MARCKS on CGE
Eggs microinjected with ms MARCKS cRNA were activated 2.5 h later by 30 ng/ml TPA. CGE intensity of the injected eggs was arbitrarily assigned the value of 1.00 and the CGE intensity of other groups was calculated relatively. Untreated MII eggs exhibited a minimal rate of CGE (0.16±0.06). The rate of CGE intensity in TPA-activated eggs microinjected with m3 MARCKS cRNA was almost half (0.57±0.02) the intensity of TPA-activated eggs microinjected with either ms cRNA (1.00) or wt cRNA (1.01±0.12) or non-injected TPA-activated eggs (0.98±0.64). Typical representative micrographs of the CGE intensity in eggs of various treatment groups are depicted in Fig. 7: non-injected MII eggs (Fig. 7A and A'); non-injected TPA-activated eggs (Fig. 7B and B') and TPA-activated eggs microinjected with ms, wt, or m3 MARCKS cRNAs (Fig. 7C, C', D, D', E and E' respectively). These results suggest that phosphorylation of MARCKS by PKC is required for CGE to occur. Injections of the various MARCKS cRNA forms did not induce RMII in the eggs.

Two out of the three subfamilies of PKC: conventional PKCs (cPKCs) and novel PKCs (nPKCs) can be activated by TPA (Eliyahu & Shalgi 2002, Quan et al. 2003). In order to determine which subfamily induced MARCKS phosphorylation, thus enabling its translocation and causing CGE, we performed another set of experiments in which MARCKS cRNAs-injected eggs were activated by ionomycin. The CGE intensity of eggs injected with wt MARCKS cRNA was arbitrarily assigned the value of 1.00, whereas the CGE intensity of the other experimental groups was calculated relatively. The CGE intensity induced by ionomycin in eggs injected with m3 MARCKS cRNA was lower (0.58±0.16; P<0.01) than the one induced by ionomycin in eggs injected with wt MARCKS cRNA (1.00). Non-injected MII eggs had only 0.03±0.02 CGE intensity and non-injected ionomycin-activated eggs had a CGE intensity of 0.65±0.21. These results suggest that, in addition to nPKCs, cPKCs are also participating in the phosphorylation of MARCKS, leading to CGE.

The CGE intensity of MARCKS cRNA-injected eggs activated by either TPA or ionomycin imply involvement of nPKCs and/or cPKCs in MARCKS phosphorylation and translocation and hence in CGE in rat eggs. To establish a direct involvement of cPKCs in the pathway leading to CGE through MARCKS, we quantified the intensity of CGE induced by ionomycin (2 µM) in the presence of myristoylated PKC{psi} (myrPKC{psi}; 35 µM), a specific cPKC pseudosubstrate that served as a PKC inhibitor (Eliyahu & Shalgi 2002), and looked for inhibition of MARCKS translocation. myrPKC{psi} reduced CGE intensity to close to 50% (Fig. 8) and inhibited translocation of MARCKS from the membrane to the cortex in almost 50% of the eggs (data not shown). The CGE intensity in myrPKC{psi} treated eggs was 0.58±0.03 (P<0.01), whereas the CGE intensity in ionomycin-activated eggs was arbitrarily assigned the value of 1.00. Untreated MII eggs exhibited a minimal rate of CGE (0.1±0.07). These results support the hypothesis that cPKCs are responsible for almost half of the Ca2+-induced CGE, probably by causing MARCKS phosphorylation, on top of the Ca2+/CaM-induced MARCKS translocation.


Figure 8
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  		Figure 8 Effect of myrPKC{psi} on CGE in ionomycin-activated eggs. Eggs were fixed at MII stage (A and A') or after 5-min incubation in the presence of 2 µM ionomycin, followed by additional 15-min incubation in fresh medium lacking the activator (B and B'). Eggs were incubated for 5 min in the presence of 35 µM myrPKC{psi} and 2 µM ionomycin, followed by additional 15-min incubation in the presence of myrPKC{psi} (C and C'). Eggs were labeled with LCA–biotin (1:100). LCA–biotin was detected by Texas Red Streptavidin (1:1000). CGE was imaged using CLSM. Light microscopy (A–C), confocal microscopy (A'–C'). Images were taken at the equatorial plane of the eggs. At least three independent experiments were performed. Results from a representative experiment are presented.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The block to polyspermy, both at the plasma membrane and at the ZP levels, is imperative for preventing incorporation of more than one sperm into the egg at fertilization, (Sun 2003, Gardner & Evans 2006). Several studies reported that CGE, a major process in preventing polyspermy, can be triggered either by [Ca2+]i rise or by PKC activation (Gangeswaran & Jones 1997, Johnson & Capco 1997, Raz et al. 1998a,1998b, Luria et al. 2000, Pauken & Capco 2000, Eliyahu & Shalgi 2002). We have previously demonstrated that upon egg activation, MARCKS could either be phosphorylated by activated PKC or be bound by Ca2+-activated CaM (Eliyahu et al. 2005, 2006, Tsaadon et al. 2006). Each pathway can independently cause translocation of MARCKS and its disassembly from the actin filaments, culminating in CGE. In the current study, our first objective was to test whether these two pathways can compensate for one another. We subjected the eggs to OAG that activates both Ca2+ and PKC pathways, in the presence of W7, a CaM inhibitor, and followed translocation of MARCKS, CGE, and RMII. The observation that W7 inhibited neither MARCKS translocation nor OAG-induced CGE led us to conclude that the PKC pathway can compensate for lack of the Ca2+/CaM pathway. There are many potential proteins that may be involved in CGE. Several studies have indicated a role for CaM in promoting CGE in eggs (reviewed by Abbott & Ducibela 2001, Eliyahu et al. 2006). Other studies claim that CaMKII activation is involved in CGE (Markoulaki et al. 2003, Sun 2003, Knott et al. 2006). As mentioned in the Introduction section, CaMKII, like CaM, is localized at the egg cortex, especially at sites of exocytosis (Shen et al. 1998, Easom 1999) and was suggested as a participant in the signal pathway leading to CGE, on top of its involvement in promoting RMII. Although it is accepted that CGE is triggered by Ca2+ increase, via activation of CaM followed by activation of CaMKII, we could not rule out the possibility that CaM activation leads to a direct interaction with MARCKS, hence to actin reorganization and CGE. The involvement of CaMKII in egg activation events has been the topic of many studies conducted lately. While CaMKII involvement in promoting RMII is well established (Markoulaki et al. 2003, Sun 2003, Madgwich et al. 2005, Ito et al. 2006, Knott et al. 2006), its involvement in CGE is still controversial.

The {alpha}CaMKII isoform could not be detected in mouse eggs (Abbott et al. 2001) but, in the current study, we could show its expression in rat eggs. In order to determine the role of CaMKII in CGE, we monitored the ability of eggs injected with either protein or cRNA of overexpressed CA-{alpha}CaMKII to undergo CGE and RMII. Our observations, supported by the work of Knott et al. (2006), led us to suggest that CaMKII is not involved in the signal transduction pathway leading to Ca2+-induced CGE. Knott et al. (2006) tested the hypothesis that CaMKII is the major integrator of Ca2+-induced egg activation events, by microinjecting a CA-{alpha}CaMKII cRNA into mouse eggs. Expression of the mutant cRNA induced a sustained rise in CaMKII activity and triggered cell cycle resumption, though CGE appearance was abnormal. Since they did not report an injection of wt cRNA or any other substance that would serve as a control for isolating the effect of the injection itself, we cannot exclude the possibility that the mechanical process of injection may be the cause for the abnormal CGE. Their results correlate with our observation of a basal CGE intensity detected in all eggs microinjected. In our work, eggs injected with both CA- and wt-overexpressed proteins, as well as those injected with {alpha}CaMKII cRNAs were unable to undergo CGE, but were able to resume meiosis. Subsequent SrCl2 activation of the microinjected eggs triggered a maximum rate of CGE, manifesting that the eggs were capable of undergoing CGE. These findings rule out a possible involvement of CaMKII in promoting Ca2+-induced CGE, but support a major role for CaMKII in promoting RMII, probably by inactivation of both cytostatic factor (CSF) and maturation-promoting factor (MPF; review by Jones 2005). These results can indirectly support our hypothesis that Ca2+-activated CaM is not only CaMKII activator, but can also act as an active link participating in the pathway leading to CGE, probably by a direct binding to MARCKS and causing MARCKS translocation.

Two articles supporting our findings have been published lately; Ito et al. (2006) have shown that the CaMKII phosphorylation occurring during egg activation can be suppressed by KN93, a CaMKII inhibitor. They claimed that this phosphorylation is involved in inactivation of p34cdc2 kinase and degradation of Mos. In addition, the fact that accumulation and phosphorylation of CaMKII that occurred in eggs undergoing spontaneous activation could be inhibited by KN93 suggests an involvement of CaMKII in spontaneous egg activation (exiting from MII arrest) in rats. We have accumulating data (unpublished) showing that rat eggs undergoing spontaneous activation do not undergo CGE. Gardner et al. (2007) showed that injection of CA-{alpha}CaMKII cRNA was not sufficient for establishing the plasma membrane block to polyspermy in mouse eggs, though it induced resumption of cell cycle and a moderate CGE. However, treating the eggs with the specific CaMKII inhibitor (myrAIP) led to a decreased block to polyspermy. They concluded that although CaMKII participates in membrane block establishment, other factors are also needed for changing the egg membrane's receptivity to sperm after fertilization.

Our main focus in the current work was to study the role of Ca2+ and PKC in inducing CGE during egg activation, and attempt to resolve whether MARCKS plays a role in inducing CGE. We have previously suggested that MARCKS may play a role in cortical F-actin disassembly and mediating CGE in rat eggs (Eliyahu et al. 2005, 2006, Tsaadon et al. 2006). We have demonstrated that, as in secretory cells, phosphorylation of MARCKS by PKC or its binding to Ca2+/CaM, causes translocation of MARCKS and disrupts its role as a cross-linker of cortical actin filaments, leading to F-actin disassembly and allowing CGs to fuse with the egg plasma membrane and undergo exocytosis (Danks et al. 1999, Vaaraniemi et al. 1999, Wohnsland et al. 2000, Arbuzova et al. 2002, Eliyahu et al. 2005, 2006, Tsaadon et al. 2006). In the current work, we compared the CGE intensity of TPA-activated eggs injected with either unphosphorylatable MARCKS mutant cRNA, wt MARCKS cRNA, or ms MARCKS cRNA. According to our findings, phosphorylation of the plasma membrane-associated MARCKS protein is essential for exocytosis of the CGs contents, induced by either TPA or ionomycin. The CGE intensity of eggs injected with mutant-unphosphorylatable MARCKS was almost half the intensity of CGE in eggs injected with ms MARCKS cRNA, indicating that phosphorylation of MARCKS by PKC is a pivotal event associated with CGE. The partial CGE in these eggs could be due to the presence of endogenous MARCKS within the eggs that can be phosphorylated by PKC and to the fact that the mutant MARCKS did not lose its CaM-binding ability; thus CGE can be induced by the Ca2+/CaM pathway in the ionomycin-activated eggs. Our results imply involvement of MARCKS during early events of egg activation, such as CGE, but they do not exclude the possibility of MARCKS involvement during later events, such as PBII formation. Michaut et al. (2005) reported the expression of phosphorylated MARCKS (pMARCKS) in mouse eggs, describing pMARCKS as a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap overlaying the egg spindle. They suggested that this localization of pMARCKS implies its role in the formation of contractile apparatus during PB emission.

PKC isoforms are classified according to their cofactor requirements (Halet 2004). cPKCs are Ca2+ and DAG dependent and nPKCs are Ca2+ independent but require DAG for activation, whereas atypical PKCs (aPKCs) are neither Ca2+ nor DAG dependent. Microinjecting eggs with wt or m3 MARCKS cRNAs prior to activation by ionomycin assisted us in sorting out the PKC isoforms responsible for MARCKS phosphorylation. The reduced CGE intensity of m3 MARCKS cRNA-injected eggs compared with that of wt MARCKS cRNA-injected eggs led us to the conclusion that nPKCs and/or cPKCs are responsible for MARCKS phosphorylation and that cPKCs contribute to the Ca2+-induced MARCKS translocation. By exposing ionomycin-activated eggs to PKC{psi}, a specific cPKC inhibitor, we were able to indicate the involvement of cPKCs in CGE induction via MARCKS translocation. MARCKS translocation from the membrane to the cortex was inhibited in almost 50% of the eggs and the CGE intensity was reduced by close to 50%, as well.

The CGE intensity of TPA or ionomycin-activated eggs injected with m3 MARCKS was reduced by 50%. Matson et al. (2006) reported similar observations, proposing stimulation of the myosin light chain kinase (MYLK2) activity by elevated [Ca2+]i at fertilization of mouse eggs, resulting in activation of the myosin motor involved in CGs translocation. The authors used ML-7, a MYLK2 antagonist, that caused inhibition of PBII formation and reduced CGE intensity by half. According to these results, it is possible that MARCKS acts in concert with MYLK2 in the signaling pathway leading to CGE. CGE is an evolutionary developed mechanism that ensures successful egg activation and embryo development. It is well accepted that a process as important as this will be secured by several pathways and mechanisms. As summarized in the Introduction section, CGE can be triggered by Ca2+ increase and/or by PKC activation, whereas RMII can be triggered only by Ca2+ increase. Collectively, the above results present direct evidence demonstrating that MARCKS, not CaMKII, is the key regulatory molecule mediating CGE. Figure 9 depicts a flow chart of our view of the possible mechanism of CGE: CGE is mediated by hydrolysis of PIP2, two second messengers, IP3 and DAG, are generated; the former induces a rise in [Ca2+]i and the latter activates PKC. Two separate pathways, compensating for one another, can bring about MARCKS translocation and lead to CGE. One is the Ca2+-independent pathway that involves activation of nPKC, which in turn causes MARCKS phosphorylation via its specific PKC phosphorylation sites. The other is the Ca2+-induced pathway that involves activation of Ca2+/CaM and its binding to MARCKS, as well as MARCKS phosphorylation by cPKC, both leading to dissociation of MARCKS from F-actin, remodeling of the actin network, and subsequently CGE.


Figure 9
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  		Figure 9 Flow chart of a possible network for CGE. Upon sperm penetration, hydrolysis of PIP2 occurs. Two important second messengers are generated: DAG and IP3. DAG activates nPKCs that cause MARCKS phosphorylation. IP3 induces a rise in [Ca2+]i that causes activation of CaM and cPKCs, both leading to dissociation of MARCKS from F-actin, the former by a direct binding and the latter by phosphorylation. Both Ca2+-dependent and Ca2+-independent pathways lead to actin reorganization through the dissociation of the MARCKS–F-actin complex, culminating in CGE. We propose that Ca2+-activated CaMKII is involved in RMII but not in CGE. Solid arrows (----{blacktriangleright}) indicate commonly accepted pathways. Dashed arrows (- - - -{blacktriangleright}) indicate pathways demonstrated in our previous and present studies.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Animals
Wistar-derived rats were housed in air-conditioned, light-controlled rooms. The study was approved by the Institutional Animal Care and Use Committee.

Collection of eggs
MII-ovulated eggs
For induction of ovulation, 25- to 27-day-old immature female rats were injected with 10 IU human chorionic gonadotropin (hCG; Sigma), 48–54 h after administration of 10 IU pregnant mares serum gonadotropin (PMSG; Syncro-part; Sanofi, Paris, France). Rats were killed 14 h after hCG administration. Cumulus-enclosed MI eggs were removed from the oviductal ampullae into Toyoda HEPES (TH; Ben-Yosef et al. 1998) medium or into Ca2+- and Mg2+-free TH (TH–/–) medium, both supplemented with 0.4% BSA (fraction V, Sigma; Talmor et al. 1998). Cumulus cells were removed by a brief exposure to 400 IU/ml of hyaluronidase (Sigma).

In vivo-fertilized eggs
PMSG- and hCG-primed immature female rats were allowed to mate and were killed 16 h after hCG administration. Eggs and cumulus cells were treated as described above for MII eggs. Chromosomes were stained with Hoechst 33342 (Sigma) to determine fertilization status.

Parthenogenetic activation
MII-ovulated eggs were parthenogenetically activated by four different activators, all of which are capable of inducing CGE in rat eggs (Eliyahu & Shalgi 2002, Tomashov-Matar et al. 2005). MII eggs were incubated in:

  1. TH medium for 5 min in the presence of 50 µg/ml 1-oleoyl-2-acetylglycerol (OAG; Sigma), followed by an additional 5-min incubation in TH medium lacking the activator. Stock solution of 1 mg/ml OAG in dimethylsulfoxide (DMSO) was kept at –20 °C.
  2. TH–/– medium for 5 min in the presence of 2 µM calcium ionophore (ionomycin 407950; Calbiochem, San Diego, CA, USA), followed by an additional 45 min incubation in TH medium lacking the activator. Stock solution of 4 mM ionomycin in DMSO was kept at –70 °C.
  3. TH–/– medium for 15 min in the presence of 2 mM SrCl2 (Merck), followed by an additional 30-min incubation in TH medium lacking the activator. Freshly prepared 2 mM SrCl2 in TH–/– medium was used.
  4. TH medium for 5 min in the presence of 30 ng/ml TPA (Sigma), followed by an additional 15 min incubation in TH medium lacking the activator. Stock solution of 1 mg/ml TPA in DMSO was stored at –20 °C.

Inhibition of CaM and PKC
CaM inhibition
Eggs were incubated for 30 min in the presence of 20 µM W7 (Sigma; Eliyahu et al. 2006) prior to activation by OAG at the same regimen as described in the previous paragraph. Stock solution of 5 mM W7 in double distilled water (DDW) was kept at 4 °C.

PKC inhibition
Eggs were incubated for 5 min in the presence of 35 µM pseudosubstrate (myristoylated PKC{psi} amino acids 19–27; Biomol, Plymouth, PA, USA; Eliyahu & Shalgi 2002), together with 2 µM ionomycin, followed by an additional 15-min incubation in TH medium containing the inhibitor. Stock solution of 1 mM pseudosubstrate in DDW was kept at –20 °C.

Molecular cloning and sequence analysis
The full-length sequence of rat cDNA encoding {alpha}CaMKII wild type (wt) was generously provided by Prof. T Meyer (Stanford University, CA, USA). Human cDNA constructs of MARCKS/80K-L (wt) and mutant (m3) were generously provided by Prof. N Saito (Kobe University, Kobe, Japan).

The {alpha}CaMKII cDNA was digested with BamHI at the 5' end and with EcoRI at the 3' end; the resulting insert of ~1.5 kb was subcloned into several similarly digested plasmid vectors. For protein overexpression, the DNA insert was subcloned into pET-28a plasmid (Novagen, Merck) with an in-frame hexahistidine tag at the 3' end. For cRNA synthesis, the DNA insert was subcloned into pCMV-Tag 4A plasmid with an in-frame FLAG epitope at the 3' end (Stratagene, La Jolla, CA, USA). Site-directed mutagenesis was performed using the wt {alpha}CaMKII clone as a template for making a CA mutant of {alpha}CaMKII. The MARCKS cDNAs (wt and m3) were digested with EcoRI at both the 5' and 3' ends and were ligated into the same pCMV-Tag 4A plasmid, in the presence of Antarctic phosphatase that catalyzes the removal of 5' phosphate groups from DNA vector to prevent self-ligation. Both strands of ten independent colonies were sequenced and analyzed for open reading frame by MacVector 6.5 (Oxford Molecular, Oxford, UK).

Point mutation and cRNA synthesis
{alpha}CaMKII
Threonine at position 286 (T286) was substituted by aspartate to form CA mutant of {alpha}CaMKII (T286D). Aspartate at position 286 mimics the auto-phosphorylated form of {alpha}CaMKII that allows the enzyme to become CA, abrogating its requirement for Ca2+/CaM.

The amino acid substitution was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The sequence of primers used to make this mutation is shown below. The mutated codon is underlined.

Sense: 5'-GCACAGACAGGAGGACGTGGACTGCCTG-3'
Antisense: 5'-CAGGCAGTCCACGTCCTCCTGTCTGTGC-3'

MARCKS
In the mutant that we have received (m3), the three serine residues at the PKC phosphorylation site of MARCKS were replaced with alanine, making the mutant m3 unsusceptible to phosphorylation by PKC. A clone, in which the insertion was ligated backward (will be regarded as missense cRNA; ms) served as another control for injection.

Sequences were verified prior to synthesis of cRNAs. cRNAs were synthesized from linearized pCMV-Tag 4A CaMKII/MARCKS (Ribomax RNA synthesis; Promega) in the presence of 3 mM m7G(5')ppp(5')G (NEB; Ipswich, MA, USA), precipitated by isopropanol and resuspended in DEPC-treated water containing 4 U/µl RNasin (Promega).

Protein synthesis
Protein overexpression of wt and CA-{alpha}CaMKII subcloned into pET-28a was carried out in BL21 Escherichia coli cells. The expression of the His-tagged protein was induced with isopropyl β-D-thiogalactopyranoside (IPTG). Cells were collected by centrifugation and lysed in lysis buffer (0.3 mM Nacl, 50 mM Tris (pH 7.6), 1 mM EDTA, 10 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulphonyl fluoride, 30 µg/ml aprotonine). Cells lysate was sonicated and clarified by spinning out the insoluble fraction. Clarified lysate was mixed with Ni-NTA His-bind resins. Protein purification was performed on a column, using a Ni-NTA binding kit (Novagen) under denaturing conditions according to the manufacturer's instructions. The refolded His-tagged protein was then eluted and the main peak pooled. The protein was dialyzed (slide-a-laser; Pierce, Rockford, IL, USA) for purification and its concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories GmbH).

Egg microinjection
The various cRNAs were microinjected into MII eggs at a volume of 3–5% of the egg volume, using the FemtoJet microinjector (Eppendorf, Germany). wt or ms cRNAs or PBS served as controls.

Antibodies
Primary antibodies
Anti-MARCKS goat polyclonal IgG (N-19; sc-6454; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-{alpha}CaMKII goat polyclonal IgG (L-15; sc-5391, Santa Cruz Biotechnology), anti-cdc p34 rabbit polyclonal IgG (C-19; sc-954, Santa Cruz Biotechnology), and anti-FLAG mouse monoclonal IgG (F-1804, Sigma).

Secondary antibodies
Donkey anti-goat IgG peroxidase (705-035-147; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), goat anti-rabbit IgG peroxidase (111-035-003; Jackson ImmunoResearch Laboratories), donkey anti-goat IgG-Cy-3 (705-165-147; Jackson ImmunoResearch Laboratories), and donkey anti-mouse IgG-Cy-3 (715-165-151; Jackson ImmunoResearch Laboratories).

Western blot analysis
Lysis buffer was added to samples of 300 MII eggs collected in 5–10 µl of TH medium (Ben-Yosef et al. 1998). After two cycles of freezing/thawing, the extracts were stored at –70 °C. Proteins were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane (BioTrace NT; Gelman Sciences, Ann Arbor, MI, USA), using a wet blotting apparatus (Hoeffer, San Francisco, CA, USA). Blots were blocked by 5% dry milk in TBS (Talmor et al. 1998) and incubated overnight at 4 °C in the presence of anti-{alpha}CaMKII antibody (1:250) or anti-cdc p34 antibody (1:1000). Bound antibody was recognized by secondary anti-goat IgG antibody (1:2500) or anti-rabbit IgG antibody (1:5000) respectively, conjugated to horseradish peroxidase. Detection was performed by an ECL detection system (Pierce). Approximate molecular masses were determined by comparison with the migration of prestained protein standards (Amersham).

Immunofluorescence staining
Eggs were fixed in 3% paraformaldehyde, supplemented with 0.01% glutaraldehyde. ZPs were removed post-fixation by 0.25% pronase (Sigma). The plasma membrane was permeabilized with 0.05% NP-40. Membrane-permeabilized eggs were incubated in the presence of anti-MARCKS (1:50) or anti-FLAG (1:1000) antibodies. Primary antibodies were detected using fluorescent-labeled anti-goat or anti-mouse Cy-3 secondary antibodies (1:250 or 1:1000 respectively).

CGE quantification and DNA staining
Fixed eggs were labeled with 5 µg/ml Lens culinaris agglutinin (LCA)–biotin (B-1045; Vector, Burlingame, CA, USA), which binds specifically to CGs exudate (Cherr et al. 1988, Ducibella et al. 1988, Eliyahu & Shalgi 2002), washed, and labeled with 1 µg/ml Texas Red Streptavidin (SA-5006; Vector) for detection of CGE, and with 1 µg/ml Hoechst 33342 (Sigma) for determining cell cycle or fertilization status. Labeled eggs were visualized and photographed with a Zeiss confocal laser scanning microscope (LSM 410 CLSM; Zeiss, Oberkochen, Germany). The intensity of CGE staining was measured in one confocal slice depicting a mean CGE response of each egg. The staining intensity was calculated using the corrected mean density values obtained by the LSM software.

Statistical analysis
Data were evaluated by t-test, one-way ANOVA, or Fisher's exact test. Differences between treatment groups were determined by post hoc test. P<0.01 was considered significant.


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We gratefully thank Dr Leonid Mittelman for his excellent technical assistance at the confocal microscope, and Dr Yehudith Ghetler for providing us with microinjection capillaries. This work was partially supported by grants from the Israel Science Foundation (695/05) and the Ministry of Health to RS. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Received 12 December 2007
First decision 7 January 2008
Accepted 4 February 2008

   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

Abbott AL & Ducibella T 2001 Calcium and the control of mammalian cortical granule exocytosis. Frontiers in Bioscience 6 D792–D806.[Web of Science][Medline]

Abbott AL, Fissore RA & Ducibella T 2001 Identification of translocation deficiency in cortical granule secretion in preovulatory mouse oocytes. Biology of Reproduction 65 1640–1647.[Abstract/Free Full Text]

Arbuzova A, Schmitz AA & Vergeres G 2002 Cross talk unfolded: MARCKS proteins. Biochemical Journal 362 1–12.[CrossRef][Web of Science][Medline]

Ben-Yosef D, Talmor A, Shwartz L, Granot Y & Shalgi R 1998 Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg. Molecular Reproduction and Development 49 1–10.[CrossRef][Web of Science][Medline]

Capco DG 2001 Molecular and biochemical regulation of early mammalian development. International Review of Cytology 207 195–235.[Web of Science][Medline]

Cherr GN, Drobnis EZ & Katz DF 1988 Localization of cortical granule constituents before and after exocytosis in the hamster egg. Journal of Experimental Zoology 246 81–93.[CrossRef][Web of Science][Medline]

Danks K, Wade JA, Batten TFC, Walker JH, Ball SG & Vaughan PFT 1999 Redistribution of F-actin and large dense-cored vesicles in the human neuroblastoma SH-SY5Y in response to secretagogues and protein Kinase C activation. Molecular Brain Research 64 236–245.[Medline]

Ducibella T, Anderson E, Albertini DF, Alberg J & Rangarajan S 1988 Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation. Developmental Biology 130 184 –197.[CrossRef][Web of Science][Medline]

Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz RM, Kopf GS, Fissore R, Madoux S & Ozil JP 2002 Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number. Developmental Biology 250 280–291.[CrossRef][Web of Science][Medline]

Ducibella T, Schultz RM & Ozil JP 2006 Role of calcium signals in early development. Seminars in Cell and Developmental Biology 17 324–332.[CrossRef]

Easom RA 1999 CaM kinase II: a protein kinase with extraordinary talents germane to insulin exocytosis. Diabetes 48 675– 684.[Abstract]

Eliyahu E & Shalgi R 2002 A role for protein kinase C during rat egg activation. Biology of Reproduction 67 189–195.[Abstract/Free Full Text]

Eliyahu E, Tsaadon A, Shtraizent N & Shalgi R 2005 The involvement of protein kinase C and actin filaments in cortical granules exocytosis in the rat. Reproduction 129 161–170.[Abstract/Free Full Text]

Eliyahu E, Shtraizent N, Tsaadon A & Shalgi R 2006 Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs. Reproduction 131 221–231.[Abstract/Free Full Text]

Gallicano GI, Mcgaughey RW & Capco DG 1995 Protein kinase M, the cytosolic counterpart of protein kinase C, remodels the internal cytoskeleton of the mammalian egg during activation. Developmental Biology 167 482–501.[CrossRef][Web of Science][Medline]

Gangeswaran R & Jones KT 1997 Unique protein kinase C profile in mouse oocytes: lack of calcium-dependent conventional isoforms suggested by RT-PCR and western blotting. FEBS Letters 412 309–312.[CrossRef][Web of Science][Medline]

Gardner AJ & Evans JP 2006 Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm. Reproduction, Fertility, and Development 18 53–61.[CrossRef][Medline]

Gardner AJ, Knott JG, Jones KT & Evans JP 2007 CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs. Journal of Cellular Physiology 212 275–280.[CrossRef][Medline]

Halet G 2004 PKC signaling at fertilisation in mammalian eggs. Biochimica et Biophysica Acta 1742 185–189.[Medline]

Halet G, Tunwell R, Parkinson SJ & Carroll J 2004 Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilisation in mouse eggs. Journal of Cell Biology 164 1033–1044.[Abstract/Free Full Text]

Hatch KR & Capco DG 2001 Colocalization of CaM KII and MAP kinase on architectural elements of the mouse egg: potentiation of MAP kinase activity by CaM KII. Molecular Reproduction and Development 58 69–77.[CrossRef][Web of Science][Medline]

Ito J, Kaneko R & Hirabayashi M 2006 The regulation of calcium/calmodulin-dependent protein kinase II during oocyte activation in the rat. Journal of Reproduction and Development 52 439– 447.[CrossRef][Web of Science]

Johnson J & Capco DG 1997 Progesterone acts through protein kinase C to remodel the cytoplasm as the amphibian oocyte becomes fertilisation-competent egg. Mechanisms of Development 67 215–226.[CrossRef][Web of Science][Medline]

Jones KT 2005 Mammalian egg activation: from Ca2+ spiking to cell cycle progression. Reproduction 130 813– 823.[Abstract/Free Full Text]

Jones KT, Matsuda M, Parrington J, Katan M & Swann K 2000 Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs. Biochemical Journal 3 743–749.

Knott JG, Gardner A, Madgwick JS, Jones KT, Williams CJ & Schultz RM 2006 Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations. Developmental Biology 296 388–395.[CrossRef][Web of Science][Medline]

Luria A, Tennenbaum T, Sun QY, Rubinstein S & Breitbart H 2000 Differential localization of conventional protein kinase C isoforms during mouse oocyte development. Biology of Reproduction 62 1564 –1570.[Abstract/Free Full Text]

Madgwick S, Levasseur M & Jones KT 2005 Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs. Journal of Cell Science 118 3849–3859.[Abstract/Free Full Text]

Markoulaki S, Matson S, Abbott AL & Ducibella T 2003 Oscillatory CaMKII activity in mouse egg activation. Developmental Biology 258 464 – 474.[CrossRef][Web of Science][Medline]

Markoulaki S, Matson S & Ducibella T 2004 Fertilisation stimulates long lasting oscillations of CaMKII activity in mouse eggs. Developmental Biology 272 15–25.[CrossRef][Web of Science][Medline]

Matson S, Markoulaki S & Ducibella T 2006 Antagonists of myosin light chain kinase and of myosin II inhibit specific events of egg activation in fertilized mouse eggs. Biology of Reproduction 74 169–176.[Abstract/Free Full Text]

Michaut MA, Williams CJ & Schultz RM 2005 Phosphorylated MARCKS: a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs. Developmental Biology 280 26–37.[CrossRef][Web of Science][Medline]

Pauken CM & Capco DG 2000 The expression and stage-specific localization of protein kinase C isotypes during mouse preimplantation development. Developmental Biology 223 411– 421.[CrossRef][Web of Science][Medline]

Quan HM, Fan HY, Meng XQ, Huo LJ, Chen DY, Schatten H, Yang PM & Sun QY 2003 Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes. Zygote 11 329–337.[CrossRef][Web of Science][Medline]

Raz T, Ben-Yosef D & Shalgi R 1998a Segregation of the pathways leading to cortical reaction and cell cycle activation in the rat egg. Biology of Reproduction 58 94–102.[Abstract/Free Full Text]

Raz T, Eliyahu E, Yesodi V & Shalgi R 1998b Profile of protein kinase C isoenzymes and their possible role in mammalian egg activation. FEBS Letters 431 415– 418.[CrossRef][Web of Science][Medline]

Runft LL, Jaffe LA & Mehlmann LM 2002 Egg activation at fertilisation: where it all begins. Developmental Biology 245 237–254.[CrossRef][Web of Science][Medline]

Shen K, Teruel MN, Subramanian K & Meyer T 1998 CaMKII beta functions as an F-actin targeting module that localizes CaMKIIalpha/beta heterooligomers to dendritic spines. Neuron 21 593–606.[CrossRef][Web of Science][Medline]

Sun QY 2003 Cellular and molecular mechanisms leading to cortical reaction and polyspermy block in mammalian eggs. Microscopy Research and Technique 61 342–348.[CrossRef][Web of Science][Medline]

Talmor A, Kinsey WH & Shalgi R 1998 Expression and immunolocalization of p59c- 393 Fyn tyrosine kinase in rat egg. Developmental Biology 194 38 – 46.[CrossRef][Web of Science][Medline]

Talmor-Cohen A, Eliyahu E & Shalgi R 2002 Signaling in mammalian egg activation: role of protein kinases. Molecular and Cellular Endocrinology 187 145–149.[CrossRef][Web of Science][Medline]

Tatone C, Iorio R, Francione A, Gioia L & Colonna R 1999 Biochemical and biological effects of KN- 93, an inhibitor of calmodulin- dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol. Journal of Reproduction and Fertility 115 151–157.[Abstract/Free Full Text]

Tatone C, Monache SD, Caserta RID, Cola MD & Colonna R 2002 Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilisation Ca(2+) signal in mouse oocyte activation. Molecular Human Reproduction 8 750–757.[Abstract/Free Full Text]

Tatone C, Monache SD, Francione A, Gioia L, Barboni B & Colonna R 2003 Ca2+-independent protein kinase C signaling in mouse eggs during the early phases of fertilisation. International Journal of Developmental Biology 47 327–333.[CrossRef][Web of Science][Medline]

Tomashov-Matar R, Tchetchik D, Eldar A, Kaplan-Kraicer R, Oron Y & Shalgi R 2005 Strontium-induced rat egg activation. Reproduction 130 467– 474.[Abstract/Free Full Text]

Tsaadon A, Eliyahu E, Shtraizent N & Shalgi R 2006 When a sperm meets an egg: block to polyspermy. Molecular and Cellular Endocrinology 252 107–114.[CrossRef][Web of Science][Medline]

Vaaraniemi J, Palovuori R, Lehto VP & Eskelinen S 1999 Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity, the confluence, and transformation state of kidney epithelial cell lines. Journal of Cellular Physiology 181 83–95.[CrossRef][Web of Science][Medline]

Viveiros M, Hirao Y & Eppig JJ 2001 Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes. Developmental Biology 235 330–342.[CrossRef][Web of Science][Medline]

Wassarman PM, Jovine L & Litscher ES 2001 Profile of fertilisation in mammals. Nature Cell Biology 3 E59–E64.[CrossRef][Web of Science][Medline]

Wohnsland F, Schmitz AAP, Steinmetz MO, Aebi U & Vergeheres G 2000 Interaction between actin and the effector peptide of MARCKS-related protein. Identifcation of functional amino acid segments. Journal of Biological Chemistry 275 20873–20879.[Abstract/Free Full Text]


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J. Backs, P. Stein, T. Backs, F. E. Duncan, C. E. Grueter, J. McAnally, X. Qi, R. M. Schultz, and E. N. Olson
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