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RESEARCH |
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel
Correspondence should be addressed to R Shalgi; Email: shalgir{at}post.tau.ac.il
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Src family kinases (SFKs) have been suggested as possible inducers of some aspects of egg activation (see reviews by Kinsey 1997, Runft et al. 2002, Sato et al. 2000b). SFKs are the products of nine known genes: c-Src, c-Fgr, c-Yes, Fyn, Lck, Lyn, Hck, Blk and Yrk (Erpel & Courtneidge 1995, Superti-Furga & Courtneidge 1995, Bjorge et al. 2000, Schlessinger 2000). They fall into two classes: those found in a broad range of tissue types, namely c-Src, Fyn, c-Yes and Yrk, and those whose expression is restricted to cells of specific hematopoietic lineages, namely Blk, c-Fgr, Hck, Lck and Lyn. Current evidence indicates that the Ca2+ rise at fertilization in marine invertebrate eggs is initiated by a process that requires the sequential activation of SFK, PLC gamma (PLC
) and IP3 receptors at the endoplasmic reticulum (Carroll et al. 1999, Giusti et al. 1999a,b, 2000a, b, 2003, Abassi et al. 2000, Kinsey & Shen 2000, Runft & Jaffe 2000, Jaffe et al. 2001, Runft et al. 2002). In vertebrate eggs, the mechanism leading to IP3 production and to Ca2+ rise during fertilization is less established. The SFKs known to be expressed in vertebrate eggs are: c-Src kinase, c-Yes kinase and a 57 kDa Src-related kinase in Xenopus laevis (Schartl & Barnekow 1984, Steele et al. 1989, Sato et al. 1996); Fyn kinase in Xenopus, zebrafish, rat and mouse (Kinsey 1996, Talmor et al. 1998, Wu & Kinsey 2001, Sette et al. 2002).
There is evidence that SFK is activated within 30 s of insemination of zebrafish eggs (Wu & Kinsey 2001). A coimmunoprecipitation study has identified receptor-like protein tyrosine phosphatase-
(rPTP) as the phosphatase complexed with Fyn in the zebrafish egg, thus raising the possibility that rPTP is part of the regulatory mechanism responsible for activating Fyn at fertilization (Wu & Kinsey 2002). Other studies have indicated that an Src-related protein tyrosine kinase (PTK) may act upstream of the Ca2+ release during fertilization of frog eggs (Sato et al. 1999, 2001, Iwao 2000). Moreover, Src-related PTK-dependent PLC activity, that acts upstream of the Ca2+ rise is required for fertilization of Xenopus eggs (Sato et al. 2000a). The tyrosine kinase inhibitor lavendustin, inhibits Ca2+ release at fertilization of frog eggs (Glahn et al. 1999), as do two other SFK-specific inhibitors, the peptide A7 and the pharmacological inhibitor pyrazolopyrimidine (PP1; Sato et al. 1999, 2000a, Sato et al. b). However, since A7 and PP1 inhibit sperm–egg fusion as well, it is still debatable whether they inhibit egg activation or a former sperm-related function.
The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs.
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Materials and Methods |
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Oocytes resuming meiosis
Cumulus-enclosed oocytes at the germinal vesicle breakdown (GVBD) or metaphase I (MI) stages were isolated from antral follicles into culture medium (Toyoda Hepes (TH) medium supplemented with 0.1% BSA; Talmor et al. 1998), 4 or 8 h after hCG administration respectively. At all times, the medium temperature was kept at 36 ± 1 °C. All manipulations were performed on a warm plate (37 °C) placed in a temperature-controlled hood.
MII eggs
MII ovulated eggs were isolated, 14 h after hCG injection, from the oviductal ampullae into TH medium and their cumulus cells were removed by hyaluronidase (Sigma). The eggs were parthenogenetically activated by either calcium ionophore (ionomycin 407950, Calbiochem, San Diego, CA, USA) or phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; Sigma). Stock solutions of 10 mM ionomycin and of 1 µg/ml TPA in dimethyl sulfoxide (DMSO) were prepared and kept at 4 °C or at -20 °C respectively. MII eggs were incubated for 3 min in the presence of 2 µM ionomycin followed by an additional 0, 2, 7, or 17 min incubation in fresh medium lacking the activator. Other MII eggs were incubated for 5 min in the presence of 30 or 50 ng/ml TPA followed by an additional 0, 5, or 15 min incubation in fresh medium lacking the activator.
Fertilized eggs
Fertilized eggs at different stages of development were isolated either from the oviductal ampullae of superovulated immature females at various time points after mating, or from the oviducts of mated rats, 40 h after hCG injection (Kaplan-Kraicer et al. 1995). The developmental stages of zygotes were classified as: sperm binding (SB), fertilization cone (FC), second polar body (PBII) and pronuclear stage (PN) (at 0–0.25, 0.25–1, 1–3 or 6–8 h after sperm attachment respectively (Eliyahu & Shalgi 2002)), followed by mitosis and two-cell embryos.
Antibodies and drugs
Anti-Yes (sc-14), anti-Src (sc-19) and anti-Fyn (sc-16) rabbit polyclonal antibodies (pAb; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Src mouse monoclonal antibody (mAb; kindly contributed by Dr Joan S Brugge, Harvard Medical School, Boston, MA, USA), goat anti-rabbit immunoglobulin G (IgG) peroxidase (Sigma), donkey anti-rabbit IgG-Cy and donkey anti-mouse IgG-Cy (Jackson Immunoresearch Laboratories, West Grove, PA, USA), anti-ß-tubulin mAb (Sigma).
Stock solutions of 13 mM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d ]pyrimidine (PP2), PP3 and of 10 mM SU6656 (529573, 529574 and 572635 respectively; Calbiochem-Novabiochem, CA, USA) were prepared in DMSO and diluted before use in TH medium to final concentrations of either 10–100 µM (PP2, PP3) or 1–5 µM (SU6656).
SDS-PAGE and Western blot analysis of proteins
Samples of 100–300 eggs were collected in 1–3 µl TH medium, mixed with 10 µl of NP-40 lysis buffer (50 mM Tris, pH 7.4, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 5 mM NaF and 10 µg/ml aprotinin (Sigma)) and stored at -70 °C until use. Upon thawing, lysates of eggs were boiled for 5 min and subjected to separation by 10% SDS-PAGE under reducing conditions. Proteins were transferred onto a Millipore membrane (Millipore Co., Bedford, MA, USA) using a wet blotting apparatus (Hoeffer, San Francisco, CA, USA). Approximate molecular masses were determined by comparison with the migration of prestained protein standards (Amersham). Western blot analysis was performed with either anti-Yes IgG or anti-Src IgG at a dilution of 1:250 or anti-Fyn IgG at a dilution of 1:1000 in blocking buffer (100 mM NaCl, 10 mM Tris, 0.1% Tween 20 and 5% non-fat dry milk, pH 7.4). Bound antibodies were recognized by horseradish peroxidase conjugated to anti-rabbit antibodies (1:1000 for c-Yes and Fyn, and 1:5000 for Src). Detection was performed by an enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA).
Immunofluorescent staining for SFK localization
Eggs were fixed for 10 min at room temperature in 3% paraformaldehyde, supplemented with 0.01% glutaraldehyde, and then washed with Dulbecco’s phosphate-buffered saline (DPBS)/FCS solution (3% FCS in DPBS; Biological Industries, Beit-Haemek, Israel). After fixation, the zonae pellucidae (ZP) were removed by a brief exposure to 0.25% pronase (Sigma) prepared in DPBS/FCS solution and washed again. The plasma membranes of the eggs were permeabilized by 0.05% NP-40 in DPBS/FCS and then washed with 0.005% NP-40 in DPBS/FCS (blocking solution). All further manipulations were performed in blocking solution.
Eggs were incubated in the presence of specific primary antibodies for 1–2 h, washed in blocking solution and reincubated for 30 min in the presence of secondary, fluorescent, antibodies. SFKs were detected within the eggs by specific antibodies: anti-c-Src mAb and anti-c-Yes pAb (1:10); anti-Fyn pAb (1:100). Microtubules were detected by anti-ß-tubulin mAb (1:200). Primary antibodies were detected by fluorescent-labeled Cy secondary antibodies: donkey anti-rabbit IgG-Cy (1:1000) for pAb and donkey anti-mouse IgG-Cy (1:1000) for mAb. The chromosomal stage was detected by a DNA-specific fluorochrome (Hoechst 33342, Sigma).
For detection of CGE, fixed eggs were labeled by Lens Culinaris Aectin (LCA) and Texas-red streptavidin (Vector; Eliyahu & Shalgi 2002). Specificity of the antibodies was determined by incubating the eggs in the presence of secondary antibodies only.
Labeled eggs were visualized and photographed with a Zeiss confocal laser-scanning microscope. The Zeiss LSM 410 (Oberkochen, Germany) is equipped with a u.v. laser (Coherent Inc. Lazer Group, Santa Clara, CA, USA) and with a 25 mW krypton–argon laser and a 10 mW helium–neon laser (488, 543 and 633 maximum lines). A x 40 numerical aperture/1.2 planapochromat water-immersion lens (Axiovert 135 M, Zeiss) was used for all imaging.
Inhibition of SFK activation
To determine the effect of selective inhibitors of SFKs (PP2 and SU6656) on parthenogenetic activation, MII eggs were incubated for 30 min in the presence of 10–100 µM PP2 or 1–5 µM SU6656, then subjected to parthenogenetic activation by either ionomycin or TPA as described previously, followed by an additional 0–15 min incubation in the presence of the inhibitors only. Eggs were fixed and monitored for morphological criteria that indicate progression through the egg activation process: RM and CGE (see previous subsection). Eggs incubated in the presence of either PP3 (a negative control for PP2), DMSO or medium devoid of PP2, served as controls.
Statistical analysis
Data are expressed as the percentage of eggs which resumed meiosis or demonstrated CGE, per total number of treated eggs. The significance of differences between treated and control eggs was determined by one-way ANOVA; P < 0.01 was considered significant.
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Results |
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). We detected a c-Src band at 60 kDa (Fig. 1A), which is in keeping with the predicted relative molecular mass, Mr, of the protein (Steele 1985). A minor band of 200 kDa was often detected by this method as well. c-Yes appeared as a single band with an apparent Mr of 62 kDa (Fig. 1A) which is consistent with the reported Mr expected for c-Yes kinase (Zhao et al. 1990). Both SFKs were detected in Jurkat cells that served as positive control (Fig. 1B; c-Src 60 kDa, c-Yes 62 kDa). Our observations demonstrated, for the first time, the expression of c-Src and c-Yes in mammalian eggs. Fyn had already been demonstrated to be expressed in mammalian eggs at the predicted Mr of 59 kDa (Talmor et al. 1998).
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All three SFKs examined were found to be expressed in MII eggs and to be distributed throughout the egg cytoplasm (Fig. 2). c-Yes and Fyn tended to concentrate at the egg cortex (Fig. 2I, Unfertilized, B' and C'), while Fyn kinase is also localized to the meiotic spindle (Fig 2I, Unfertilized, C'). The subcellular distribution of the three kinases did not change upon fertilization, as can be seen at the SB (Fig. 2I, Sperm binding), FC (Fig. 2I, Fertilizing cone) or PBII stages. As a control, eggs were incubated in the presence of the second antibody only. As seen in Fig 2II, Control, no fluorescence was observed in control eggs.
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Ionomycin elevates [Ca2+]i in a manner that resembles the first [Ca2+]i transient of fertilization, and induces a full egg activation (Raz & Shalgi 1998). Initially, we established the dynamics of ionomycin-induced meiotic resumption. Non-activated MII eggs were exposed, in vitro, to 2 µM ionomycin for 1, 3, 5 or 10 min and then were further incubated in the absence of ionomycin (Fig. 3). Exposing the eggs to ionomycin for 1–10 min induced RM, although the 10 min exposure presented different dynamics of RM. A 1 min exposure to the activator plus an additional 9 min in fresh culture medium caused a transition of 76.5 ± 13.7% (mean±S.E.) of the eggs to the anaphase stage, as compared with 19.3 ± 5.6% (mean±S.E.) of the eggs that were exposed to the activator for 10 min (Fig. 3A). The results infer that a short exposure to ionomycin is advantageous. A 1 min exposure to ionomycin is too short for manipulation of a large number of eggs. We chose to expose the eggs to ionomycin for 3 min, as the Ca2+ transient within the eggs is already detectable after a 1–2 min exposure to 2 µM ionomycin (Raz & Shalgi 1998). Under these conditions (Fig. 3B), 91% of the eggs reached the anaphase stage within 15 min of culture (3 min exposure to ionomycin plus an additional 12 min incubation in fresh medium). After 30 min of culture (3 min exposure to ionomycin plus an additional 27 min), 41% were still at anaphase whereas 51% had already progressed to telophase and the rest extruded the PBII. After 45 min (3 min exposure to ionomycin plus an additional 42 min), the majority of eggs had already extruded PBII.
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To examine the possible role of SFKs in CGE and/or in RM we used SFK inhibitors (PP2 and SU6656). Exposing the eggs to 100 µM PP2 caused a dose-dependent inhibition of ionomycin-induced cell cycle resumption in 75% of the eggs, but had no effect on CGE (Fig. 4). PP3 had no effect on ionomycin-induced RM or CGE. In order to verify the effect of PP2 on egg activation we used another specific SFK inhibitor, SU6656 (Blake et al. 2000, Bowmann et al. 2001). Exposing the eggs to 5 µM SU6656 significantly inhibited ionomycin-induced cell cycle resumption, in a dose-dependent manner with no effect on CGE, compared with controls (Fig. 5).
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Discussion |
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Our study has demonstrated that c-Yes and c-Src are distributed throughout the egg cytoplasm, but c-Yes, like Fyn (Talmor et al. 1998), tends to concentrate at the egg cortex as well. The egg cortex is known to be rich in cortical structures such as actin cytoskeleton and cortical vesicles. Of the three SFKs studied, Fyn kinase is unique in that it is also localized to the meiotic and mitotic spindles (Talmor et al. 1998). Localization of c-Src, c-Yes and Fyn to different compartments within the egg indicates that these proteins may have different functions within the egg. No change in the subcellular distribution of the three kinases has been observed throughout the stages of the fertilization process, or after parthenogenetic activation. It is possible that the intracellular distribution of c-Src, c-Yes and Fyn imply their association with the cytoskeleton. SFKs have been shown to be associated with a wide range of cytoskeletal components and/or to phosphorylate them (Thomas & Brugge 1997). Microfilaments play a role in many dynamic events that take place during mammalian egg maturation and fertilization, such as sperm incorporation, CGE, PB emission, etc. These processes, among others, are accompanied by reorganization of the actin and tubulin cytoskeleton (Diamaggio et al. 1997). We may speculate that SFKs are involved in signaling events that implicate cytoskeletal reorganization via association with activated cytoskeletal proteins. The reorganization of the cytoskeleton could play a physiological role during fertilization. Although our findings may hint towards involvement of SFKs in reorganization of the cytoskeleton, further studies are needed to determine whether and how this might occur.
Fertilization is known to stimulate the SFKs in eggs of some vertebrates such as Xenopus and zebrafish (Sato et al. 1996, 1999, Wu & Kinsey 2001, 2002). Furthermore, SFKs inhibitors interrupt sperm–egg fusion in frogs (Sato et al. 1999, 2000a,b). In the mouse, several tyrosine kinase inhibitors delay Ca2+ release at fertilization and reduce the number of Ca2+ oscillations (Dupont et al. 1996). A recent study has demonstrated the existence of a sperm protein capable of inducing activation of a Src-like kinase in mouse eggs (Sette et al. 2002). However, Kurokawa et al. (2004, this issue) suggest that activation of an SFK is neither necessary, nor sufficient for triggering fertilization-induced [Ca2+]i oscillations.
To examine whether SFKs participate in the signal transduction pathways during egg activation, we have employed selective inhibitors of the SFKs activity. PP1 is a potent, selective inhibitor (Hanke et al. 1996) that has been used to study a number of Src-type mediated signaling processes in a variety of cells (Briddon et al. 1999, Mocsai et al. 1999). SU6656, a potent SFK specific inhibitor (Blake et al. 2000), inhibits c-Src (concentration giving 50% of maximum inhibition, IC50 = 280 nM) as well as the closely related kinases, Fyn (IC50 = 170 nM) and c-Yes (IC50 = 20 nM). Both inhibitors bind the kinase at its ATP binding site and insert a methylphenyl group into an adjacent hydrophobic pocket, thus inhibiting the kinase activity (Schindler et al. 1999, Bowman et al. 2001). Incubation of sea urchin eggs in 10 µM PP1 had resulted in a significant delay in Ca2+ release, implying the importance of SFKs for the initiation of Ca2+ release (Abassi et al. 2000). Recent evidence, from studies engaging domain-specific fusion proteins and constitutively active Src kinase mutants, have indicated a requirement for SFKs, such as c-Src or Fyn, for initiating Ca2+ release at fertilization of marine invertebrate eggs (Giusti et al. 1999b, 2000b, Kinsey & Shen 2000). PP2, similar to PP1, can selectively inhibit the Src-family tyrosine kinase activity (Hanke et al. 1996). An Src-like kinase inhibitor, PP2, has been demonstrated to suppress trkit-induced resumption of the cell cycle in mouse eggs (Sette et al. 2002). We found that PP2 and SU6656 inhibit RM, in a dose-dependent manner, in ionomycin-activated MII eggs, implying the dependency of RM, at least in part, on the activity of SFKs (Fig. 6).
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) at fertilization of the eggs of marine invertebrates, their role in mammalian eggs remains doubtful (Mehlman et al. 1998). PLC zeta (PLC
), a suspected sperm factor that has recently been discovered, initiates Ca2+ oscillations during mammalian egg fertilization (Cox et al. 2002, Saunders et al. 2002, Swann et al. 2004 (this issue)). The fact that PLC lacks Src-homology domains, implies that any involvement of SFKs in mammalian egg activation is downstream of Ca2+ release. We have demonstrated that SFKs play a role downstream of Ca2+ elevation leading to resumption of the cell cycle, but are not required for the completion of CGE. It has been shown that CGE can be triggered, independently, either by Ca2+ release or by PKC activation (Eliyahu & Shalgi 2002). In the current work, CGE could be induced either by TPA, which activates PKC (Nishizuka 1986), or by ionomycin, even in the presence of a SFKs inhibitors. These findings imply that under the conditions described, the CGE is not dependent on SFKs when it is triggered either by Ca2+ elevation, or by PKC (Fig. 6
). The possibility that SFKs play a role, at some step upstream of the Ca2+ transient, during egg activation by a spermatozoon, remains plausible (Fig. 6).
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Footnotes |
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This work was supported by a grant from the Israel Science Foundation to R. Shalgi.
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References |
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L. Meng, J. Luo, C. Li, and W. H Kinsey Role of Src homology 2 domain-mediated PTK signaling in mouse zygotic development. Reproduction, September 1, 2006; 132(3): 413 - 421. [Abstract] [Full Text] [PDF]
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L. M Mehlmann and L. A Jaffe SH2 domain-mediated activation of an SRC family kinase is not required to initiate Ca2+ release at fertilization in mouse eggs Reproduction, May 1, 2005; 129(5): 557 - 564. [Abstract] [Full Text] [PDF]
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A Talmor-Cohen, R Tomashov-Matar, W B Tsai, W H Kinsey, and R Shalgi Fyn kinase-tubulin interaction during meiosis of rat eggs Reproduction, October 1, 2004; 128(4): 387 - 393. [Abstract] [Full Text] [PDF]
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K Swann, M G Larman, C M Saunders, and F A Lai The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLC{zeta} Reproduction, April 1, 2004; 127(4): 431 - 439. [Abstract] [Full Text] [PDF]
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M. Kurokawa, K.-i. Sato, J. Smyth, H. Wu, K. Fukami, T. Takenawa, and R. A Fissore Evidence that activation of Src family kinase is not required for fertilization-associated [Ca2+]i oscillations in mouse eggs Reproduction, April 1, 2004; 127(4): 441 - 454. [Abstract] [Full Text] [PDF]
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); 3 min (
); 5 min (
); 10 min (x). (B) MII eggs were activated by ionomycin for 3 min, washed and cultured in vitro for additional periods of time to complete 10, 15, 30 or 45 min in culture. The y-axis indicates the percentage of eggs at: MII (
