Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Developmental Biology 322 (2008) 21–32

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / d eve l o p m e n t a l b i o l o g y

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos Anna Bielak-Zmijewska a,b,⁎, Agnieszka Kolano a, Katarzyna Szczepanska a, Marek Maleszewski a, Ewa Borsuk a a b

Department of Embryology, Institute of Zoology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland Laboratory of Molecular Bases of Aging, Department of Biochemistry, Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland

a r t i c l e

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Article history: Received for publication 15 February 2008 Revised 26 June 2008 Accepted 30 June 2008 Available online 9 July 2008 Keywords: Actin cytoskeleton Cleaving embryo Meiotic spindle Oocyte Maturation Cdc42 IQGAP1 Toxin B Cytokinesis Oogenesis

a b s t r a c t Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte. © 2008 Elsevier Inc. All rights reserved.

Introduction Cell polarization is crucial for morphogenesis and differentiation of many types of animal cells. It is critical for the formation and function of epithelial and neuronal cells, for the chemotactic reaction of individual cells or their growth in a defined direction, as well as for the asymmetrical divisions of female gametes. During meiotic divisions correct orientation of the meiotic spindle and its positioning near the oocyte cortex, as well as the proper segregation of chromosomes are necessary for the formation of functional haploid oocyte. The correct positioning of the spindle is a result of its migration to the oocyte cortex (Longo and Chen, 1985), but the mechanisms engaged in this process are still poorly understood. Several studies suggest that Rho GTPases, particularly Cdc42 (cell division cycle 42) protein, play a central role in establishing the polarity of the cell. It is also known that this protein is important for regulation of cell division. There are two potential functions of Cdc42 in cell division: regulation of chromosomes alignment and spindle ⁎ Corresponding author. Laboratory of Molecular Bases of Aging, Department of Biochemistry, Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland. Fax: +4822 8225342. E-mail address: [email protected] (A. Bielak-Zmijewska). 0012-1606/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2008.06.039

assembly (Oceguera-Yanez et al., 2005; Tatsumoto et al., 2003; Yasuda et al., 2004), and regulation of the formation of contractile ring through the regulation of actin cytoskeleton (Drechsel et al., 1997; Dutartre et al., 1996; Jantsch-Plunger et al., 2000; Ma et al., 2006; Merla and Johnson, 2000). First evidence of Cdc42 involvement in establishing the polarity of the cell came from the studies on Saccharomyces cerevisiae (Adams et al., 1990). Now it is known that also in the multicellular organisms the activation of Cdc42 is the key event leading to cell polarization (Etienne-Manneville and Hall, 2002; Schlessinger et al., 2007; Wu et al., 2007; Yamana et al., 2006). Rho GTPases are also required for many microfilament-dependent cellular processes, such as the regulation of endocytosis (Qualmann and Mellor, 2003), vesicle trafficking (Symons and Rusk, 2003), neuronal morphogenesis (Luo, 2000) and lymphocyte and fibroblast adhesion (Hall, 1998; Takaishi et al., 1995). There are also evidences that Cdc42 and Rac1 influence dynamics of microtubules (Grigoriev et al., 2006). There are some data suggesting that Rho GTPases, such as Cdc42, RhoA and Rac1, play also an important role in the polarization and asymmetrical cell division of vertebrate oocytes. For example, Cdc42 is involved in the polar body formation in Xenopus laevis oocytes. Inhibition of Cdc42 activity blocks the emission of polar body due to improper formation of a contractile ring (Ma et al., 2006). Cdc42 may

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also play a role in the asymmetric divisions in maturing mouse oocytes (Cui et al., 2007; Na and Zernicka-Goetz, 2006). Silencing of Cdc42 disrupts microtubules of the spindle as well as distribution of actin filaments and reduces the rate of first polar body extrusion (Cui et al., 2007). The expression of mutated form of Cdc42 protein totally inhibits the emission of the first polar body (Na and Zernicka-Goetz, 2006). The expression of both dominant-negative and constitutively active mutants of Cdc42 causes elongation of meiotic spindle and prevents its movement to the cortex of the oocyte. Na and ZernickaGoetz (2006) postulated that Cdc42 might participate in the organization and movement of the meiotic spindle in mouse oocytes. The elongation of the spindle and inhibition of polar body extrusion was also observed in mouse oocytes after the inhibition of Rac1 activity (Halet and Carroll, 2007). Rac1 is also involved in the regulation of spindle stability and its anchoring to the oocyte cortex (Halet and Carroll, 2007). Another Rho GTPase, RhoA, which regulates organization of microfilaments in mouse oocytes and embryos, influences rotation of the spindle and emission of the polar body, but not the organization of the spindle or chromosomes separation (Zhong et al., 2005). The establishment of cell polarity requires cooperation of many proteins. One of such proteins is IQGAP1 (IQ-domain GTPaseactivating protein 1). It is a conserved protein, engaged in the organization of microfilament and microtubule cytoskeleton during cell adhesion, migration and cytokinesis. There are some evidences suggesting that IQGAP1 is a scaffold protein that links components of different signaling cascades (Ho et al., 1999). IQGAP1 participates in several cellular signaling pathways by binding to selected partners such as F-actin (Bashour et al., 1997; Fukata et al., 1997), E-cadherin, βcatenin (Fukata et al., 1999; Kuroda et al., 1998; Shannon and Li, 1999), APC and CLIP-170 (Fukata et al., 2002; Yasuda et al., 2006). It interacts with Cdc42 and is involved in the processes leading to proper polarization of the cell (Noritake et al., 2005; Yasuda et al., 2006). IQGAP1 binds to Cdc42 and Rac1 in their active, GTP-bound form, and does not bind RhoA (Kuroda et al., 1996). IQGAP1 functions downstream or upstream of Cdc42, depending on the cell type or the type of signaling pathway. It increases the level of active Cdc42 during Cdc42dependent cell migration (Mataraza et al., 2003). On the other hand active Cdc42 is necessary to promote interaction of IQGAP1 with CLIP170 (Fukata et al., 2002). Until now nothing was known about the localization and function of IQGAP1 during maturation of mouse oocytes or in early embryonic development, and its possible interactions with Cdc42 during these events. In the present study we investigated the localization of Cdc42 and IQGAP1 proteins in oocytes and preimplantation embryos of the mouse. We have also examined the role of Cdc42 in establishing the polarity of the oocyte and its role during meiotic divisions of oocytes and mitotic cleavages of embryos and analyzed embryonic development after treatment with toxin B. Toxin B is an inhibitor of the binding activity of Rho family proteins such as Cdc42, Rac1 and RhoA (Just et al., 1995; Wex et al., 1997; Yasuda et al., 2004). We also studied the effect of toxin B treatment on the localization of IQGAP1. We showed that Cdc42 protein is localized on the microtubules of meiotic and mitotic spindles of oocytes and cleaving embryos. The localization pattern of IQGAP1 protein in mouse oocytes and cleaving embryos was highly dynamic and changed during growth and maturation of oocyte and embryo development. We showed that both proteins cooperate with each other in regulating the process of cytokinesis during meiotic and mitotic divisions, probably through the regulation of microfilament cytoskeleton. Our results indicate that in mouse oocytes and embryos IQGAP1 acts downstream of Cdc42. Materials and methods Approval for this study was obtained from the Local Ethic Committee No.1 in Warsaw (Poland). Oocytes and zygotes used in all

experiments described below were obtained from F1(C57Bl/6xCBA/H) female mice. Reagents and media All chemicals were obtained from Sigma-Aldrich (Poznan, Poland), unless stated otherwise. The oocytes and embryos were cultured in M2 medium (M16 medium buffered with HEPES; (Fulton and Whittingham, 1978). Oocytes obtained from 13-day-old females were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium; Invitrogen, Eugene, USA) supplemented with 10% FBS. Collection and culture of oocytes, parthenogenetic one-cell embryos, zygotes and cleaving embryos Growing oocytes Growing oocytes were obtained by puncturing the ovaries (isolated from 13-day-old females) in M2. The zonae pellucidae were removed by incubation of oocytes in 0.5% pronase in Ringer's solution for 10–20 min, at 37 °C with 5% CO2 in the air. Zona-free oocytes were fixed immediately and processed for immunostaining. To inhibit transcription, growing oocytes were cultured for 24 h in DMEM supplemented with 10% FBS and 10 µg/ml of α-amanitin. In each experiment, a control group of oocytes was cultured in the same medium without α-amanitin. After 24 h zonae pellucidae were removed and oocyte were fixed for immunocytochemistry. Fully-grown oocytes Fully-grown oocytes were obtained from the ovaries of 2–3month-old females injected with 10 IU of pregnant mares' serum gonadotrophin (PMSG, Intervet, Boxmeer, Netherlands) to stimulate the growth of the follicles. Forty eight hours after PMSG administration the ovaries were placed in M2 supplemented with dibutyryl cyclic AMP (dbcAMP; 150 µg/ml) to prevent spontaneous resumption of meiosis (Cho et al., 1974) and oocytes were released from the largest follicles by puncturing them with hypodermic needle. Isolated oocytes were placed in droplets of M2 or M2 + dbcAMP under mineral oil and were incubated at 37 °C with 5% CO2 in the air. Selection of SN oocytes Fully-grown GV oocytes were individually classified into SN (surrounded nucleoli) and NSN (nonsurrounded nucleoli) type (Debey et al., 1993; Zuccotti et al., 1995), according to the method described by Debey et al. (1989). Oocytes were cultured in droplets of M2+dbcAMP containing 2 ng/ml of Hoechst 33342 dye (Riedel-deHaen, Seelze, Germany) and after 30 min they were transferred into individual droplets of the same medium under mineral oil placed in glass bottom dish (Willco Wells B. V., Amsterdam, Netherlands). Classification of oocytes was performed under fluorescent microscope (Axiovert 135, Carl Zeiss, Jena, Germany) equipped with the UV attenuation filter block (Carl Zeiss, 0.03). To achieve non-harmful level of radiation, exposure of oocytes to the UV light lasted no longer than 0.5 s. Images were captured with CCD camera (Pixelfly, PCO, Kelheim, Germany). Only transcriptionally inactive SN oocytes (Bouniol-Baly et al., 1999) were used for microinjections. Metaphase II-arrested (MII) oocytes Oocytes arrested in MII were obtained from females induced to superovulate by injection of 10 IU of PMSG followed 48 h later by the injection of 10 IU of human chorionic gonadotrophin (hCG, Intervet, Boxmeer, Netherlands). MII oocytes were collected from the oviducts 16–17 h after hCG injection and treated with hyaluronidase solution (500 µg/ml in Ca2+/Mg2+-free PBS) to remove cumulus cells. Subsequently oocytes were washed in M2 and were either fixed for immunofluorescence or kept in M2 and used for parthenogenetic activation.

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Parthenogenetic one-cell embryos Ovulated MII-arrested oocytes (obtained as described above, 18 h after hCG injection) were incubated in 8% ethanol (POCh, Poland) in M2 for 8 min at room temperature (Cuthbertson, 1983; Cuthbertson et al., 1981) Subsequently, they were washed twice in M2 and cultured under standard conditions. They were fixed for immunostaining 40 min or 4 h after activation. Zygotes Zygotes (one-cell embryos) were obtained from females, which were induced to superovulate as described above and mated with F1 males immediately after hCG injection. Zygotes were collected from the oviducts 21, 26 or 28 h after hCG injection. Cumulus cells surrounding zygotes were removed by brief exposure to hyaluronidase and zygotes were stored in M2. Two-, four- or eight-cell embryos Females induced to superovulate were mated with F1 males. Zygotes, collected from the oviducts 24 h after hCG were cultured in vitro in M2 under standard culture conditions. Two-cell embryos were obtained after 18 h of culture and four-cell embryos after 42 h. Some females were sacrificed at specific time points after hCG injection in order to obtain cleaving embryos at desired stages of development. Oviducts were placed in M2 medium and cut into pieces and collected embryos were transferred to M2. Two-cell embryos were obtained 36–42 h, four-cells embryos 48–62 h and eight-cells embryos 63–67 h after hCG injection. Immunostaining Zonae pellucidae were removed from oocytes or embryos with 0.5% pronase in Ringer's solution or with acidic Tyrode's solution (pH 2.5; Nicolson et al., 1975). The oocytes and embryos were fixed and permeabilized as described by Szczepanska and Maleszewski (2005) and then they were incubated with the primary antibodies: antiCdc42 mouse monoclonal antibody or rabbit polyclonal antibody, anti-IQGAP1 rabbit polyclonal antibody (both diluted 1:50; Santa Cruz Biotechnology Inc., Santa Cruz, USA) and mouse monoclonal anti-β tubulin antibody (1:800; BD Pharmingen, Warsaw, Poland) in PBS containing 3% BSA and 0.05% Tween 20, overnight at 4 °C. After washing (1× in PBS and 2× in PBS containing 3% BSA and 0.05% Tween 20) oocytes and embryos were incubated with secondary antibodies: TRITC-conjugated goat anti-mouse IgG, FITC-conjugated donkey antirabbit IgG (both from Jackson Immunoresearch Laboratories, Bar Harbor, ME, USA) or Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen, Molecular Probes, Eugene, Oregon, USA) for 1 h, at room temperature and then washed three times in PBS. For chromatin visualization, cells were incubated in DRAQ5 (10 µM in PBS without Mg2+ and Ca2+ ions; Biostatus, Leicestershire, UK) for 10 min at 37 °C. Samples were analyzed with confocal microscope (LSM 510, Carl Zeiss, Jena, Germany). F-actin staining F-actin staining was performed using phalloidin conjugated with TRITC (final concentration 0.02 µg/ml). After fixation cells were incubated 30 min at room temperature in phalloidin solution, and then 10 min at 37 °C in DRAQ5 solution. Samples were then analyzed with confocal microscope as described above. Western blotting Protein lysates from oocytes and zygotes (900 GV oocytes, 900 MII oocytes and 900 zygotes for detection of Cdc42 and 180 GV oocytes and 180 MII oocytes, and 180 zygotes for detection of IQGAP1) were mixed with 4× NuPage LDS sample Buffer and 10× NuPage Sample

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Reducing Agent (Invitrogen, Carlsbad, CA, USA) and were heated for 10 min in 70 °C. The samples were subjected to NuPage Novex 10% Tris–Acetate gels in MOPS SDS Running buffer (Invitrogen, Carlsbad, CA, USA). Extracts from mouse embryonic fibroblasts and 13.5-day-old embryos were prepared according to Ciemerych et al. (2002). Separated proteins were transferred onto PVDF membranes (Hyperbond-P, Amersham Biosciences, Little Chalfont Buckinghamshire, UK). Cdc42 and IQGAP1 were immunodetected by incubating the membranes at 4 °C overnight with following primary antibodies: antiCdc42 rabbit polyclonal antibody (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, USA) and anti-IQGAP1 rabbit polyclonal antibody (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, USA). Subsequently a peroxidase-conjugated secondary antibodies goat anti-rabbit, (1:10 000, Pierce, Rockford, USA) were used. Detection was performed using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, USA). Detection of actin, with anti-β actin polyclonal rabbit antibody (1:1000, Santa Cruz Biotechnology Inc., Santa Cruz, USA) and secondary anti-rabbit antibody (1:10 000, Pierce, Rockford, USA) was used as a loading control. Microinjection of oocytes and zygotes with toxin B Fully-grown GV oocytes For intracytoplasmic injection of toxin B only the oocytes with SN chromatin configuration were used to avoid misinterpretation of the results, caused by less effective maturation of NSN oocytes (Zuccotti et al., 2002). The toxin B from Clostridium difficile (Calbiochem, Germany) in concentration 40 or 200 µg/ml (dissolved in the buffer containing 50 mM Tris pH 7.5, 50 mM NaCl and 5 mM UDP-glucose) was microinjected using Eppendorf microinjector (type 5242, Eppendorf AG, Hamburg, Germany) connected to Leitz micromanipulator (Leitz, Germany). Control oocytes were microinjected with buffer, which was used as a solvent for toxin B. During injections the oocytes were kept in M2 + dbcAMP to avoid resumption of meiosis. Injected oocytes were either cultured in the same medium for 18 h or were transferred into dbcAMP-free medium 1 h after toxin B injection, to allow meiotic maturation and then cultured for 18 h in M2. Zygotes Zygotes were microinjected with toxin B (200 µg/ml) 21, 26 or 28 h after hCG treatment, and cultured for 18 or 42 h. Injections and culture were performed in M2. Control zygotes were microinjected with buffer only and cultured in M2 for the same period of time. Results Verification of the specificity of primary antibodies To verify the specificity of the primary antibodies, the presence of Cdc42 and IQGAP1 in oocytes and zygotes as well as in 13.5-day-old mouse embryos and embryonic mouse fibroblasts was examined by Western blotting. The same antibodies were used for immunocytochemistry (see below) and for immunoblotting. Cdc42 Protein extracts from 13.5-day-old mouse embryos, embryonic mouse fibroblasts, GV oocytes, metaphase II oocytes and zygotes were examined. The anti-Cdc42 antibody recognized single 21 kDa protein band, which corresponds to predicted molecular weight of Cdc42 in control extract derived from embryonic fibroblasts and HCT116 (not shown). However, in protein extract obtained from 13.5-day-old mouse embryo two bands and in GV oocytes, metaphase II-arrested oocytes, and zygotes three bands were detected (Fig. 1A). This suggests that depending on the cell type more than one isoform of Cdc42 protein are present. Two murine isoforms of Cdc42, being a result of alternative splicing of a single gene product, have been already

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Fig. 1. Western blot analysis of Cdc42 and IQGAP1 proteins. (A) Cdc42 protein in: mouse embryonic fibroblasts (lane 1); metaphase II oocytes (lanes 2); GV oocytes (lane 3); zygotes isolated 26 h after hCG injection (lane 4); 13.5-day-old mouse embryos (lane 5); (B) IQGAP1 protein in: GV-arrested oocytes (lane 1); zygotes isolated 26 h after hCG injection (lane 2); metaphase II oocytes (lane3). Upper band corresponds to IQGAP1 and lower band to actin.

described in adult mouse tissues (Marks and Kwiatkowski, 1996; Miki et al., 1993). One of these isoforms, Cdc42a, is expressed in variety of tissues and the other, Cdc42b, is brain specific. Here we showed for the first time the presence of three isoforms of Cdc42 in mouse oocytes and zygotes. IQGAP1 Protein extracts from GV oocytes, metaphase II oocytes and zygotes were examined by Western blotting. Anti-IQGAP1 antibody recognized a protein of molecular weight about 190 kDa, which corresponds to predicted molecular weight of IQGAP1 and single 190 kDa band was detected in GV oocytes, metaphase II-arrested oocytes, and in zygotes (Fig. 1B). Localization of Cdc42 and IQGAP1 proteins Localization of Cdc42 The localization of Cdc42 protein was examined in following samples: • growing and fully-grown oocytes • maturing oocytes • one-cell embryos obtained after parthenogenetic activation of MII oocytes

• zygotes • two-, four-, and eight-cell embryos. Cdc42 in growing, fully-grown and maturing oocytes. In growing and in fully-grown NSN and SN type oocytes Cdc42 protein was visible in the oocyte cortex and undetectable in the cytoplasm (Fig. 2A). Two hours after germinal vesicle breakdown (GVBD) Cdc42 presence was detected as a diffused staining around condensing chromosomes (Fig. 2B), and 4 h after GVBD on the microtubules of forming meiotic spindle and in the oocyte cortex. This pattern of localization persisted through the metaphase of the first meiotic division (Fig. 2C). During anaphase I and telophase I, Cdc42 was observed in the equatorial region of the spindle (Fig. 2D, arrow) and around two separated groups of chromosomes. During metaphase of the second meiotic division Cdc42 was again observed on the microtubules of the meiotic spindle and in the oocyte cortex (Fig. 2E). Cdc42 in parthenogenetic one-cell embryos, zygotes and cleaving embryos. Parthenogenetically activated oocytes were used to examine the localization of Cdc42 during telophase of the second meiotic division. In early telophase II, 40 min after activation, Cdc42 was present on the meiotic spindle and in the oocyte cortex (Fig. 2F). After extrusion of the second polar body Cdc42 was observed on the remnants of the spindle visible in the polar body

Fig. 2. Localization of Cdc42 protein in fully-grown oocytes, during meiotic maturation and in one-cell embryos. Chromatin shown in red, Cdc42 shown in green. (A) Fully-grown SN oocyte; (B) maturing oocyte 2 h after GVBD; (C) metaphase I; (D) extrusion of the first polar body; (E) metaphase II; (F) parthenogenetic oocyte in telophase II; (G, H) zygotes during first mitotic division. Cdc42 protein is present in the cortex of GV oocytes (A) and after GVBD it localizes in the vicinity of condensing chromosomes (B). During first and second meiotic divisions Cdc42 accumulates on the spindle (C, E, F). In telophase of first and second meiotic division Cdc42 accumulates in the equatorial part the spindle (D, F, arrows). During the first mitotic division Cdc42 is observed on the microtubules of the spindle (G, H). During anaphase/telophase transition Cdc42 is also present in the equatorial part of the mitotic spindle (H, arrow). Scale bar: 20 µm.

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Fig. 3. Localization of IQGAP1 protein in growing and fully-grown NSN and SN oocytes and after inhibition of transcription in growing oocytes. Chromatin shown in red, IQGAP1 shown in green. (A, A′) growing oocyte isolated from 13.5-day-old mouse; (B, B′) fully-grown NSN oocyte; (C, C′) fully-grown SN oocyte; (D, D′) growing oocyte treated with αamanitin. (A–D) chromatin and IQGAP1, (A′, B′, C′, D′) IQGAP1 only. In all types of oocytes IQGAP1 is present in the cytoplasm and accumulates in the cortex (A, A′–C, C′). In transcriptionally active growing and fully-grown NSN oocytes IQGAP1 is present in the nuclei and forms a ring around nucleoli (A′, B′ — arrows). In fully-grown SN oocytes only the cytoplasmic and cortical signal of IQGAP1 is present. The cytoplasmic signal seems to be lower than in transcribing oocytes. In growing oocytes treated with α-amanitin chromatin is condensed around nucleolus and IQGAP1 is not visible in the nucleus (D, D′). The cytoplasmic signal of IQGAP1 is weaker than in transcribing oocytes (compare A′ and D′). Scale bar: 20 µm.

and in the oocyte cytoplasm in the vicinity of the polar body. Additionally Cdc42 was present in the cortex of oocyte and polar body (supplement Fig. S1). In zygotes in the interphase, Cdc42 was detected exclusively in the cortex. During the first mitotic division, Cdc42 was additionally localized on the microtubules of the mitotic spindle (Figs. 2G, H). In telophase the stronger signal of Cdc42 was observed in the equatorial part of the mitotic spindle (Fig. 2H, arrow). In two-,

four- and eight-cell embryos in interphase Cdc42 was visible exclusively in the cortex (not shown). Localization of IQGAP1 The localization of IQGAP1 was examined in: • growing and fully-grown GV oocytes • maturing oocytes • one, two-, four- and eight-cell embryos.

Fig. 4. Localization of IQGAP1 protein in maturing oocytes, in parthenogenetically activated oocytes, zygotes and cleaving embryos. Chromatin shown in red, IQGAP1 shown in green. (A) GV oocyte SN type; (B) 2 h after GVBD; (C) oocyte during extrusion of the first polar body; (D) metaphase II; (E) parthenogenetically activated oocyte extruding the second polar body; (F) zygote in interphase. 2 pb — second polar body; fPNu —female pronucleus; male pronucleus not visible (located at different focal plane); (G) early 2-cell embryo; the cytokinesis is not finished and the contractile ring is still visible. (H) late 2-cell embryo; (I) four-cell embryo (one blastomere not visible, located at different focal plane); (J) eight-cell embryo (only four blastomeres well visible, the other are located at different focal plane). In GV oocytes IQGAP1 protein localizes in the cortex of the oocytes and is also present in the cytoplasm (A). After GVBD, the level of IQGAP1 protein in the cytoplasm and cortex decreases (B). During extrusion of the first polar body IQGAP1 is present in contractile ring (C, arrow) but not in cortex. In metaphase II oocytes IQGAP1 is visible only in the cytoplasm (D). During the extrusion of the second polar body IQGAP1 is localized in the contractile ring (E, arrow). In zygotes during interphase IQGAP1 is localized at the contact site between the zygote and second polar body (F, arrow). During mitotic cytokinesis IQGAP1 is localized in the contractile ring (G). In two-, four- and eight-cell embryos IQGAP1 protein is present in the cytoplasm of the blastomeres and it is localized at the cell–cell contact sites (H–J, arrows). In two-cell embryos nuclear localization is absent (H, insert), while in four- and eight-cells embryos IQGAP1 accumulates around the nucleoli (I, J, inserts — arrowheads). Scale bar: 20 µm.

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IQGAP1 localization in growing, fully-grown and maturing oocytes. In oocytes in the middle of growth period, obtained from 13-day-old females, strong cytoplasmic, and evenly distributed cortical signal of IQGAP1 protein was observed. Additionally, IQGAP1 was found in the oocyte nucleus, forming a ring surrounding the nucleolus (Fig. 3A′, arrow). The same distribution of IQGAP1 was also observed in transcriptionally active fully-grown oocytes (NSN type oocytes) (Fig. 3B′, arrow). In fully-grown oocytes of SN type (Fig. 3C), in which transcription has been already silenced, and in which the majority of the chromatin in the germinal vesicle was condensed and formed characteristic rim surrounding the nucleolus, distribution of IQGAP1 differed from that observed in growing and NSN oocytes. Cytoplasmic signal of the protein was significantly diminished, and there was no visible staining around nucleoli. The intensity of the signal of IQGAP1 in the cortex of the SN oocytes was similar to that in growing and fullygrown NSN oocytes (Fig. 3C′). The fact that IQGAP1 is visible around the nucleolus in transcriptionally active oocytes and vanishes in transcriptionally silent oocytes suggests a correlation between its presence and RNA synthesis. To verify this hypothesis, the transcription in growing oocytes was inhibited by treatment with α-amanitin. Inhibition of RNA synthesis in these oocytes resulted in condensation of their chromatin (compare Figs. 3A and D), similar to that observed in fully-grown SN oocytes. The signal of IQGAP1 in the cytoplasm was much lower than in control growing oocytes (non-treated with α-amanitin), and there was no signal around nucleoli (Fig. 3A′ arrow and D′). In maturating oocytes IQGAP1 disappeared from the cortex and remained dispersed in the cytoplasm (compare Figs. 4A and B; negative control is shown in supplement, Fig. S2). Interestingly, during cytokinesis of the first meiotic division IQGAP1 was concentrated at the region corresponding to the contractile ring (Fig. 4C). This signal weakened in the final steps of the polar body extrusion and subsequently the signal was visible only at the contact site between the oocyte and the first polar body (not shown). In oocytes arrested at the metaphase of the second meiotic division IQGAP1 was present in the cytoplasm. The signal of IQGAP1 in the cortex was very weak (Fig. 4D). IQGAP1 localization during second polar body extrusion, during interphase in zygotes and in cleaving embryos. In order to follow the localization of IQGAP1 during second polar body extrusion the ovulated oocytes were activated parthenogenetically. Forty minutes after oocyte activation (telophase II) IQGAP1 started to accumulate at the region corresponding to the contractile ring (Fig. 4E, arrow). However, during subsequent stages of the second polar body extrusion this staining became weaker (not shown) and finally IQGAP1was present only at the contact site between activated oocyte and the second polar body (which was similar to its localization pattern after first polar body extrusion). In zygotes with visible pronuclei IQGAP1 protein was distributed evenly in the cytoplasm and was concentrated at the contact site Table 1 Localization of IQGAP1 protein in growing, fully-grown and maturing oocytes and in one-, two-, four-cell embryos IQGAP1 localization

Growing oocytes

Cytoplasm Cortex Nucleus (around nucleoli)

+++ ++ +

Fullygrown oocytes

Maturing oocytes

Embryos

NSN

SN

GVBD

MII

1-cell

2-cell

4-cell

+++ ++ +

++ ++ −

+ + −

+ +a −

+ +b −

+ +c −

+ +c +

“+” reflects the relative strength of the signal. a In the contractile ring and at the contact site between the oocyte and the first polar body. b At the contact site between the zygote and the second polar body. c At the contact site between blastomeres.

Fig. 5. Effect of toxin B on oocyte maturation. (control) non injected oocytes; (buffer) oocytes injected with buffer used to dissolve toxin B; (ToxB 40) oocytes injected with toxin B in concentration of 40 µg/ml; (ToxB 200) oocytes injected with toxin B in concentration of 200 µg/ml. (GV) oocytes arrested at the GV stage; (GVBD) oocytes which have undergone GVBD; (Ipb) oocytes which extruded first polar body. Number of oocytes in each group: ToxB 40 n = 115; ToxB 200 n = 53; buffer n = 60; control n = 132.

between the zygote and the second polar body (Fig. 4F). During first mitotic division IQGAP1 was found at the region corresponding to the contractile ring (Fig. 4G). In the two-cell embryos (46–48 h after hCG administration, S/G2 phase of the second cell cycle), besides being accumulated in the cytoplasm, IQGAP1 was also present at the blastomeres contact site (Fig. 4H; arrow). In four- and eight-cell embryos this protein was detected in the cytoplasm and at the contact sites between blastomeres (Figs. 4I, J; arrows), and also in the nuclei of blastomeres, where it formed a rim around nucleoli, similar to that observed in transcribing ovarian oocytes (Figs. 4I, J, and inserts; arrowheads). Localization of IQGAP1 did not change during the compaction taking place in the eight-cell embryos. In both, noncompacted (early) and compacted (late) eight-cell embryos the localization and the intensity of the fluorescent signal, indicating the presence of IQGAP1 was similar (not shown). Table 1 summarizes the localization pattern of IQGAP1 observed in growing, fully-grown and maturing oocytes and in early embryos. Specificity of anti-Cdc42 and anti-IQGAP1 antibodies was additionally confirmed by immunocytochemistry performed on somatic HCT116 cells (see Supplementary data). Inhibition of the binding activity of Cdc42 Toxin B was used to inhibit binding activity of Cdc42 in oocytes and zygotes. Cdc42, similarly to all Rho GTPases, cycles between inactive, GDP-bound, and active, GTP-bound form. Toxin B is a glycosyltransferase that inactivates Rho GTPases via transfer of a sugar. Glucosylated proteins are present in the cell in GDP form only, and they do not have binding activity (Just et al., 1995; Voth and Ballard, 2005). The effect of toxin B on meiotic maturation of oocytes Toxin B (in concentration 40 and 200 µg/ml) was microinjected into the cytoplasm of GV oocytes of SN type. At these doses, the lethality of Toxin B treated and control (injected with buffer) oocytes was comparable. To analyze the effect of toxin B on the process of meiotic maturation, 1 h after the microinjection oocytes were transferred into dbcAMP-free medium and cultured in vitro for 18 h. This period of time was sufficient for control oocytes to finish the first meiotic division and extrude the first polar body. Toxin B in concentration of 40 µg/ml had almost no effect on meiotic maturation of oocytes (Fig. 5). About 80% of injected and control oocytes extruded first polar body. However, toxin B in concentration of 200 µg/ ml effectively inhibited first polar body extrusion. More than 90% of injected oocytes did not finish the first meiotic division (Fig. 5). However, in all these oocytes the meiotic spindle was formed properly and its migration to the oocyte cortex was unaffected.

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Fig. 6. Actin cytoskeleton in oocytes treated with toxin B. Chromatin shown in red, actin shown in green. (A) control GV SN oocyte; (B) control oocyte in metaphase II; (C, D) oocyte, injected at GV stage with toxin B in concentration 40 µg/ml, which completed maturation after 18 h of culture (MII); (E) GV oocyte, treated with 200 µg/ml of toxin B and cultured in the presence of dbcAMP for 18 h; (F) oocyte injected at GV stage with toxin B in conc. 200 µg/ml, which didn't extruded first polar body after 18 h of culture. In control, untreated GV (A) and metaphase II-arrested (B) oocytes actin is accumulated in the cortex. In metaphase II oocytes actin additionally forms a cap above the meiotic spindle, and is also visible in the cortex of the first polar body. In oocytes treated with low dose of toxin B (40 µg/ml) cortical actin is absent, but actin cap and actin in the first polar body is still present (C, D). In GVarrested oocytes treated with high dose of toxin B (200 µg/ml) actin is present in the cytoplasm and the signal of cortical actin is diminished and irregular in comparison with control oocyte (compare: A and E). In oocytes which did not complete the first meiotic division, after treatment with toxin B in conc. 200 µg/ml, neither cortical actin, nor the actin cap are visible. Signal in the cytoplasm suggests the presence of cytoplasmic pool of actin (F). Scale bar: 20 µm.

The effect of toxin B on microfilament cytoskeleton and Cdc42 localization in maturing oocytes. To verify whether microinjection of toxin B affected the microfilament cytoskeleton, the distribution of actin was analyzed in oocytes with intact GV (GV oocytes) and in maturing oocytes. Staining of F-actin with phalloidin (Wulf et al., 1979) showed, that in control GV oocytes cortical actin was distributed uniformly (Fig. 6A). In MII oocytes F-actin was present in the cortex of oocyte and first polar body and was also concentrated (in the form of a cap) above the spindle (Fig. 6B). F-actin was not detected in the cytoplasm of GV or MII control oocytes. Treatment with toxin B caused an intense rearrangement of cortical actin. Toxin B in concentration of 40 µg/ml induced disappearance of cortical actin in MII oocytes, but actin cap was still present above the spindle (Figs. 6C and D), and accumulation of F-actin was still observed in the cortex of the first polar body (Fig. 6C). F-actin was undetectable in the cytoplasm of these oocytes. At this concentration, toxin B did not disrupt the organization of meiotic spindle or did not change the localization of Cdc42 which, as in control oocytes, was present on the microtubules of the spindle (not shown). Treatment with toxin B in concentration of 200 µg/ml caused more profound changes in the arrangement of actin cytoskeleton. Cortical actin and actin forming the cap over the spindle disappeared (Figs. 6E and F). Instead, uniform staining, suggesting the presence of some

amount of F-actin, was observed in the cytoplasm. Such staining was never observed in control oocytes. The staining of β-tubulin revealed the changes in the shape of the first meiotic spindle (Fig. 7A). However, the localization of Cdc42 protein remained unchanged. (Fig. 7B). The effect of toxin B on IQGAP1 localization in maturing oocytes. As it was described above, in control GV oocytes strong signal of IQGAP1 was observed in the cortex and in the cytoplasm. During meiotic maturation the protein gradually disappeared from the cortex. During emission of the first polar body it localized at the region corresponding to the contractile ring, and finally in MII oocytes IQGAP1 was almost undetectable (see: Fig. 4D). In oocytes treated with toxin B (in concentration of 200 µg/ml), and allowed to mature, IQGAP1 also disappeared from the cortex but the intensity of cytoplasmic signal was lower than in control oocytes. Surprisingly, in those oocytes treated with toxin B, which achieved the metaphase II stage, IQGAP1 accumulated on the meiotic spindle (Fig. 7C), This was never observed in the metaphase II-arrested control oocytes. To verify, whether the disappearance of cortical localization of IQGAP1 in toxin B treated and maturing oocytes was the effect of inhibition of Cdc42 activity or only the result of oocyte maturation, the GV-intact oocytes injected with toxin B were cultured for 18 h in the

Fig. 7. Changes in the morphology of the meiotic spindle and the effect of toxin B on the localization of Cdc42 and IQGQP1. Chromatin shown in red, β-tubulin, Cdc42, IQGAP1 shown in green. (A) tubulin; (B) Cdc42; (C) IQGAP1. The shape of meiotic spindle in oocytes treated with toxin B and allowed to mature, is changed and exhibit “blunt-ended” poles (A). Cdc42 protein (B) as well as IQGAP1 (C) accumulate on the microtubules of the spindle. Scale bar: 20 µm.

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Fig. 8. Changes in the localization of IQGAP1 protein in oocytes treated with toxin B and arrested in GV stage. Chromatin shown in red, IQGAP1 shown in green. Control and injected with toxin B (200 µg/ml) oocytes were cultured in the presence of dbcAMP. (A) Control GV oocyte cultured for 18 h; (B) GV oocyte treated with toxin B and cultured for 4 h; (C) GV oocyte treated with toxin B and cultured for18 h. In control oocytes IQGAP1 is accumulated in the cortex and is present in the cytoplasm (A). In oocytes treated with toxin B and cultured for 4 h (B) cortical localization of IQGAP1 is irregular (B), and after 18 h of culture some regions of the oocyte cortex are totally deprived of IQGAP1 (C, arrows). Scale bar: 20 µm.

presence of dbcAMP. In control, GV-intact oocytes cultured under the same conditions, localization and intensity of the fluorescent signal of IQGAP1 were similar to observed in freshly obtained oocytes (Fig. 8A). In contrast, in oocytes treated with toxin B, already after 4 h of culture the distribution of IQGAP1 in the oocyte cortex was aberrant (Fig. 8B). After 18 h of culture some areas of oocyte cortex were completely lacking of IQGAP1 (Fig. 8C, arrows). The effect of toxin B on mitotic divisions The zygotes were injected with toxin B (conc. 200 µg/ml) 21, 26 or 28 h after hCG administration and cultured in vitro to follow their ability to undergo cleavage divisions. Results of this experiment (Fig. 9) clearly showed the correlation between the age of zygotes (the phase of the first cell cycle) at the moment of toxin B injection and the frequency of first cleavage. None of the zygotes injected at G1 phase of the first cell cycle (21 h after hCG) cleaved to the twocell stage. When microinjection of toxin B was performed few hours later, at late S/early G2 phase, or at G2 phase of the first cell cycle (26 or 28 h after hCG, respectively), 60% to 80% of zygotes reached the two-cell stage. However, none of these two-cell embryos cleaved again. Meanwhile, 98% of control embryos reached the four-cell stage (Fig. 9). The morphology of zygotes arrested at one-cell stage after toxin B treatment was analyzed after 18 h of culture, when control embryos reached already the two-cell stage. The analysis revealed the presence of two nuclei residing in the cytoplasm of such embryos (Fig. 10B). However, these nuclei differed from the male and female pronuclei present in the cytoplasm of control zygotes (Fig. 10A), where the pronuclei were big and round, with highly dispersed chromatin, without the heterochromatin areas. Often, in late zygotes only one, large nucleolus-like body (Kopecny et al., 1989) was present in each pronucleus (Fig. 10A). The nuclei present in toxin B treated zygotes (Fig. 10B) were less round, visibly heterochromatic and contained few nucleoli. Such morphology is typical for the nuclei of control twoblastomere embryos (Fig. 10C). This observation suggested, that in this group of toxin B treated zygotes the karyokinesis of the first cleavage was successfully finished while the cytokinesis was inhibited. Two-cell embryos, derived from zygotes treated with toxin B 26 or 28 h after hCG, did not differ morphologically from control embryos cultured for 24 h (compare Figs. 10C and D). However, 48 h after microinjection, when control embryos already reached the four-cell stage (Fig. 10E), experimental embryos were still at the two-cell stage (Fig. 10F). Two nuclei were present in each blastomere of these arrested embryos. This phenotype was similar to that observed in embryos blocked at one-cell stage after toxin B treatment, and suggested that karyokinesis of the second mitotic division was successfully finished, while the cytokinesis had not occurred. Additionally, we observed that after zonae pellucidae removal the sister blastomeres of arrested two-cell embryos had the tendency to separate from each other. In contrast, sister blastomeres of control

two-cell embryos always stayed attached to each other. This implies the impairment of blastomere adhesion in toxin B treated embryos. The effect of toxin B on microfilament cytoskeleton and Cdc42 distribution. Results presented above showed that toxin B affected the microfilament cytoskeleton of cleaving embryos. Embryos derived from zygotes injected with toxin B, showed pronounced changes in F-actin distribution. They lacked of the typical cortical layer of F-actin, present in control zygotes or embryos (Figs. 10A, C and E). Instead F-actin formed irregular patches scattered randomly in the cortex. In addition, in contrast to control embryos, the experimental embryos showed intense cytoplasmic staining, which suggested the presence of F-actin in the cytoplasm (Figs. 10B, D and F). Despite of the reorganization of actin cytoskeleton, the localization of Cdc42 protein did not change in embryos derived from toxin B treated zygotes. In all control and toxin B treated zygotes and embryos the Cdc42 remained localized in the cortex. The effect of toxin B on IQGAP1 distribution. In control zygotes IQGAP1 was not detected in the cortex. However, in embryos arrested at one-cell stage after toxin B treatment, IQGAP1 accumulated in the cortical layer (Fig. 11A). In all two-cell embryos derived from zygotes injected with toxin B, IQGAP1 was detected in the cytoplasm and in some embryos at the contact site between sister blastomeres. The intensity of the fluorescent signal of IQGAP1 protein at the contact site was different in different embryos, from almost undetectable to very strong (compare Figs. 11B, and C, and D). In two-cell-arrested embryos,

Fig. 9. Effect of toxin B on cleavage ability of zygotes. (21 h) zygotes injected with toxin B 21 h after hCG injection (n = 32); (26 h) zygotes injected with toxin B 26 h after hCG injection (n = 103); (28 h) zygotes injected with toxin B 28 h after hCG injection (n = 46); (control) noninjected zygotes obtained 21 h after hCG injection and cultured in vitro (n = 138); (buffer) zygotes injected with buffer 21 h after hCG injection (n = 48); (1-cell) embryos, which did not undergo first mitotic division; (2-cell) embryos that reached two-cell stage; (4-cell) embryos that reached four-cell stage.

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Fig. 10. Actin cytoskeleton and the morphology of embryos derived from zygotes treated with toxin B. Chromatin shown in red, actin shown in green. (A) Control zygote (21 h after hCG); (B) one-cell arrested embryo derived from zygote injected with toxin B 21 h after hCG and cultured for 24 h; (C) control two-cell embryo; (D) two-cell embryo derived from zygote injected with toxin B 28 h after hCG and cultured for 24 h; (E) control four-cell embryo; (F) two-cell embryo derived from zygote injected with toxin B 28 h after hCG, and cultured for 48 h. 2pb — second polar body. In control zygotes and embryos actin forms a regular ring in cell cortex. Actin filaments are also present at the blastomere contact sites (C, E). In embryos derived from zygotes treated with toxin B polymerized actin was observed in the cytoplasm and formed irregular patches scattered randomly in the cortex (B, D, F). Scale bar: 20 µm.

like in control four-cell embryos (see Fig. 4B), IQGAP1 was also detected around the nucleoli (Figs. 11B, - D, arrowheads). Is the actin depolymerization responsible for changes in localization of IQGAP1 protein? To verify, whether the changes in IQGAP1 distribution were a consequence of the toxin B inhibition of the binding activity of Rho family proteins, and Cdc42 in particular, or of the rearrangement of actin cytoskeleton, the localization of IQGAP1 was analyzed in zygotes cultured in the presence of cytochalasine D (CD), which, like toxin B, induces actin depolymerization, but does not influence the activity of Cdc42 (Palazzo et al., 2001). In zygotes cultured in the presence of CD the cytokinesis of the first mitosis was inhibited,

resulting in one-cell embryos with two nuclei (the morphology of nuclei resembled that of blastomere nuclei; Fig.12A). Similar effect was observed in zygotes treated with toxin B (Fig. 12B). However, in contrast to toxin B, the CD treatment did not affect the distribution of IQGAP1 (Fig. 12A), which localization was identical to that observed in control zygotes (Fig. 12C) or early two-cell embryos (see Fig. 4G). Discussion We analyzed in mouse oocytes and early cleaving embryos the localization pattern and the relation between Cdc42 and IQGAP1. Although, it has been known that these two proteins are involved in

Fig. 11. Changes in the localization of IQGAP1 protein in embryos derived from zygotes treated with toxin B. Chromatin shown in red, IQGAP1 shown in green. (A, A′) One-cell arrested embryo derived from zygote treated with toxin B 21 h after hCG and cultured for 24 h; (B, B′, C, C′,D, D′) two-cell arrested embryos derived from zygotes treated with toxin B 28 h after hCG and cultured for 48 h. Two nuclei are present in each blastomere of these embryos. IQGAP1 is localized in the cortex of the one-cell arrested embryo (A). In two-cell arrested embryos IQGAP1 was detected in the cytoplasm (B, B′), or in the cytoplasm and at blastomere contact sites (C, C′ and D, D′). IQGAP1 was detected also around the nucleoli (B′, C′ and D′ arrowheads). Scale bar: 20 µm.

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Fig. 12. Comparison of the localization of IQGAP1 protein in zygotes treated with cytochalasin D and toxin B. Chromatin shown in red, IQGAP1 shown in green. (A, B) One-cell arrested embryos derived from zygotes: cultured in the presence of cytochalasin D for 24 h (A); injected with toxin B and cultured for 24 h (B). (C) control zygote. 2pb — second polar body. In zygote cultured in the presence of cytochalasin D IQGAP1 remains localized in the cytoplasm (A) similarly as in the control zygotes (C). After toxin B treatment IQGAP1 accumulates in the cortex (B). Scale bar: 20 µm.

the establishment of cell polarity and regulation of cell divisions in somatic cells, the knowledge about their role and localization in embryonic cells was limited or absent (like in case of IQGAP1), and sometimes presented results were inconsistent with that obtained for somatic cells. Our present studies showed that in fully-grown GV oocytes Cdc42 protein was localized exclusively in the cortex. After GVBD it accumulated around condensing chromosomes and finally was localized on the microtubules of the meiotic spindle. We also observed the localization of Cdc42 on the spindle in early embryos at mitosis. Accumulation of this protein on the mitotic spindle was reported previously for HeLa cells (Ban et al., 2004; Minoshima et al., 2003; Oceguera-Yanez et al., 2005). Our study has confirmed spindle localization of Cdc42 in mitotic HCT116 epithelial cells (see Supplementary material). Recently Cui et al. (2007) reported that in mouse oocytes and embryos Cdc42 is localized exclusively in the cytoplasm. This result remains in contradiction with data described previously for somatic cells and with described in the present studies. The reasons for such discrepancy remain unclear. One of the possible explanations may be the difference between anti-Cdc42 antibodies used. However, the Western blot analysis performed on somatic cell lysates and on oocytes and zygotes confirmed that the antibody used in our studies specifically recognized Cdc42 protein. In addition, the fact that Cdc42 accumulated on mitotic and meiotic spindles suggests its common or similar role in meiotic and mitotic cell division. Several studies in Xenopus and mouse have suggested the involvement of Cdc42 in establishing the oocyte polarity and regulation of polar bodies extrusion (see Introduction). However, it is also known that the establishment of the cell polarity is a very complex process usually requiring the cooperation between many different protein partners. Because IQGAP1 protein is known to interact with Cdc42 in somatic cells (Swart-Mataraza et al., 2002; Briggs and Sacks 2003; Watanabe et al., 2004) it was logical to predict that it also acts as a Cdc42 partner in oocytes and embryos. We analyzed, for the first time, IQGAP1 distribution during mouse oogenesis and early embryonic development. The behavior of this protein during meiotic maturation of mouse oocytes and during extrusion of polar bodies, suggests its possible involvement in the regulation of cytokinesis. We found that the localization of IQGAP1 changes from cortical in GV-intact oocytes, to cytoplasmic in maturing oocytes and that during the extrusion of polar bodies, it localizes in the region corresponding to the contractile ring. After the extrusion of the polar bodies IQGAP1 localizes at the contact site between the oocyte and the polar body. The localization of IQGAP1 in the region corresponding to the contractile ring during cytokinesis and in cell– cell contact sites was observed also in zygotes and early cleaving embryos (two- to eight-cell). In HCT116 epithelial cells we also observed IQGAP1 in the contact site between sister cells, and such localization was also described for somatic MDCKII epithelial cells, where its presence was related to the regulation of cell–cell adhesion through the cadherin–catenin pathway (Kuroda et al., 1998).

To verify the hypothesis that Cdc42 and IQGAP1 interact in the regulation of cytokinesis in both meiosis and mitosis, we inhibited the binding activity of Cdc42 using toxin B (Just et al., 1995; Wex et al., 1997). Toxin B inhibits the binding activity not only of Cdc42, but also of the other Rho proteins (Just et al., 1995; Wex et al., 1997; Yasuda et al., 2004). However, the data obtained for HeLa cells confirmed that toxin B effectively inactivates Cdc42. In this cell line toxin B blocks progression of mitosis in prometaphase and prevents chromosomes alignment in the metaphase plate. Similar effect was obtained when the activity of Cdc42 was inhibited by N17-Cdc42 dominant-negative mutant (Yasuda et al., 2004). Additionally, it is well documented that only two proteins from Rho family interact directly with IQGAP1, namely Cdc42 and Rac1 (Kuroda et al., 1996). We observed, that the treatment with toxin B affected both meiotic and mitotic divisions inducing changes in IQGAP1 distribution and provoked abnormalities in microfilament cytoskeleton. The effect of toxin B on maturing oocytes was clearly dose dependent. The lower dose (40 µg/ml) influenced actin cytoskeleton, but not the ability of oocytes to finish the first meiotic division. Despite the disappearance of cortical actin (which remained only in actin cap), the meiotic spindle migrated to the cortex and the first polar body was extruded. Apparently, the presence of actin cap was sufficient for successful completion of the first meiotic division. Higher dose of toxin B (200 µg/ml) effectively inhibited extrusion of the first polar body although, like in control oocytes, the meiotic spindle migrated to the cortex. This higher dose of toxin B also affected the cytokinesis during mitotic divisions of cleaving embryos. In both oocytes and embryos, we observed a deep rearrangement of actin cytoskeleton coupled with depolymerization of cortical actin. The presence of polymerized actin filaments, as well as the presence of IQGAP1 and active Cdc42 seems to be indispensable for the proper formation of a contractile ring during cytokinesis. It has been shown in somatic cells and also in in vitro systems (Fukata et al., 1997; Bashour et al., 1997), that the process of IQGAP1 binding to F-actin and cross-linking of Factin is enhanced by the GTP-bound form of Cdc42. Mentioned above accumulation of IQGAP1 in the region corresponding to the contractile ring in meiotically dividing oocytes and mitotically dividing zygotes suggests that it may play a role in proper arrangement of actin filaments. It seems also that active Cdc42 is necessary for the formation of contractile ring. On the other hand, the disassembly of a contractile ring and completion of cytokinesis is possible only after the decrease of the level of active Cdc42 (Schweitzer and D'SouzaSchorey, 2004). The inhibition of active form of Cdc42 by toxin B treatment of GV oocytes and early zygotes (21 h after hCG), many hours before expected cytokinesis, most probably disturbs the proper connections between actin filaments and IQGAP1 resulting in massive depolymerization of cortical actin and, in a consequence, inhibition of contractile ring formation. It seems possible, that in late zygotes (injected with toxin B 26–28 h after hCG) the actin cytoskeleton had been already stabilized, and the lack of active Cdc42 did not interfere

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with the formation of the contractile ring thus allowing for a successful completion of the first mitosis. However, during the subsequent cell cycle, due to the permanent lack of GTP-bound form of Cdc42 and the lack of binding between IQGAP1 and the actin filaments the cytokinesis was inhibited. We showed that the treatment with toxin B changed the distribution of IQGAP1. In oocytes treated with toxin B and allowed to mature in vitro, IQGAP1 lost its cortical localization, similar as in control oocytes. The treatment of GV-arrested oocytes with toxin B confirmed that the gradual disappearance of IQGAP1 from the oocyte cortex was, at least partially, caused by the inhibition of Cdc42 binding activity. In contrast, in zygotes arrested at one-cell stage after toxin B injection, IQGAP1 accumulated in the cortex, what was never observed in control one-cell embryos. This result was rather surprising considering the fact that in these zygotes the cortical actin was depolymerized. Moreover, the inhibition of actin polymerization with cytochalasin D did not induced the accumulation of IQGAP1 in the cortex. It remained distributed evenly in the cytoplasm like in control zygotes. This result confirmed that in toxin B treated zygotes changes in IQGAP1 localization were specifically related to the inhibition of Cdc42 binding activity and not to the rearrangement of actin filaments. In summary, our studies suggest that in cells which will pass through cytokinesis in near future (maturing oocytes, cleaving embryos, dividing somatic cells), IQGAP1 is accumulated in the cytoplasm, where its presence is necessary to arrange properly actin filaments during formation of the contractile ring. In contrast, in cells in which the cytokinesis is not expected (GV oocytes), or inhibited (embryos arrested at one-cell stage after toxin B treatment) IQGAP1 localizes to the cortex. The fact, that inhibition of Cdc42 binding activity causes changes in the localization of IQGAP1 suggests that in mouse oocytes and cleaving embryos IQGAP1 acts downstream of Cdc42. Na and Zernicka-Goetz (2006) postulated that Cdc42 plays an important role in spindle migration during meiotic maturation of mouse oocytes. In their studies the inactivation of Cdc42 by ectopic expression of its GTPase-defective mutants affected migration of the spindle, which remained in the center of the oocyte. On the other hand, the inhibition of Cdc42 activity by siRNA, did not block the migration of the meiotic spindle (Cui et al., 2007). Our current study also indicates that there is no direct link between Cdc42 activity and the movement of the meiotic spindle to the cortex (the inhibition of Cdc42 active form by toxin B did not inhibit migration of the spindle). It is known that toxin B inhibits the binding activity not only of Cdc42, but also of other Rho family members such as Rac1 and RhoA (Just et al., 1995; Wex et al., 1997; Yasuda et al., 2004), which are engaged in the organization and migration of the spindle (Narumiya et al., 2004; Yasuda et al., 2004, 2006), or its attachment to the cortex (Halet and Carroll, 2007). Taking this into account one could expected that the effect of toxin B on spindle organization and migration should be even more pronounced than that observed after inhibition of Cdc42 only. As it turned out, treatment with toxin B affected only the shape of the meiotic spindle, but not its movement to the oocyte cortex. It was reported previously that spindle migration is an actin-dependent process and that it is accompanied by local reorganization of the cortex (Brunet and Maro, 2005; Simerly et al., 1998). Our results suggest that cortical F-actin is not necessary for meiotic spindle migration, because in toxin B treated oocytes the spindle still moved to the cortex, despite the fact that cortical actin disappeared. The conclusion that actin is responsible for spindle migration was based on the experiments in which actin cytoskeleton was totally destroyed by cytochalasin D treatment (Brunet and Maro, 2005; Simerly et al., 1998). In our study the cytoplasm of toxin B treated oocytes contained F-actin, which suggests that not all actin filaments were depolymerized, although their localization was changed, from cortical to cytoplasmic. We cannot exclude the possibility that in toxin B treated oocytes, actin filaments present in the cytoplasm were sufficient to support the migration of meiotic spindle.

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It seems that the main function of IQGAP1 is the regulation of cytokinesis in embryonic and somatic cells. However, the nuclear localization of this protein during specific periods of oogenesis and embryogenesis suggests that it may also play another role, not related to the regulation of divisions. It has been reported previously that IQGAP1 may be involved in the regulation of gene expression (Hogan et al., 2003; Willingham et al., 2005), acting as a repressor of transcription factor NFAT. In the present studies nuclear localization of IQGAP1 was observed in the stages of oogenesis and embryonic development in which RNA polymerase I-dependent transcription was active. IQGAP1 formed characteristic ring around nucleoli in transcribing growing and fully-grown (NSN) oocytes. Nuclear signal of IQGAP1 disappeared after experimental inhibition of RNA synthesis in growing oocytes and the signal was absent in GVs of fully-grown SN oocytes which are transcriptionally silent (BouniolBaly et al., 1999) and in the pronuclei of zygotes, in which the nucleolus-like bodies (Kopecny et al., 1989) are transcriptionally inactive (Bouniol et al., 1995). IQGAP1 appeared again in the form of ring around nucleoli, in the nuclei of cleaving embryos, in which nucleoli exhibit typical pol I-dependent RNA synthesis. Similar changes in the nuclear localization were described for SURF6, a protein probably involved in ribosome biogenesis (Romanova et al., 2006). Our results suggest, that also IQGAP1 may be involved in the regulation of ribosomal genes transcription during oogenesis and early mouse embryogenesis. However, additional studies are needed to address this hypothesis. Acknowledgments This work was financed by grants from the Ministry of Science and Higher Education (grant K107/P04/05 and grant 2P04C 136 29). Authors thank: Dr. Anna Ciemerych-Litwinienko for fruitful and inspiring discussions, the colleagues from Laboratory of Molecular Bases of Aging from Nencki Institute for their help in performing experiments on somatic cells and Prof. M. Kloc for her valuable comments on the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2008.06.039. References Adams, A.E., Johnson, D.I., Longnecker, R.M., Sloat, B.F., Pringle, J.R., 1990. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J. Cell. Biol. 111, 131–142. Ban, R., Irino, Y., Fukami, K., Tanaka, H., 2004. Human mitotic spindle-associated protein PRC1 inhibits MgcRacGAP activity toward Cdc42 during the metaphase. J. Biol. Chem. 279, 16394–16402. Bashour, A.M., Fullerton, A.T., Hart, M.J., Bloom, G.S., 1997. IQGAP1, a Rac- and Cdc42binding protein, directly binds and cross-links microfilaments. J. Cell. Biol. 137, 1555–1566. Bouniol-Baly, C., Hamraoui, L., Guibert, J., Beaujean, N., Szollosi, M.S., Debey, P., 1999. Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes. Biol. Reprod. 60, 580–587. Bouniol, C., Nguyen, E., Debey, P., 1995. Endogenous transcription occurs at the 1-cell stage in the mouse embryo. Exp. Cell. Res. 218, 57–62. Briggs, M.W., Sacks, D.B., 2003. IQGAP1 as signal integrator: Ca2+, calmodulin, Cdc42 and the cytoskeleton. FEBS Lett. 542 (1–3), 7–11. Brunet, S., Maro, B., 2005. Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space. Reproduction 130, 801–811. Cho, W.K., Stern, S., Biggers, J.D., 1974. Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J. Exp. Zool. 187, 383–386. Ciemerych, M.A., Kenney, A.M., Sicinska, E., Kalaszczynska, I., Bronson, R.T., Rowitch, D.H., Gardner, H., Sicinski, P., 2002. Development of mice expressing a single D-type cyclin. Genes Dev. 16, 3277–3289. Cui, X.S., Li, X.Y., Kim, N.H., 2007. Cdc42 is implicated in polarity during meiotic resumption and blastocyst formation in the mouse. Mol. Reprod. Dev. 74, 785–794. Cuthbertson, K.S., Whittingham, D.G., Cobbold, P.H., 1981. Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature 294, 754–757. Cuthbertson, K.S., 1983. Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol. J. Exp. Zool. 226, 311–314.

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Debey, P., Renard, J.P., Coppey-Moisan, M., Monnot, I., Geze, M., 1989. Dynamics of chromatin changes in live one-cell mouse embryos: a continuous follow-up by fluorescence microscopy. Exp. Cell. Res. 183, 413–433. Debey, P., Szollosi, M.S., Szollosi, D., Vautier, D., Girousse, A., Besombes, D., 1993. Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics. Mol. Reprod. Dev. 36, 59–74. Drechsel, D.N., Hyman, A.A., Hall, A., Glotzer, M., 1997. A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curr. Biol. 7, 12–23. Dutartre, H., Davoust, J., Gorvel, J.P., Chavrier, P., 1996. Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs. J. Cell. Sci. 109 (Pt 2), 367–377. Etienne-Manneville, S., Hall, A., 2002. Rho GTPases in cell biology. Nature 420, 629–635. Fukata, M., Kuroda, S., Fujii, K., Nakamura, T., Shoji, I., Matsuura, Y., Okawa, K., Iwamatsu, A., Kikuchi, A., Kaibuchi, K., 1997. Regulation of cross-linking of actin filament by IQGAP1, a target for Cdc42. J. Biol. Chem. 272, 29579–29583. Fukata, M., Kuroda, S., Nakagawa, M., Kawajiri, A., Itoh, N., Shoji, I., Matsuura, Y., Yonehara, S., Fujisawa, H., Kikuchi, A., Kaibuchi, K., 1999. Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin. J. Biol. Chem. 274, 26044–26050. Fukata, M., Nakagawa, M., Kuroda, S., Kaibuchi, K., 2002. Effects of Rho family GTPases on cell-cell adhesion. Methods Mol. Biol. 189, 121–128. Fulton, B.P., Whittingham, D.G., 1978. Activation of mammalian oocytes by intracellular injection of calcium. Nature 273, 149–151. Grigoriev, I., Borisy, G., Vorobjev, I., 2006. Regulation of microtubule dynamics in 3T3 fibroblasts by Rho family GTPases. Cell. Motil. Cytoskeleton 63, 29–40. Halet, G., Carroll, J., 2007. Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes. Dev. Cell 12, 309–317. Hall, A., 1998. Rho GTPases and the actin cytoskeleton. Science 279, 509–514. Ho, Y.D., Joyal, J.L., Li, Z., Sacks, D.B., 1999. IQGAP1 integrates Ca2+/calmodulin and Cdc42 signaling. J. Biol. Chem. 274, 464–470. Hogan, P.G., Chen, L., Nardone, J., Rao, A., 2003. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes. Dev. 17, 2205–2232. Jantsch-Plunger, V., Gonczy, P., Romano, A., Schnabel, H., Hamill, D., Schnabel, R., Hyman, A.A., Glotzer, M., 2000. CYK-4: a Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis. J. Cell. Biol. 149, 1391–1404. Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., Aktories, K., 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500–503. Kopecny, V., Flechon, J.E., Camous, S., Fulka Jr., J., 1989. Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine-structural autoradiographic study. Mol. Reprod. Dev. 1, 79–90. Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., Kaibuchi, K., 1996. Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1. J. Biol. Chem. 271, 23363–23367. Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., Kaibuchi, K., 1998. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherinmediated cell–cell adhesion. Science 281, 832–835. Longo, F.J., Chen, D.Y., 1985. Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus. Dev. Biol. 107, 382–394. Luo, L., 2000. Rho GTPases in neuronal morphogenesis. Nat. Rev. Neurosci. 1, 173–180. Ma, C., Benink, H.A., Cheng, D., Montplaisir, V., Wang, L., Xi, Y., Zheng, P.P., Bement, W.M., Liu, X.J., 2006. Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation. Curr. Biol. 16, 214–220. Marks, P.W., Kwiatkowski, D.J., 1996. Genomic organization and chromosomal location of murine Cdc42. Genomics 38, 13–18. Mataraza, J.M., Briggs, M.W., Li, Z., Entwistle, A., Ridley, A.J., Sacks, D.B., 2003. IQGAP1 promotes cell motility and invasion. J. Biol. Chem. 278, 41237–41245. Merla, A., Johnson, D.I., 2000. The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe. Eur. J. Cell. Biol. 79, 469–477. Miki, T., Smith, C.L., Long, J.E., Eva, A., Fleming, T.P., 1993. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature 362, 462–465. Minoshima, Y., Kawashima, T., Hirose, K., Tonozuka, Y., Kawajiri, A., Bao, Y.C., Deng, X., Tatsuka, M., Narumiya, S., May Jr., W.S., Nosaka, T., Semba, K., Inoue, T., Satoh, T., Inagaki, M., Kitamura, T., 2003. Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis. Dev. Cell 4, 549–560. Na, J., Zernicka-Goetz, M., 2006. Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function. Curr. Biol. 16, 1249–1254. Narumiya, S., Oceguera-Yanez, F., Yasuda, S., 2004. A new look at Rho GTPases in cell cycle: role in kinetochore-microtubule attachment. Cell. Cycle 3, 855–857. Nicolson, G.L., Yanagimachi, R., Yanagimachi, H., 1975. Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. J. Cell. Biol. 66, 263–274.

Noritake, J., Watanabe, T., Sato, K., Wang, S., Kaibuchi, K., 2005. IQGAP1: a key regulator of adhesion and migration. J. Cell. Sci. 118, 2085–2092. Oceguera-Yanez, F., Kimura, K., Yasuda, S., Higashida, C., Kitamura, T., Hiraoka, Y., Haraguchi, T., Narumiya, S., 2005. Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis. J. Cell. Biol. 168, 221–232. Palazzo, A.F., Joseph, H.L., Chen, Y.J., Dujardin, D.L., Alberts, A.S., Pfister, K.K., Vallee, R.B., Gundersen, G.G., 2001. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr. Biol. 11, 1536–1541. Qualmann, B., Mellor, H., 2003. Regulation of endocytic traffic by Rho GTPases. Biochem. J. 371, 233–241. Romanova, L.G., Anger, M., Zatsepina, O.V., Schultz, R.M., 2006. Implication of nucleolar protein SURF6 in ribosome biogenesis and preimplantation mouse development. Biol. Reprod. 75, 690–696. Schlessinger, K., McManus, E.J., Hall, A., 2007. Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity. J. Cell. Biol. 178, 355–361. Schweitzer, J.K., D'Souza-Schorey, C., 2004. Finishing the job: cytoskeletal and membrane events bring cytokinesis to an end. Exp. Cell. Res. 295, 1–8. Shannon, K.B., Li, R., 1999. The multiple roles of Cyk1p in the assembly and function of the actomyosin ring in budding yeast. Mol. Biol. Cell. 10, 283–296. Simerly, C., Nowak, G., de Lanerolle, P., Schatten, G., 1998. Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos. Mol. Biol. Cell. 9, 2509–2525. Swart-Mataraza, J.M., Li, Z., Sacks, D.B., 2002. IQGAP1 is a component of Cdc42 signaling to the cytoskeleton. J. Biol. Chem. 277 (27), 24753–24763. Symons, M., Rusk, N., 2003. Control of vesicular trafficking by Rho GTPases. Curr. Biol. 13, R409–R418. Szczepanska, K., Maleszewski, M., 2005. LKB1/PAR4 protein is asymmetrically localized in mouse oocytes and associates with meiotic spindle. Gene Expr. Patterns 6, 86–93. Takaishi, K., Sasaki, T., Kameyama, T., Tsukita, S., Tsukita, S., Takai, Y., 1995. Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell–cell adhesion sites and cleavage furrows. Oncogene 11, 39–48. Tatsumoto, T., Sakata, H., Dasso, M., Miki, T., 2003. Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cellfree extracts. J. Cell. Biochem. 90, 892–900. Voth, D.E., Ballard, J.D., 2005. Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247–263. Watanabe, T., Wang, S., Noritake, J., Sato, K., Fukata, M., Takefuji, M., Nakagawa, M., Izumi, N., Akiyama, T., Kaibuchi, K., 2004. Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev. Cell 7 (6), 871–883. Wex, C.B., Koch, G., Aktories, K., 1997. Effects of Clostridium difficile toxin B on activation of rat peritoneal mast cells. Naunyn. Schmiedebergs Arch. Pharmacol. 355, 328–334. Willingham, A.T., Orth, A.P., Batalov, S., Peters, E.C., Wen, B.G., Aza-Blanc, P., Hogenesch, J.B., Schultz, P.G., 2005. A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science 309, 1570–1573. Wu, X., Li, S., Chrostek-Grashoff, A., Czuchra, A., Meyer, H., Yurchenco, P.D., Brakebusch, C., 2007. Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development. Dev. Dyn. 236, 2767–2778. Wulf, E., Deboben, A., Bautz, F.A., Faulstich, H., Wieland, T., 1979. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc. Natl. Acad. Sci. U. S. A. 76, 4498–4502. Yamana, N., Arakawa, Y., Nishino, T., Kurokawa, K., Tanji, M., Itoh, R.E., Monypenny, J., Ishizaki, T., Bito, H., Nozaki, K., Hashimoto, N., Matsuda, M., Narumiya, S., 2006. The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src. Mol. Cell. Biol. 26, 6844–6858. Yasuda, S., Oceguera-Yanez, F., Kato, T., Okamoto, M., Yonemura, S., Terada, Y., Ishizaki, T., Narumiya, S., 2004. Cdc42 and mDia3 regulate microtubule attachment to kinetochores. Nature 428, 767–771. Yasuda, S., Taniguchi, H., Oceguera-Yanez, F., Ando, Y., Watanabe, S., Monypenny, J., Narumiya, S., 2006. An essential role of Cdc42-like GTPases in mitosis of HeLa cells. FEBS Lett. 580, 3375–3380. Zhong, Z.S., Huo, L.J., Liang, C.G., Chen, D.Y., Sun, Q.Y., 2005. Small GTPase RhoA is required for ooplasmic segregation and spindle rotation, but not for spindle organization and chromosome separation during mouse oocyte maturation, fertilization, and early cleavage. Mol. Reprod. Dev. 71, 256–261. Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C.A., 1995. Chromatin organization during mouse oocyte growth. Mol. Reprod. Dev. 41, 479–485. Zuccotti, M., Ponce, R.H., Boiani, M., Guizzardi, S., Govoni, P., Scandroglio, R., Garagna, S., Redi, C.A., 2002. The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst. Zygote 10, 73–78.