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RESEARCH |
1 State Key Laboratory of Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, No. 25 Beisihuanxi Road, Beijing 100080, China, 2 Department of Animal Science and Technology, Northeast Agricultural University, Harbin, China and 3 Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
Correspondence should be addressed to Q-Y Sun; Email: sunqy{at}ioz.ac.cn; sunqy1{at}yahoo.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsExperimental designResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsExperimental designResultsDiscussionAcknowledgementsReferences |
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The Ran GTPase affects many cellular processes, including the regulation of cell cycle progression, nuclear envelope structure and function, and nucleo–cytoplasmic transport. Ran is regulated by a cytosolic-activating protein, RanGAP1, and by a chromatin-bound nucleotide exchange factor, RCC1. The distribution of RanGTP provides important spatial information that directs cellular activities during different parts of the cell cycle (Sazer & Dasso 2000, Dasso 2001).
During interphase, the localization of RCC1 and RanGAP1 implies that nuclear Ran is GTP-bound and cytosolic Ran is GDP-bound. This compartmentalization determines the direction of nuclear transport by promoting the loading and unloading of transport receptors in a manner that is appropriate to the nucleus or cytosol, that is, the nuclear RanGTP promotes the dissociation of import complexes – and hence the release of nuclear proteins in the nucleoplasm – as well as the assembly of export complexes, which, conversely, mediate transport of cytoplasmic proteins and RNAs to the cytoplasm (Gorlich & Kutay 1999).
In mitosis the nuclear envelope breaks down and condensed chromosomes are released into the cytoplasm, and then spindle microtubules are nucleated by centrosomes and chromosomes (Compton 2000, Wittmann et al. 2001). Strikingly, addition of RanGTP or mutant forms of Ran locked in a GTP-like conformation to meiotic Xenopus egg extracts stimulates microtubule assembly and can even induce the formation of centrosome-independent bipolar spindles in the presence or absence of chromatin (Carazo-Salas et al. 1999, Kalab et al. 1999, Ohba et al. 1999, Zhang et al. 1999). The role of Ran in spindle formation has also been demonstrated in yeast and Caenorhabditis elegans (Ouspenski 1998, Fleig et al. 2000, Quimby et al. 2000, Bamba et al. 2002).
When adding chromatin to Xenopus egg extract in vitro, it was found that either RanGTP or RanGDP together with RCC1 is necessary for early membrane fusion steps in nuclear envelope assembly (Hetzer et al. 2000). Similarly, beads coated with wild-type Ran formed a pseudonucleus surrounded by nuclear envelope, whereas beads attached to mutant forms of Ran incapable of cycling between GTP- and GDP-bound conformations failed to do so (Zhang & Clark 2000). More recently, it was reported that perturbing the Ran system in C. elegans by double-stranded RNA-mediated interference (RNAi) gave rise to embryos where the chromatin no longer associated with nucleoporins, indicative of either a defect in nuclear envelope assembly or nuclear pore complex (NPC) assembly or insertion into the nuclear envelope (Bamba et al. 2002).
Mammalian oocytes are arrested at the G2/M phase transition of the first meiotic division. In vitro, fully grown oocytes liberated from their follicles spontaneously reinitiate meiosis I, characterized by germinal vesicle (GV) breakdown (GVBD), chromatin condensation, spindle assembly, emission of the first polar body and progression to metaphase of the second meiotic division (MII), at which stage they undergo a second arrest until fertilization. After sperm penetrates the oocyte, the second polar body extrudes, male and female pronuclei form and syngamy occurs to start early embryo development. Ran is highly conserved in eukaryotes from yeast to humans, but we still do not know whether such an important GTPase regulates cellular processes from a follicular oocyte to an early embryo. In the present study, we investigated: (i) the distribution of Ran during meiotic maturation, fertilization and subsequent early embryo development; (ii) the correlation of microtubule assembly and Ran localization in mouse oocytes after different treatments with microtubule regulators or after anti-Ran antibody microinjection; and (iii) Ran’s distribution in pseudo-cleavage of GV-intact oocytes. All these experiments were aimed at clarifying the possible correlation between Ran and microtubule assembly as well as cell cycle progression in mouse oocytes.
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Materials and Methods |
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Anti-Ran antibody
Anti-Ran antibody (c-20) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). It was an affinity-purified goat polyclonal antibody raised against a peptide mapping to the C-terminus of Ran of human origin, and it reacts with Ran of mouse, rat and human origin.
Oocyte collection and culture
Kunming strain mice at 4–6 weeks of age were purchased from the Laboratory Animal Core, Institute of Genetics, Chinese Academy of Sciences. Animals were housed in a temperature-controlled room with proper darkness–light cycles, fed with a regular diet, and maintained under the care of the Laboratory Animal Unit, State Key Laboratory of Reproductive Biology, Institute of Zoology, the Chinese Academy of Sciences. Following 5 days of acclimation, GV-stage oocytes were collected from ovaries of the mice 48 h after the females were injected with 10 IU equine gonadotropin (eCG). GV-intact follicular oocytes were released from the large antral follicles by puncturing with a needle in M2 medium with 60 µg/ml penicillin and 50 µg/ml streptomycin. Oocytes were denuded by pipetting and normal-looking oocytes were cultured for maturation in M2 at 37°C in a humidified atmosphere of 5% CO2.
Cumulus cell-enclosed MII-arrested eggs were obtained from mice of the same strain. Females were superovulated by i.p. injection with 10 IU eCG, and 48 h later they were injected with 10 IU human gonadotropin (hCG). Mice were killed by cervical dislocation 15 h after hCG injection. The cumulus cell masses surrounding the eggs were removed by brief exposure to 300 IU/ml hyaluronidase in M2 medium. Oocytes were subjected to in vitro fertilization and parthenogenetic activation by 7% alcohol for 5 min, and then cultured in M2 medium for 6 h.
Zygote and embryo collection
In vivo fertilized zygotes were collected 16 h after hCG from the oviduct ampullae of superovulated females that had been mated with the same strain of males. After removing cumulus cells with 300 IU/ml hyaluronidase in M2 medium, zygotes were cultured in M16 medium (Sigma) until use. Two cell embryos were flushed from the oviducts of copulated mice 44–46 h after hCG injection and cultured in M2 medium. Embryos at different stages of mitosis were collected for confocal microscopy.
Confocal microscopy
Ran detection was based on the procedures reported previously (Melchior et al. 1995). After removal of zona pellucidae (ZP) in acidified Tyrode’s solution (pH 2.5) eggs were fixed in 4% formaldehyde in PBS for 30 min, and then incubated in incubation buffer (0.5% Triton X-100 in 20 mM HEPES, pH 7.4, 3 mM MgCl2, 50 mM NaCl, 300 mM sucrose, 0.02% NaN3) for 30 min, washed in PBS with 0.1% Tween 20 and 0.01% Triton X-100, then incubated with 1:100 of the polyclonal anti-Ran antibody at 4 °C overnight. The eggs were rinsed three times, each for 5 min, and incubated with 1:100 FITC-conjugated rabbit anti-goat IgG for 1 h, followed by staining with 10 µg/ml propidium iodide. Finally the eggs were mounted on glass slides and examined using a TCS-4D laser scanning confocal microscope (Leica Microsystems, Bensheim, Germany).
Tubulin detection was conducted by fixing and permeabilizing the eggs in methanol for 5 min at –20 °C, and incubating the eggs in 1:50 diluted FITC-anti-
-tubulin for 1 h, then staining with propidium iodide as described above.
Microinjection of anti-Ran antibody
Anti-Ran antibody (200 µg/ml in 1 ml PBS containing 0.1% NaN3 and 0.2% gelatin) was injected into the cytoplasm of fully grown GV oocytes as described by Dai et al.(2000). The injection was repeated three times, and 35 oocytes were used each time. An Eppendorf microinjector (Hamburg, Germany) was used for these experiments. All microinjections were performed by using a beveled micropipette to minimize damage and were finished within 30 min. A microinjection volume of about 7 pl per oocyte was used in all the experiments. The same amount of goat IgG diluted in PBS containing 0.1% NaN3 and 0.2% gelatin was injected as control. After injection, eggs were washed thoroughly with M2 medium and cultured in the same medium.
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Experimental design |
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Experiment 2
To further reveal the relationship between the spindle assembly/disassembly and the location of Ran, MII oocytes were treated with 1 µM microtubule disassembly inhibitor taxol for 10 min or 10 µg/ml microtubule polymerization inhibitor colchicine for 1 h at 37 °C. After each treatment, oocytes were collected for confocal microscopy.
Experiment 3
To investigate the role of Ran in cytoplasmic cleavage (cytokinesis), induction of pseudo-cleavage was performed as reported (Wassarman et al. 1977). Briefly, cumulus-free and GV-intact oocytes were cultured in M2 medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX) (this drug is used to prevent spontaneous meiotic resumption) and 5 µM cytochalasin B (CB) overnight. To increase the rate of pseudo-cleaved oocytes, some ZP-free oocytes were cultured under the same conditions.
Experiment 4
The possible roles of Ran in meiosis spindle organization were revealed by antibody microinjection. Anti-Ran antibody was injected into GV-intact oocytes. The GVBD rate was recorded 2 h after culture. The spindle formation and polar body extrusion rate was recorded after 14 h of injection. The spindle structure of the oocytes undergoing GVBD was examined at 8 h and extrusion of the first polar body was examined at 14 h after injection by confocal microscopy.
Statistical analysis
All data on the GVBD rate of oocytes after antibody microinjection were evaluated by 
2 analysis. Eggs showing degenerative signs were not included.
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Results |
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TopAbstractIntroductionMaterials and MethodsExperimental designResultsDiscussionAcknowledgementsReferences |
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). After GVBD, Ran dispersed throughout the entire cell, but it was not evenly distributed. There were numerous concentrated dots around the condensed chromatin or chromosomes (Fig. 1B–D). With the organization of chromosomes to the equatorial plate, Ran staining was preferentially distributed to the spindle. It was associated with the spindle microtubules, while it distributed evenly to other areas of the oocyte (Fig. 1E and F). At anaphase I and telophase I following the separation of chromosomes, Ran accumulated in the separating region between the first polar body and the oocyte, but staining was not observed in the midbody (Fig. 1G). Immediately after meiosis I, Ran concentrated to the MII spindle microtubules (Fig. 1H).
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). With the expansion of sperm chromatin, Ran began to move to the region around the expanding sperm chromatin and oocyte chromatin, and concentrated to early male and female pronuclei (Fig. 2B). With the growth of pronuclei the green fluorescent staining of Ran became increasingly stronger in the pronuclei as compared with the cytoplasm (Fig. 2C). When the male and female pronuclei became apposed, a significant signal of Ran was observed in the pronuclei (Fig. 2D and E). After pronuclear membrane breakdown, Ran dispersed through the entire zygote cytoplasm, but again it was not evenly distributed. Numerous concentrated dots were observed in the chromosome region (Fig. 2F).
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). When chromosomes became organized to the equatorial plate, Ran was stained in association with the entire mitotic spindle (Fig. 3C). Then Ran concentrated between the separated chromosome clusters and formed two brighter green bands during anaphase and telophase of mitosis (Fig. 3D and E). After completion of the first mitotic cell cycle, two blastomeres formed and both of them entered interphase. At this time, Ran concentrated again in the assembling nuclei and the intensity of the nuclear staining of Ran compared with the cytoplasm became more evident during the completion of nuclear formation (Fig. 3F and G). Throughout the remainder of the cleavage stage, Ran showed the same pattern of dispersion: distribution to the cytoplasm at pro-metaphase, concentration to the spindle, localization between the separated chromosomes at telophase, and nuclear localization in the interphase nucleus (Fig. 3H–L).
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-tubulin detection. We could find that in 100% of the oocytes Ran concentrated to the expanded spindle around the chromosomes (Fig. 4A), in the same configuration as microtubule distribution (Fig. 4B). Asters were observed in all taxol-treated oocytes, but we could not see any aster-like foci stained positively for Ran (Fig. 4A) as that shown for tubulin (Fig. 4B).
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-tubulin detection. It was found that in 100% of the oocytes Ran protein distributed evenly in the cytoplasm, with a concentrated distribution beneath the cell membrane near the chromosomes (Fig. 4C
), corresponding to the disappearance of the spindle (Fig. 4D).
When ZP-intact mouse oocytes at the GV stage were cultured overnight in the presence of both IBMX and CB, 82% (41 of 50) slowly underwent pseudo-cleavage, which included the division of the oocytes into two approximately equal portions, one of which contained an intact GV and nucleolus. Ran was localized mainly in the GV except for the nucleolus, and no staining was observed in the cytoplasm of the two cleaved portions (Fig. 4E).
When MII oocytes were activated by alcohol, and cultured for 6 h in M2 medium, Ran was concentrated to the pronuclei (Fig. 4F).
Microinjection of anti-Ran antibody affects cell cycle progression and spindle assembly
Interphase (GV-intact) oocytes were injected with anti-Ran antibody or with non-specific IgG for control, then observed by light-field microscopy 2 and 14 h after injection; a proportion were fixed after 8 h and stained for confocal microscopy. As shown in Table 1, the GVBD rate of oocytes 2 h after antibody injection was significantly lower than that of the IgG-injected control group. The rate of the first polar body extrusion 14 h after antibody injection was significantly lower than that of the IgG-injected control group. Other oocytes did not extrude the first polar body, even when cultured for 16–18 h. After 8 h culture, abnormal spindles were observed in most oocytes injected. The spindles were significantly enlarged and the chromosomes could not be organized to the equatorial plate (Fig. 5A). A few oocytes did not even form spindles (Fig. 5B). At 14 h, half of the oocytes (14 of 27) with the first polar body extruded showed abnormal spindles with broader poles (Fig. 5C) compared with controls (Fig. 5D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsExperimental designResultsDiscussionAcknowledgementsReferences |
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During mouse oocyte maturation, the spatial organization of the microtubule cytoskeleton undergoes profound changes. The mitotic roles of Ran have been firmly established in Xenopus oocyte extracts. In our experiments, Ran was freed from the nucleoplasm to the cytoplasm and aggregated around the chromosomes. With the establishment of a bipolar spindle, it was obvious that the Ran distribution pattern was similar to that of the spindle microtubules. Ran was shown to be associated with the chromosomes in Xenopus, starfish and mouse oocytes (Hinkle et al. 2002). Our result is different from Ran’s immunofluorescent staining in C. elegans where Ran localizes to kinetochore regions of metaphase and ana-phase chromosomes (Bamba et al. 2002). This may be because Ran concentrated in the spindle in mouse oocytes, which could prevent us seeing its localization on the kinetochore region clearly. Experiments using Xenopus egg extracts showed that generation of RanGTP by RCC1 played roles in mitotic spindle assembly. In somatic cells, when Ran network members, such as RCC1 (Moore et al. 2002), RanBP1 (Di Fiore et al. 2003) or targets (TPX2) (Gruss et al. 2002) were experimentally perturbed, the spindle pole formation was primarily affected. After anti-Ran antibody was injected into the GV oocytes, we found abnormally large spindles without poles or with broad poles and the chromosomes could not be organized at the equatorial plate. In addition, some oocytes did not form spindles. The changes in spindle morphology are like those found in a TPX2 (a major regulatory target of Ran GTPase)-immunodepleted Xenopus system (Wittmann et al. 2000). Ran plays a central role in spatial and temporal organization microtubules of the vertebrate cell (Dasso 2002). Some Ran network members regulated by ubiquitin-like protein SUMO-1 (Joseph et al. 2002) or NPC subunit Nup358 (Salina et al. 2003, Joseph et al. 2004), localize transiently at kinetochores and act in the spindle attachment checkpoint. In our experiment, about one-third of injected oocytes eventually extruded polar bodies, suggesting that the spindle in these oocytes remains functional, while the others did not extrude a polar body in prolonged culture, which may be caused by the spindle’s functional failure. The abnormal chromosome alignment and subsequent non-disjunction of chromosomes caused by Ran depletion may contribute the failure of cytokinesis.
Mouse oocytes and early embryos lack centrioles until the blastocyst stage. When microtubule organization was disturbed by taxol in mouse MII oocytes, all taxol-treated oocytes stained for -tubulin showed an expanded spindle and many aster-like foci in the cytoplasm, consistent with 
-tubulin distribution previously reported (Gueth-Hallonet et al. 1993). A fraction of Ran had been shown tightly associated with the centrosome throughout the cell cycle in HeLa cells, suggesting that Ran participated in the control of centrosome activity (Guy et al. 2003). The concentrated Ran distribution was only observed in the expanded spindle, while no clear distribution was observed in aster-like foci in our study. This may be because the antibody we used could not distinguish GTP-and GDP-bound Ran and/or RanGTP concentration in aster-like foci was not high enough. When oocytes were treated with colchicine, the typical Ran distribution disappeared. The results provide further evidence showing the important roles of Ran in meiotic spindle microtubule assembly.
Ran has been implicated in cell cycle control, including the transition from mitosis (or meiosis) to interphase when the compartmentalization of the nucleus is established. In an experiment using a cell-free system of Xenopus egg extracts to examine the function of Ran during the assembly of pronuclei from demembranated Xenopus sperm heads, it was found that increased concentration of RanGTP produced a large, stable microtubule aster nucleated from the sperm centrosome and arrested nuclear assembly, blocking chromatin decondensation. In contrast to RanGTP, excess RanGDP had no inhibitory effects on aster formation and nuclear assembly. The RanGDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the re-assembly of the nucleus associated with the transition from mitosis (or meiosis) to interphase (Zhang et al. 1999).
We found that Ran was concentrated in the pronuclei in fertilized mouse eggs and in the nuclei of early embryos. Ran’s concentration in the interphase nuclei is either required directly to allow nuclear envelope assembly/disassembly to occur, or needed to modify chromatin. Ran and its cofactors RanBP2, RanGAP and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins (Askjaer et al. 2002). Depleting Ran from C. elegans embryos by RNAi leads to defective nuclear envelope assembly (Kahana & Cleveland 1999). Ran’s direct role in nuclear envelope vesicle fusion came from experiments in which beads coupled to wild-type Ran could nucleate the assembly of functional, NPC-containing nuclear envelopes in extracts of either Xenopus eggs or mammalian cultured cells (Zhang & Clark 2000). Recent data have shown that Ran associated with meiotic or mitotic chromatin both in nuclear assembly extracts in vitro and in several types of cells in vivo (Hetzer et al. 2002). In Xenopus egg extracts, it has been shown that RanGDP is essential for proper nuclear assembly and DNA replication (Hughes et al. 1998). The involvement of Ran in pronucleus formation from a very early stage as revealed in this study suggests that RanGDP may promote pronuclear formation in fertilized eggs. Further studies are required to prove this hypothesis.
The phenomenon of Ran’s concentration in the nucleoplasm seems to require abundant nuclear binding sites, which apparently largely originate from the importin ß family transport receptor (Ribbeck et al. 1998, Zhang et al. 2002). In our experiments we observed Ran increased in intensity within the pronuclei with the growth and apposition of pronuclei, while Ran was almost absent in the cytoplasm. This also occurred in parthenogenetically activated eggs. When the chromosomes began to recondense just before pronuclear envelope breakdown, Ran accumulated in the region where the two pronuclei mixed. During first and second mitosis, the subcellular localization of concentrated Ran was associated with the chromatin and spindle microtubules throughout the entire cell cycle, just like its distribution pattern during meiosis. These results showed that Ran could regulate microtubule assembly during early mitosis of mouse embryo development. Ran did not show any specific distribution in meiotic and mitotic furrows as well as in pseudo-cleaved oocytes induced by IBMX and CB treatment, suggesting that it does not play functional roles in cytoplasmic division (cytokinesis).
Taken together, Ran has a cell cycle-dependent localization and may have regulatory roles in cell cycle progression and microtubule organization in mouse oocytes, fertilized eggs and early embryos.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsExperimental designResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsExperimental designResultsDiscussionAcknowledgementsReferences |
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