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Regular Article |
a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
b Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, fertilization, kinases, meiosis, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been found that all the Plks so far examined share the common property of associating with the spindle pole bodies early in mitosis [2, 8, 10]. In Drosophila and mice, Plks also distribute to the centromeres from prophase until anaphase [11, 12]. At the onset of anaphase, Plks accumulate in the central spindle and remain localized in midbody at telophase. The localization of Plks to specific components of spindle apparatus at different stages of mitosis suggests the potential role of Plks in spindle organization. Mutations in plks genes cause the appearance of abnormal mitotic cells, characterized by monopolar spindles or bipolar spindles with one of the poles broadened. Plk-specific antibody microinjection also results in the formation of monopolar spindles. Recently, a study [13] has shown that 
-, ß-, and 
-tubulins are stably associated with Plk in both interphase and mitotic cells and that all three tubulins are phosphorylated by Plk in vitro. Furthermore, the kinase domain of Plk alone is necessary and sufficient for the interaction with the tubulins. These findings propose that Plk affects the organization of the spindle in part through its association with and phosphorylation of tubulins.
Besides the organization of spindle, Plx1 plays multiple essential roles during mitosis in Xenopus. It is necessary for activation of the APC [7, 8, 14]. In Xenopus, the initial phosphorylation and activation of the phosphatase Cdc25, whose activity is prerequisite for the activation of MPF, results from activation of the Plx1 [6, 15]. In both Saccharomyces cerevisiae and Drosophila, Plks are localized to the midbody at telophase, and Plk function appears to be required for cytokinesis [16–18].
Although numerous studies have shown that Plks are necessary for mitotic spindle organization, their functions in meiotic microtubule organization are not fully known. From the viewpoint of the cell cycle, meiosis differs from mitosis in several aspects. For example, oocyte undergoes a period of extended meiotic arrest after entering the G2 phase while quiescence in mitotic cells is imposed by progression from G1 to a G0 phase of the cell cycle. Furthermore, meiosis lacks an intervening S phase between two meiotic divisions while mitosis has S phase and M phase alternating invariantly. Mammalian oocytes, ideal models in the study of meiotic cell cycle, undergo considerable chromosomal and cytoplasmic changes during maturation and fertilization, including chromosome condensation, germinal vesicle breakdown (GVBD), spindle organization, polar body emission, pronuclear formation, and syngamy. The organization of microtubules is well known to be involved in the regulation of these dynamic events [19, 20]. Since Plks are important in mitotic microtubule organization, their interaction with meiotic microtubules is very possible. A study in S. cerevisiae cdc5 mutations shows that a complete spindle does not form during the first meiotic division [21], suggesting a role of Plks in meiotic microtubule organization. Recent studies [22] in mice also found that, during oocyte maturation, Plk1 is localized to the meiotic spindle. Plk1 was activated through phosphorylation as early as 30 min before GVBD, and its activity was maintained throughout meiotic maturation. The phosphorylated form of Plk1 gradually disappeared after exit from meiosis. However, the dynamics of Plk1 during fertilization and the following early embryo development has not been studied.
In the present study, we, for the first time, investigated 1) the dynamics of the polo-like kinase1 (Plk1) at fertilization and syngamy, 2) the role of Plk1 in spindle organization during early embryonic mitosis, and 3) the correlation of microtubule assembly and Plk1 localization in mouse oocyte after different treatments with microtubule regulators or after Plk1 antibody microinjection. All these experiments were aimed at elucidating the possible roles of this kinase in meiotic and mitotic microtubule assembly and its regulation in mouse eggs and early embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Germinal vesicle (GV)-stage oocytes were collected from ovaries of 4- to 6-wk-old Kunming mice at 48 h after the females were injected with 10 IU eCG. Cumulus-free and GV-intact follicular oocytes were released from the large antral follicles by puncturing with a needle in M2 (Sigma, St. Louis, MO) medium with 60 µg/ml penicillin and 50 µg/ml streptomycin. All cultures were maintained in M2 medium at 37°C in a humidified atmosphere of 5% CO2.
Cumulus cell-enclosed metaphase II-arrested eggs were obtained from mice of the same strain. Females were superovulated by i.p. injection with 10 IU of eCG, and 48 h later, they were injected with 10 IU of hCG. Mice were killed by cervical dislocation at 15 h post-hCG injection. The cumulus cell masses surrounding the eggs were removed by brief exposure to 300 IU/ml hyaluronidase in M2 medium.
In Vivo and In Vitro Fertilization
In vivo fertilized zygotes were collected 16 h post-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 (Sigma) medium until use. Two-cell embryos were flushed from the oviducts of copulated mice 44–46 h after hCG injection and were cultured in M2 medium. Embryos at different stages of mitosis were collected for confocal microscopy.
In vitro fertilization was performed using 1 x 106/ml motile cauda epididymal sperm, which had been previously capacitated in M16 medium with 2.5 mM taurine for 1 h. Zona pellucida (ZP)-free eggs were used to achieve a more synchronous timing of fertilization within each stage group and to minimize the lag period of sperm-egg interaction. The emission of the second polar body and the formation of the pronuclei were observed with an inverted microscope. The eggs were collected at different stages for confocal microscopy or Western blot analysis.
Western Blot Analysis
Morphologically normal cells (100 eggs/sample) were collected in SDS sample buffer after treatment with different stimuli or different times, heated to 100°C for 4.5 min, and frozen at -20°C until use. The proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel at 90 V, 0.5 h and 120 V, 2.0 h, respectively, and electrically transferred to PDVF membrane (Sino-American Biotec, Beijing, China; pore size 0.45 µm) for 2 h, 200 mA, at 4°C. Following transfer, the membrane was immersed in methanol for 1 min and dried overnight at room temperature. The membrane was then incubated for 2 h at 37°C with monoclonal mouse anti-Plk antibody (Zymed Laboratories Inc., South San Francisco, CA) and monoclonal mouse anti-ß tubulin antibody (Sigma) diluted 1:500 in TBST (TBS containing 0.1% Tween-20) with 5% skimmed milk, respectively. After washing three times in TBST, 10 min each, the membrane was incubated for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:1000 in TBST. The membrane was washed three times in TBST, 10 min each, and processed by using the ECL detection system (Amersham International, Buckinghamshire, U.K.). All experiments were repeated at least three times.
Confocal Microscopy
Plk1 detection was based on the procedures reported previously [23]. After removal of ZP in acidified Tyrode solution (pH 2.5), eggs were fixed in 3% formaldehyde, 2% sucrose 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. After placed in methanol for 5 min at -20°C, the eggs were washed in PBS with 0.1% Tween 20 three times and then incubated with 1:100 monoclonal mouse anti-Plk antibody (Zymed Laboratories Inc.) for 1 h. The eggs were rinsed three times and incubated with 1:100 FITC-conjugated goat anti-mouse IgG for 45 min, 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).
The spindle organization was determined by incubating the eggs in 1:50 diluted FITC-anti- tubulin for 1 h after fixation and permeabilization as described above.
Microinjection of Plk1 Antibodies
Plk1 antibody (0.5 mg/ml in PBS without Ca2+, pH 7.4) was injected into the cytoplasm of fully grown GV oocytes as described by Dai et al. [24]. The injection was repeated three times, and 30 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 in 30 min. Isobutylmethylxanthine (IBMX; 0.2 µM) was added to the medium to prevent GVBD. A microinjection volume of about 7 pl per oocyte was used in all the experiments. The same amount of mouse IgG diluted in PBS was injected as control. After microinjection, eggs were washed thoroughly with M2 medium and cultured in the same medium. GV oocytes incubated in 0.2 µM IBMX for 30 min, and then washed and cultured for the evaluation of GVBD and MI spindle formation were also used as control.
Chemicals
All the chemicals used in this experiment were purchased from Sigma Chemical Company except for those specifically mentioned. Drugs were prepared as stock solution by dissolving in dimethyl sulfoxide (DMSO) and were stored in a dark box at -20°C. The stock solutions were diluted with M2 medium prior to use.
Experimental Design
Experiment 1 To detect the changes in Plk1 expression during the meiotic maturation and fertilization, the eggs were collected at different stages for Western blot analysis.
Experiment 2 To investigate the possible roles of Plk1 in microtubule organization during meiotic maturation, fertilization, and early embryonic mitosis, eggs of different stages of meiosis, fertilization, or early embryos at the first and second cleavages were collected for Plk1 localization with confocal microscopy.
Experiment 3 To further reveal the relationship between the spindle assembly/disassembly and the localization of Plk1, 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. In another experiment, MII oocytes were treated with 30 µM protein kinase inhibitor staurosporine for 30 min to destruct the meiotic spindle. Some eggs treated with staurosporine were further exposed to 1 µM taxol for 10 min. After each treatment, oocytes were collected for confocal microscopy.
Experiment 4 The possible roles of Plk1 in the meiotic spindle organization were revealed by antibody microinjection. The Plk1 antibody was injected into the GV intact oocytes. The GVBD rate was recorded 2 and 4 h after culture. The oocytes undergoing GVBD within 4 h after culture were fixed at 8 h after injection, and the spindle structure was examined with 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. The relative Plk1 quantity in different development stages was determined by the relative Plk1 intensity obtained by densitometric scan of the band, and the values were analyzed by Student t-test. Differences at P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to detect Plk1 protein expression in mouse oocytes during meiotic maturation and fertilization, samples were taken from GV stage until 8 h after fertilization. Western blot analysis showed that Plk1 protein was expressed in mouse oocytes, and its quantity seemed unchanged during meiotic maturation (Fig. 1, A and B), but the Plk1 expression decreased gradually following fertilization in vitro (Fig. 2, A and B). These results were compared on the same membrane to the blots of ß-tubulin, whose expression remained unchanged during the same periods.
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Subcellular Localization of Plk1 in Mouse Oocytes and Early Embryos
During oocyte maturation, fertilization, and syngamy, the localization of Plk1 varied at different developmental stages. In GV oocytes, Plk1 distributed diffusely, being detected in both the cytoplasm and the germinal vesicle (Fig. 3A). There was no obvious difference in the green fluorescence intensity in the GV and the cytoplasm as judged by the TCS-NT system. Shortly after GVBD, Plk1 concentrated around the condensed chromatin (Fig. 3, B and C) and then Plk1 staining focused as several dots near the chromosomes (Fig. 3, D and E). With the organization of chromosomes to the equatorial plate, the dots of Plk1 associated with the spindle poles as two dots until the metaphase of the first meiosis, and then Plk1 began to migrate to the middle region of spindles (Fig. 3, F and G). In anaphase I and telophase I, following the separation of chromosomes, the kinase accumulated to the middle region of the spindle and associated with the midbody between the first polar body and the oocyte (Fig. 3, H and I). Immediately after meiosis I, the oocytes entered meiosis II without interphase. After the formation of the second meiotic spindle at the early MII stage, Plk1 appeared at the spindle poles and then translocalized to the middle region of the spindle until the exit from MII arrest induced by fertilization (Fig. 3, J and K). In our experiments, the eggs extruded their second polar body 2 h after fertilization, and complete pronuclear formation was observed 8 h following insemination. As shown in Figure 4A, the chromosomes of oocytes moved to the spindle poles 2 h after insemination, but the Plk1 was still present in the middle region of the spindle. With the extrusion of the second polar body, Plk1 concentrated in the midbody (Fig. 4B). Following the completion of meiosis II and the decondensation of chromosomes, Plk1 redistributed into the cytoplasm, but its expression could not be detected in the pronuclei (Fig. 4, C–E). When the chromatin began to condense, just before pronuclear membrane breakdown, a significant signal of Plk1 accumulation was detected between the male and female pronuclei (Fig. 4F). With the pronuclear breakdown, Plk1 was found in association with the first mitotic spindle and then concentrated in the midbody during the anaphase and telophase of the first mitosis (Fig. 5, A–D). After the completion of the first cell cycle, two blastomeres formed and both of them entered interphase. At this time, Plk1 distributed evenly in the cytoplasm. Following further cleavage, Plk1 showed the same pattern of migration, with the spindle pole localization at the beginning of metaphase, midbody localization at telophase, and even distribution in interphase (Fig. 5, E–I).
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Localization of Plk1 When the Microtubule Organization Was Disturbed
After treatment of eggs with colchicine, a potent microtubule polymerization inhibitor, the meiotic spindle disappeared, -tubulin distributed evenly in the eggs. Plk1 protein also diffused into the cytoplasm (Fig. 6, A and A'). When MII oocytes were treated with staurosporine, a broad-spectrum protein kinase inhibitor that disturbs the spindle organization as we reported before [25], the spindle was partially disorganized and -tubulin could be found around the chromosomes. The localization pattern of Plk1 was the same for -tubulin (Fig. 6, B and B'). When MII eggs were treated with taxol, the meiotic spindle expanded and many cytoplasmic asters were observed. Plk1 disappeared from the middle region of the spindle and accumulated as numerous clusters in the cytoplasm (Fig. 6, C and C'). In oocytes treated with taxol following pretreatment with staurosporine, multiple cytoplasmic asters could no longer be found and the clusters of Plk1 distribution could not be detected either. Overall, the distribution of -tubulin and Plk1 in the oocytes treated with taxol following pretreatment with staurosporine (Fig. 6, D and D') were similar to those treated with staurosporine alone (Fig. 6, B and B').
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Microinjection of Plk1 Antibody Affects GVBD and Spindle Assembly
The GVBD rates of oocytes 2 or 4 h after antibody injection were 35.29% (24/68) and 45.31% (29/64), respectively, significantly lower than that of the IgG injection control group (64.58% [31/48] and 68.75% [33/48], respectively). In control oocytes, a typical MI spindle was formed 8 h after culture (25/25) (Fig. 7D), while three types of microtubule organizations were observed in the oocytes injected with Plk1 antibody. Normal spindles with two poles existed in 3 of the total 13 oocytes examined (Fig. 7A). Abnormal spindles with one pole were observed in most injected oocytes (8 of 13) (Fig. 7B). Spindles were absent in the other two oocytes, and microtubules were aggregated randomly around the condensed chromosomes in these oocytes (Fig. 7C). Treatment of oocytes with IBMX for 30 min had no effect on GVBD (68.62% [35/51] 4 h after culture) and normal MI spindle formation after IBMX removal when compared with the nontreatment group (GVBD rate after 4 h culture, 70.16% [181/258]).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Plk1 protein distributed uniformly in mouse oocytes at the GV stage. After GVBD, before the establishment of a bipolar spindle, Plk1 aggregated to form several condensed dots surrounding the chromosomes. At this stage of meiosis, several microtubule organizing centers (MTOCs) formed, and the multiarrayed microtubules were tightly associated with the chromatin mass [26]. It is obvious that the Plk1 distribution was similar to that of the MTOC arrangement at this stage. At the MI stage, the Plk1 concentrated to the spindle poles, the loci of MTOC at metaphase, and lined as two opposite thick bands until the transition from metaphase to anaphase. All these results indicated that Plk1 took part in not only the maintenance but also the initiation of spindle microtubule organization.
There was evidence that Plk1 began to translocate from spindle poles to the equatorial region of the spindles at MII stage. This phenomenon is also found in mitotic cells. It is proposed that Plks play a role in regulating the APC, which directs the degradation of mitotic cyclins and the separation of sister chromatids in mitosis. Such a conclusion was obtained in budding yeast and Xenopus [2, 8, 14]. However, the effects of perturbing Plk function in some other organism appear less supportive of such a role. In Drosphila polo1 mutants, cyclin B degradation appears to take place normally in mutant cells that show defects both in chromosome separation and cytokinesis during spermatogenesis [16]. This contradiction suggested Plk might play roles in other ways. It was reported that the microtubule nucleation capacity of centrosomes is diminished at this time [27] and kinetochores function as active MTOCs. The migration of Plk1 from spindle poles to the equator may reflect the changes of microtubule organizing activity within the metaphase spindles.
At telophase I and II, as the cleavage furrow contracts, the compact microtubule-containing structure known as the midbody is formed. Plk1 is localized to the midbody at telophase. Previous results suggest that Plk can bind and phosphorylate the kinesin CHO1/MLKP1 [28] and is important for ensuring the correct assembly of the midzone and midbody microtubule complex as well as completion of cytokinesis [29]. Several researchers have provided direct evidence for plk's roles in cytokinesis. In the absence of fission yeast plo1, multinucleate cells are produced, in which neither an actin ring nor a septum is formed [2]. Furthermore, overexpression of yeast or human Plks leads to formation of multiple septa at any stage of the cell cycle [17, 30]. We can infer from this phenomenon that Plk1 is involved in the extrusion of both the first and the second polar bodies.
After fertilization, Plk1 accumulated between the two pronuclei as they were closely apposed. Our previous work showed that several foci of centrosomes existed in association with the pronuclei, and typically a pair resided between the adjacent pronuclei when the pronuclei were moved into close apposition at the cell center. Furthermore, the microtubule-organizing centrosome increases in size to fill the egg cytoplasm with a dense matrix of assembled tubulin protein [31]. Plk1 may accumulate at the centrosome and serve as a key factor involved in the microtubule organization at this stage. In our experiment, the subcellular localization of Plk1 during the first and second mitosis was also studied. Plk1's association with metaphase spindle and midbody was similar to its distribution pattern during meiosis, so we suggest that Plk1 functions as a microtubule-associated protein kinase in both meiosis of oocytes and mitosis of zygotes or blastomeres.
Since Plk1 accumulated at the spindle pole, the middle of the spindle, and midbody at different stages of the cell cycle, we propose that this kinase is always localized to the subcellular area where microtubule organization takes place. It has been shown that, in mouse oocytes, 16 cytoplasmic centrosomes can be detected [32]. However, Plk1 has only been found on the spindle poles at early metaphase. Previous research suggested that the accumulation of Plk1 is a specific feature of the spindle poles [12], but in our experiments, when MII oocytes were treated with taxol, the MII spindle enlarged and several microtubule asters were induced in the oocytes. Correspondingly, Plk1 was found to exist as several dots in the cytoplasm. This result further supports our suggestion that the distribution of Plk1 is associated with the organization of microtubules.
The localization of Plk1 suggests its functional roles in microtubule organization. However, Plk1 cannot account for microtubule organization by itself. Our results showed that the MII spindle was destroyed after staurosporine treatment and taxol could no longer induce microtubule organization or Plk1 aggregation in cytasters. Staurosporine, a broad-spectrum protein kinase inhibitor, could inhibit the activity of protein kinase C (PKC). A recent report showed that PKC was localized to the meiotic spindle during MI/MII transition and then to the chromosomes at MII [33]. Our previous work also suggested that PKC cross-talks with MAPK [34, 35]. It is very possible that a staurosporine-sensitive kinase, such as PKC, is involved in the regulation of Plk1 localization.
In GV oocytes, GVBD was partly inhibited when the Plk1 antibody was microinjected into the cytoplasm. It is well known that maturation promoting factor (MPF) is activated at GVBD and its activation triggers chromatin condensation and nuclear envelope breakdown. Compelling data [6, 22, 36] implicate that Plk participates in the MPF amplification loop in meiotic maturation of oocytes of several species, although it might not be the only trigger for MPF activation. Perhaps just because the activation of MPF was disturbed after Plk1 antibody injection, the GVBD was inhibited to a substantial extent. Microinjection of Plk1 or Plx antibody into Hela cells and Xenopus embryos resulted in the formation of monopolar spindles [37, 38]. We also found that, in the oocytes that went through GVBD, the assembly of meiotic spindles at the MI stage was disturbed, which strongly suggested that Plk1 was involved in the meiotic apparatus organization, just as it was in mitosis.
Taken together, our results suggest that Plk1 is associated with the microtubule organizing centers or cytokinetic apparatus during meiotic progression, polar body extrusion, pronucleus apposition, and blastomere cleavage in mouse oocytes, fertilized eggs, and early embryos. The subcellular localization of this kinase may reflect its specific roles in the regulation of microtubule organization by centrosomes. Further studies are necessary to identify the substrates and upstream kinases of Plk1 in the MTOCs.
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FOOTNOTES |
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1 Supported by grants from the Special Funds for Major State Basic Research (973) Project (G1999055902) and Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-SW-303). 
2 Correspondence. FAX: 8610 6256 5689; sunqy{at}panda.ioz.ac.cn
Accepted: March 6, 2002.
Received: December 20, 2001.
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-, ß- and -tubulins in a stable complex. Biochem J 1999; 339:435-442This article has been cited by other articles:
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