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-Tubulin and Nuclear Mitotic Apparatus Protein Distribution During Meiosis in Pig and Mouse Oocytes1
a Laboratory of Protein Function, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom
b The Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe 657–8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and 
-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, -tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, -tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but -tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while -tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several -tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and -tubulin at the spindle poles in mitosis, NuMA and -tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, meiotic spindles in mammalian oocytes lack centrioles, which are present only up to the pachytene stage during oogenesis [3]. In mouse oocytes, several PCM foci have been found at the acentriolar meiotic spindle poles as well as in the cytoplasm [4]. -Tubulin, a new member of the tubulin superfamily [5] that functions in microtubule nucleation [6, 7], has been found at the spindle poles and cytoplasmic MTOCs in mouse metaphase II-arrested oocytes [8, 9]. Therefore, despite the absence of a definitive centrosome, mouse meiotic spindles contain foci of PCM including -tubulin, which probably nucleates meiotic spindle microtubules at the poles. In contrast, metaphase II-arrested oocytes of the pig [10], sheep [11], and cow [12] do not have cytoplasmic MTOC. Further, the patterns of centrosome inheritance during fertilization differ among different mammalian species, especially between murine and other mammalian eggs: soon after sperm incorporation, microtubules assemble to form a sperm aster in association with the male pronucleus in most mammals, whereas microtubules assemble at first in association with the maternal PCM foci in murine species [13]. Despite those differences, -tubulin localization during meiotic maturation of oocytes has scarcely been reported in mammals other than the mouse.
Recently, the pathway of meiotic spindle assembly has been extensively investigated in Drosophila and Xenopus oocytes, in addition to mammalian oocytes. In both species, microtubules start to grow randomly around a mass of meiotic chromosomes, then are self-organized into bipolar arrays [14, 15]. In Xenopus oocytes, -tubulin is bound along the length of the spindle microtubules during the formation of the spindle, and it is only after prometaphase that -tubulin becomes heavily concentrated to the spindle poles [16]. In Drosophila oocytes, the surprising feature is that -tubulin is not detected at the poles of the meiosis I spindle [17]. In those oocytes, the mechanism that changes the random arrays of microtubules into bipolar arrays requires the sorting of microtubules according to their polarity by microtubule motors and their associated proteins [15]. Nuclear mitotic apparatus protein (NuMA), an essential component of the motor complex, has been observed in all interphase nuclei and at all mitotic spindle poles in every type of somatic cell in humans and in a human/hamster hybrid cell [18]. NuMA has also been found in all other mammalian cells examined and in some nonmammalian cells [19]. Immunodepletion of NuMA reveals that NuMA forms a complex with cytoplasmic dynein and dynactin in Xenopus eggs [20]. Moreover, NuMA is essential for microtubule aster formation in Xenopus egg extracts [20], in human cells, and in the human cell-free system [21]. Therefore, NuMA is one of the candidates for the components of meiotic spindle poles in mammalian oocytes. However, the localization of NuMA during oocyte maturation has not been reported in mammalian oocytes so far. Further, the localization of NuMA during meiosis I to meiosis II transition has not been reported in any animals, although it is of particular interest because of the omission of formation of the interphase nucleus during that period.
The purpose of the reported study was to investigate how the meiotic spindle is formed and transformed during the meiosis I to meiosis II transition and to determine the dynamics of both -tubulin and NuMA localization throughout meiotic maturation in pig oocytes. To this end, we conducted an immunofluorescent confocal analysis with antibodies against -tubulin, -tubulin, and NuMA in pig oocytes. For comparative purposes, we also determined the localization patterns of NuMA and -tubulin in pig somatic cells and mouse oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pig ovaries were obtained from prepubertal gilts at a local slaughterhouse. After three washes in Dulbecco's PBS containing 0.1% polyvinyl alcohol (PBS-PVA), intact healthy antral follicles of 4–6 mm in diameter were dissected in PBS-PVA from ovaries according to the technique described by Moor and Trounson [22]. After being opened in 25 mM Hepes-buffered Medium 199 (Earle's salt; Gibco, Paisley, UK) containing glutamine and 0.08 mg/ml kanamycin sulphate (Sigma, St. Louis, MO), cumulus-oocyte complexes with a piece of parietal granulosa tissue (cumulus-oocyte-granulosa cell complexes) were isolated from the follicles [23]. After two washes in Hepes-buffered Medium 199, cumulus-oocyte-granulosa cell complexes were cultured in 2 ml of bicarbonate-buffered Medium 199 supplemented with 10% fetal calf serum (FCS; Biocell Co. Ltd., Carson, CA), 0.1 mg/ml sodium pyruvate, 0.08 mg/ml kanamycin sulphate, 0.1 IU/ml human menopausal gonadotropin (Pergonal; Serono, London, UK), and two everted theca shells with gentle agitation in an atmosphere of 5% CO2 in humidified air at 38.5°C [24]. Pig oocytes were cultured for various times to obtain oocytes at the germinal vesicle (GV; directly from the follicle at 0 h), early diakinesis (18 h), late diakinesis (21 h), metaphase I (24–27 h), anaphase I, telophase I (30–33 h), and metaphase II (42 h) stages.
Nocodazole and Taxol Treatment
To examine the effects of inhibition of polymerization and depolymerization of microtubules on pig meiotic spindles, after 42 h of maturation culture, metaphase II-arrested pig oocytes were cultured for 3 h in media containing 10 µg/ml nocodazole (Sigma) and 10 µg/ml taxol (Sigma), respectively.
Culture of Mouse Oocytes
Mouse oocytes were released from large antral follicles of F1 CFLP a/a females (1–2 mo old) that had received injections 48 h previously of 5 IU of eCG. The cumulus cells were removed by pipetting, and the oocytes were cultured for 8 h in Medium 199 supplemented with 0.1 mg/ml sodium pyruvate, 25 µg/ml gentamicin, and 3 mg/ml BSA in an atmosphere of 5% CO2 in humidified air at 37°C.
Culture of Pig Somatic Cells
Pig aortic endothelial cells were grown in nutrient mixture Ham's F-12 with L-glutamine (Gibco) containing 10% FCS to 30% confluence on glass chamber slides (Lab-Tek 2-well; Nalge Nunc International, Naperville, IL). To obtain a relatively higher population of mitotic cells, cells were cultured for 21–24 h without serum (serum starvation) and then for a further 23 h in the medium containing 10% FCS, before fixation. The cells were cultured in an atmosphere of 5% CO2 in humidified air at 38.5°C.
Immunofluorescent Confocal Microscopy
After being washed twice in PBS-PVA, denuded oocytes were fixed in 4% paraformaldehyde either in PBS-PVA or in PHEM (60 mM PIPES pH 6.9, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2) containing 0.2% Triton X-100 for 40 min. Both solutions gave essentially the same results. The fixed oocytes were washed twice in PBS-PVA for 15 min each, then stored in 1% BSA-supplemented PBS-PVA (BSA-PBS-PVA) at least overnight. The oocytes were blocked with 10% goat serum in BSA-PBS-PVA for 45 min, and incubated with either rat monoclonal anti--tubulin antibody (1:100 dilution, MAS 078; Harlan Sera Lab Ltd, Sussex, UK), rabbit polyclonal anti--tubulin antibody (1:1000 dilution; T3559, Sigma), or rabbit polyclonal anti-NuMA antibody (1:1000 dilution, described in [25]) at 4°C overnight. After being washed 3 times in BSA-PBS-PVA for 15 min each, the oocytes were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG or anti-rat IgG (1:40; Dako A/S, Glostrup, Denmark) for 40 min at room temperature. After being washed 3 times in BSA-PBS-PVA for 15 min each, DNA was counterstained with propidium iodide (Sigma). The oocytes were washed 3 times in BSA-PBS-PVA before being mounted on slide glasses. Determination of the meiotic stages was based on the criteria of Motlik and Fulka [26]. At least 25 oocytes incubated with each antibody were observed at each meiotic stage, and the representative confocal images are shown in the figures cited in Results.
Somatic cells were simply washed once with PBS-PVA, then fixed in 4% paraformaldehyde in PHEM for 10 min, washed twice in PBS-PVA for 5 min each, and permeabilized with 0.2% Triton X-100-supplemented PHEM for 5 min. After being washed twice again, cells were blocked with 10% goat serum in BSA-PBS-PVA and incubated with the first antibodies in BSA-PBS-PVA at appropriate dilutions for 1 h at room temperature. After two washes, cells were incubated with the secondary antibodies, and DNA was counterstained with propidium iodide.
Confocal analysis was carried out using either the Bio-Rad (Hercules, CA) MRC 500 or 1024 system. Control oocytes and somatic cells were treated exactly as those described above excepting that the first antibodies were replaced by BSA-PBS-PVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In pig somatic cells and oocytes, changes of microtubule localization during mitosis and meiosis were examined using anti--tubulin antibody (Fig. 1). Somatic cells had an MTOC/centrosome in interphase (Fig. 1a). By prophase, the centrosome had duplicated and separated (Fig. 1b). Some of the microtubules that were nucleated from the separated centrosomes were captured by chromosomes (Fig. 1c). At the completion of the capture process, chromosomes became aligned at the equator of the spindle (Fig. 1d). As the aligned chromosomes separated and moved polewards, the spindle elongated by cross-linking interdigitating microtubules at the center of spindle (Fig. 1, e and f). Finally, chromosomes and centrosomes were distributed into two daughter cells by cytokinesis (Fig. 1, g and h).
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Fully grown pig oocytes at the GV stage did not have a detectable microtubule network in the cytoplasm (Fig. 1i). In early diakinesis when bivalent chromosomes were formed and just before GV breakdown (GVBD), microtubules were polymerized around periphery of the GV and in the cell cortex (Fig. 1j). In late diakinesis after GVBD and when chromosomes were aggregated into a chromatin cluster, randomly arrayed microtubules were tightly associated with the chromatin cluster (Fig. 1k). The microtubules were in the process of becoming two polarized arrays as chromosomes became aligned on the metaphase plate (Fig. 1l). The meiotic spindle pole at metaphase I was composed of several bundles of fusiform microtubule arrays, with the spindle axis perpendicular to the cell surface. During chromosome segregation at anaphase I and telophase I (Fig. 1, m–o), the spindle elongated and rotated, with the axis becoming parallel to the cell surface. After first polar body emission, the formation of the meiosis II spindle followed the same process as that for the meiosis I spindle (Fig. 1p).
In the control cells, which were treated with only anti-rat (for -tubulin) or anti-rabbit (for -tubulin and NuMA) secondary antibody, no preferential labeling associated with the spindle or the nucleus appeared, although very faint staining was observed in the whole cytoplasm.
Localization of -Tubulin During Mitosis and Meiosis
In pig somatic cells, -tubulin was detected as a spot that delineated the centrosome in the interphase nucleus (Fig. 2a). By prophase, the -tubulin spot was split into two foci (Fig. 2b). During the convergence of chromosomes on the metaphase plate, -tubulin was detected at the spindle poles (Fig. 2, c and d). From anaphase to telophase (Fig. 2, e–g), -tubulin was continuously detected at the spindle poles. At cytokinesis, -tubulin was localized at spindle midbody, as previously reported [27] (Fig. 2h).
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During meiosis, -tubulin was diffusely localized in the GV of fully grown G2-arrested pig oocytes (Fig. 2i) and was also sometimes detected as cytoplasmic aggregates. Just before GVBD in early diakinesis, strong -tubulin signals were detected in the GV, especially near the nucleolus (Fig. 2j). In late diakinesis after GVBD, amorphous -tubulin staining was detected, with the higher concentrations surrounding the chromatin clusters (Fig. 2k). At metaphase I, -tubulin was detected along spindle microtubules (Fig. 2l). At early anaphase I, -tubulin became aggregated around the spindle poles (Fig. 1m). In late anaphase I and telophase I (Fig. 1, n and o), -tubulin became progressively more intensely localized around the spindle midzone, but was completely excluded from the midbody itself. At metaphase II (Fig. 1p), -tubulin was again localized along spindle microtubules in a pattern similar to that of metaphase I. In addition to its spindle-associated localization, -tubulin was also present near the cortex of maturing pig oocytes in a manner analogous to that in Xenopus oocytes [28].
Localization of NuMA During Mitosis and Meiosis
In pig somatic cells, NuMA was detected in the nucleus during interphase (Fig. 3a). By prometaphase, NuMA had become translocated at the spindle poles (Fig. 3, b and c), where it localized in a characteristic crescent shape (Fig. 3d). During anaphase (Fig. 3, e and f), NuMA was still localized at the spindle poles, but by telophase and during cytokinesis, the intensity of NuMA staining at the spindle poles diminished, and it became uniformly distributed throughout the nucleus (Fig. 3, g and h).
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During meiotic maturation of pig oocytes, NuMA was evenly spread across the nucleoplasm in the GV stage (Fig. 3i). In early diakinesis before GVBD, when individual bivalents start to form, some discrete NuMA foci were first detected on or close to chromosomes; however, most of the NuMA protein was still diffusely localized in the nucleus at this time (Fig. 3j). After GVBD, when chromosomes assembled to form chromatin clusters at the late diakinesis stage, NuMA became tightly associated with the chromosome cluster (Fig. 3k). At both meiotic metaphases, NuMA was localized at the spindle poles as discrete spots (Fig. 3, l and p). In anaphase and telophase, when chromosomes separated, NuMA translocated to the spindle midzone (Fig. 3, m–o).
Effects of Inhibition of Microtubule Dynamicson the Metaphase II Spindle in Pig Oocytes
When pig metaphase II oocytes were cultured for 3 h in the medium containing 10 µg/ml nocodazole, spindle microtubules disappeared (data not shown). In two thirds (18 of 29) of the oocytes, chromosomes were scattered in the ooplasm. In other oocytes, chromosomes still remained aligned on a line as if there had been a metaphase plate (Fig. 4a), but NuMA foci representing spindle poles disappeared (Fig. 4b). -Tubulin along spindle microtubules also disappeared (data not shown).
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When pig metaphase II oocytes were cultured for 3 h in the medium containing 10 µg/ml taxol, more concentrated microtubules were observed in the spindle (data not shown). In two thirds of these oocytes (18 of 28), the spindle was largely deformed. In other oocytes, the bipolar shape of the spindle was preserved, and chromosomes were still aligned at metaphase plate. In the oocytes, additional NuMA foci appeared around the polar regions of the spindle (Fig 5, a and b). In addition, more concentrated -tubulin was detected around the polar regions of the spindle, although the signal was amorphous and did not form aggregates as did NuMA foci (Fig. 5, c and d).
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Species-Specific Localization Patterns of -Tubulinand NuMA During Meiosis
For comparative purposes, mouse metaphase I oocytes were fixed and stained with anti--tubulin antibody (Fig. 6a) and anti-NuMA antibody (Fig. 6b). In contrast to the pig metaphase I spindle, the axis of the mouse metaphase I spindle was parallel to the cell surface. -Tubulin was detected as several foci at the meiotic spindle poles as well as in cytoplasmic foci as previously reported [8, 9]. NuMA was localized along spindle microtubules. When the localization pattern of -tubulin and NuMA in mouse oocytes is compared with the localization pattern in pig oocytes, it is evident that major differences exist in the distribution of both -tubulin and NuMA during meiotic maturation, even between different mammalian species (see Figs. 2l and 3l).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin [8] foci are also observed in the cytoplasm of mouse metaphase II-arrested oocytes in addition to the MTOCs that are associated with the meiotic spindle poles. Therefore, the origin of the mouse meiotic spindle pole seems to be associated with those MTOCs near the condensed chromosomes that are recruited from the cytoplasm during the process of spindle assembly [4, 29]. In pig oocytes, Kim et al. [10] have reported that microtubules are not detected at the GV stage, that microtubule asters are first observed near the condensed chromatin after GVBD, and that microtubules in the meiotic spindle become apparent only during the later meiotic stages. -Tubulin localization during pig oocyte maturation has not been reported. In the present article, we found that cytoplasmic free microtubules first appeared around the periphery of the GV and in the cell cortex just before, or at the time of, GVBD in pig oocytes. After GVBD, multi-arrayed microtubules were found tightly associated with the condensed chromosomes. These findings raise the question of how microtubule polymerization is induced in the vicinity of the chromosomes. Very recently, it has been proposed that RCC1, a component of chromatin, generates a high local concentration of Ran-GTP around chromatin, which in turn induces the local nucleation of microtubules around chromosomes in Xenopus egg extracts [30]. In the present study, amorphous staining of -tubulin was detected after GVBD around condensed chromosomes where microtubule polymerization was detected. -Tubulin has been found to form a ring-like complex at the centrosomes of Drosophila [31] and in Xenopus egg extracts [7], and it has been shown to act as a microtubule-nucleating unit that can cap the minus ends of microtubules in vitro [7]. Therefore, the high local concentration of -tubulin in its ring-like configuration, in association with Ran-GTP, might also play an important role in the microtubule nucleation in the vicinity of the condensed chromosomes in pig oocytes. In metaphase I pig oocytes, -tubulin becomes relocated along spindle microtubules in a pattern similar to that in the meiotic spindle of Xenopus oocytes [16]. Lajoie-Mazenc et al. [32] have shown by immunoelectron microscopy that -tubulin is localized not only at the centrosome but also on the surface of spindle microtubules in mitotic cells, suggesting that -tubulin might act as a microtubule-stabilizing protein. Therefore, -tubulin might contribute to the polymerization of spindle microtubules in pig oocytes by capping the microtubule-minus ends, by stabilizing the microtubule lattice, or by performing both functions.
NuMA has been shown to complex with certain other proteins, including minus end-directed motors, and to be essential for the assembly and the stabilization of the mitotic spindle pole in Xenopus [20] and the human cell-free system [21]. In the present study, NuMA was detected diffusely in the GV of pig oocytes; and after GVBD, it was shown to localize in several discrete spots in the vicinity of the condensed chromosomes. The NuMA foci were translocated from the condensed chromosomes and became aligned at both poles of the barrel-shaped spindle at metaphase I. These results suggest that the microtubules, which polymerize randomly around the condensed chromosomes, are bundled at their minus ends by the NuMA/minus end-directed motor complex at late diakinesis. Thereafter, the bundles of minus ends break away from the chromosomes and assemble at both meiotic spindle poles at metaphase I. By carrying out comparative studies, we found that some essential differences existed in meiotic spindle pole formation between rodents and ungulates: the pig meiotic spindle pole was composed of several NuMA foci while the mouse meiotic spindle pole was composed of -tubulin foci. Furthermore, the pig meiotic spindle poles, represented by NuMA foci, disappeared with the depolymerization of the spindle microtubules by nocodazole treatment, whereas the mouse meiotic spindle poles represented by -tubulin foci are resistant to the nocodazole or cold treatment that depolymerizes microtubules [8, 9]. In taxol-treated pig metaphase II oocytes, additional NuMA aggregates around meiotic spindle poles were observed; but -tubulin did not form foci, although it became more concentrated around the poles. These differences probably reflect differences in the origins of the pig and the mouse meiotic spindle poles: the pig meiotic spindle pole results from bundling of spindle microtubules by NuMA whereas the mouse meiotic spindle pole results from the gathering of pre-existing cytoplasmic MTOCs. In other words, formation of the meiotic spindle poles in pig oocytes is totally dependent on microtubule assembly, while the meiotic spindle poles in mouse oocytes, like centrosomes in somatic cells, can exist independently of the spindle microtubules.
The rotation of the meiotic spindle has been reported in Xenopus oocytes [33]. The Xenopus meiotic spindle elongates parallel to the oocyte surface during prometaphase, but at metaphase, one of the spindle poles anchors to the oocyte cortex and rotates into alignment with the animal-vegetal axis. In pig oocytes, the axis of the meiotic spindle was perpendicular to the oocyte surface at metaphase, but the spindle rotated with the axis parallel to the oocyte surface during telophase. Therefore, the rotation of the pig meiotic spindle occurs in a manner and at a time different from those of the Xenopus meiotic spindle. Although the mechanism of spindle rotation is unclear, it is possible that it is mediated by interaction with actin filaments, since Xenopus meiotic spindle rotation is inhibited by cytochalasin B [16] and a thick microfilament area exists near the cortex in which the pig meiotic spindle is located [10].
We have shown that the localizations of -tubulin and NuMA during anaphase I and telophase I of meiosis in pig oocytes differ greatly from their localizations in pig somatic cells at equivalent stages of mitosis. In mitosis, -tubulin and NuMA consistently localized at the spindle poles before -tubulin was translocated to the midzone and NuMA was distributed throughout the nucleoplasm at the end of M phase. These patterns of distribution are the same as have been reported for other somatic cell types [27, 34]. By contrast, during meiosis both these proteins were localized at the spindle midzone during anaphase I and telophase I in pig oocytes. Interestingly, NuMA appeared to stay during chromosome segregation and spindle elongation in anaphase I at the site that the spindle poles had occupied during metaphase I (see Fig. 3, l–n). As a result, NuMA became localized between the segregated chromosomes within the spindle. In pig oocytes, NuMA may play an important role in the bundling of spindle microtubules not only at spindle poles at metaphase I but also at the spindle midzone during anaphase I and telophase I. -Tubulin localization was similar to the localization of NuMA during early and late anaphase I (compare Fig. 2, l and m, with Fig. 3, l and m). Since -tubulin and NuMA are both minus end-bound proteins [35, 36], the localization of these proteins at the spindle midzone during anaphase I and telophase I provokes our hypothesis that the polarity of meiotic spindle microtubules might become inverted during the spindle elongation. We suggest that the inversion occurs as the segregated chromosomes go past the position at which the spindle poles used to be present at metaphase I (Fig. 7). This hypothesis is supported by the observation in the present study that the chromosomes reached the distal ends of spindle microtubules at late anaphase I and telophase I (Fig. 1, n and o). Furthermore, Endow and Komma [37] have recently proposed that the formation of meiosis II spindle poles in the center of the meiosis I spindle in Drosophila oocytes requires a sorting out of microtubule ends so that minus ends replace plus ends in the center of the meiosis I spindle. Therefore, the reassortment of spindle microtubules during meiotic anaphase and telophase might be a common mechanism that induces chromosome segregation and spindle elongation in a wide variety of centrosome-free meiotic spindles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work is supported in part by a grant for "Research for the Future" Program from The Japan Society for the Promotion of Science (JSPS-RFTF97L00905). 
2 Correspondence. FAX: 81 78 803 5807; miyano{at}ans.kobe-u.ac.jp
Accepted: December 29, 1999.
Received: September 20, 1999.
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