BIOLOGY OF REPRODUCTION 62, 1184–1192 (2000)
Spindle Formation and Dynamics of g-Tubulin and Nuclear Mitotic Apparatus
Protein Distribution During Meiosis in Pig and Mouse Oocytes1
Jibak Lee,3,4 Takashi Miyano,2,4 and Robert M. Moor3
Laboratory of Protein Function,3 The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom
The Graduate School of Science and Technology,4 Kobe University, Nada-ku, Kobe 657–8501, Japan
ABSTRACT
This work focuses on the assembly and transformation of the
spindle during the progression through the meiotic cell cycle.
For this purpose, immunofluorescent confocal microscopy was
used in comparative studies to determine the spatial distribution
of a- and g-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, g-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, g-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 g-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 g-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 g-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 g-tubulin at the spindle poles in mitosis,
NuMA and g-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.
INTRODUCTION
The establishment of a bipolar spindle is essential for
the accurate segregation of chromosomes in mitosis and
This work is supported in part by a grant for ‘‘Research for the Future’’
Program from The Japan Society for the Promotion of Science (JSPSRFTF97L00905).
2
Correspondence. FAX: 81 78 803 5807; e-mail: [email protected]
1
Received: 20 September 1999.
First decision: 19 October 1999.
Accepted: 29 December 1999.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
meiosis. In somatic cells, a centrosome comprising a pair
of centrioles surrounded by pericentriolar material (PCM)
defines both the location of the microtubule organizing center (MTOC) and the polarity of microtubule arrays with
their growing plus ends extending away from the MTOC.
During S phase, the centrosome is duplicated, and just before mitosis the duplicated centrosomes are separated to
enable them to serve as two mitotic spindle poles. Microtubules, which are nucleated from the centrosomes, search
the nucleo- and cytoplasm by alternating rapidly between
growing and shrinking phases until some are captured and
stabilized by interacting with kinetochores in the so-called
‘‘search and capture’’ mechanism of mitotic spindle formation [1]. Thereafter, a centrosome together with an accompanying set of chromosomes is distributed into each
daughter cell at the end of mitosis, thus ensuring that both
the centrosome and chromosome numbers are accurately
conserved through successive cell generations [2].
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]. g-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 g-tubulin, which probably nucleates meiotic spindle microtubules at the poles. In contrast, metaphase IIarrested 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,
g-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, g-tubulin is
bound along the length of the spindle microtubules during
the formation of the spindle, and it is only after prometaphase that g-tubulin becomes heavily concentrated to the
spindle poles [16]. In Drosophila oocytes, the surprising
feature is that g-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 bi-
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g-TUBULIN AND NuMA IN MAMMALIAN OOCYTES
polar 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 g-tubulin and NuMA localization throughout
meiotic maturation in pig oocytes. To this end, we conducted an immunofluorescent confocal analysis with antibodies against a-tubulin, g-tubulin, and NuMA in pig oocytes. For comparative purposes, we also determined the
localization patterns of NuMA and g-tubulin in pig somatic
cells and mouse oocytes.
MATERIALS AND METHODS
Culture of Pig Oocytes
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-oocytegranulosa 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 38.58C [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
1185
pig oocytes were cultured for 3 h in media containing 10
mg/ml nocodazole (Sigma) and 10 mg/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 mg/ml gentamicin, and 3 mg/ml BSA
in an atmosphere of 5% CO2 in humidified air at 378C.
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 38.58C.
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-a-tubulin antibody (1:100 dilution, MAS 078; Harlan Sera Lab
Ltd, Sussex, UK), rabbit polyclonal anti-g-tubulin antibody
(1:1000 dilution; T3559, Sigma), or rabbit polyclonal antiNuMA antibody (1:1000 dilution, described in [25]) at 48C
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 BioRad (Hercules, CA) MRC 500 or 1024 system. Control
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LEE ET AL.
FIG. 1. Imunofluorescence localization of a-tubulin in pig aortic endothelial cells during mitosis (a–h) and in pig oocytes during meiosis (i–p). aTubulin appears in green and DNA appears in red. Yellow reflects the overlapping of a-tubulin and DNA images. Mitotic stages are a) interphase, b)
prophase, c) prometaphase, d) metaphase, e) early anaphase, f) late anaphase, g) telophase, h) cytokinesis. The comparable meiotic stages are i) G2 or
the so-called GV stage, j) early diakinesis, k) late diakinesis, l) metaphase I, m) early anaphase I, n) late anaphase I, o) telophase I, p) metaphase II.
Bar 5 10 mm.
oocytes and somatic cells were treated exactly as those described above excepting that the first antibodies were replaced by BSA-PBS-PVA.
RESULTS
Change in Spindle Microtubules During Mitosis and Pig
Oocyte Maturation
In pig somatic cells and oocytes, changes of microtubule
localization during mitosis and meiosis were examined using anti-a-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).
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 asso-
g-TUBULIN AND NuMA IN MAMMALIAN OOCYTES
1187
FIG. 2. Immunofluorescence localization of g-tubulin in pig aortic endothelial cells during mitosis (a–h) and in pig oocytes during meiosis (i–p). gTubulin appears in green and DNA appears in red. Panels show identical mitotic and meiotic stages as presented in the Figure 1 legend. The arrow in
o indicates the position of other segregated chromosomes that are out of focus in the figure. Bar 5 10 mm.
ciated 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 antirat (for a-tubulin) or anti-rabbit (for g-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 g-Tubulin During Mitosis and Meiosis
In pig somatic cells, g-tubulin was detected as a spot
that delineated the centrosome in the interphase nucleus
(Fig. 2a). By prophase, the g-tubulin spot was split into
two foci (Fig. 2b). During the convergence of chromosomes on the metaphase plate, g-tubulin was detected at
the spindle poles (Fig. 2, c and d). From anaphase to telophase (Fig. 2, e–g), g-tubulin was continuously detected
at the spindle poles. At cytokinesis, g-tubulin was localized at spindle midbody, as previously reported [27] (Fig.
2h).
During meiosis, g-tubulin was diffusely localized in the
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FIG. 3. Immunofluorescence localization of NuMA in pig aortic endothelial cells during mitosis (a–h) and in pig oocytes during meiosis (i–p). NuMA
appears in green and DNA appears in red. Yellow reflects the overlapping of NuMA and DNA images. Panels show identical mitotic and meiotic stages
as presented in the Figure 1 legend. Bar 5 10 mm.
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 g-tubulin
signals were detected in the GV, especially near the nucleolus (Fig. 2j). In late diakinesis after GVBD, amorphous g-tubulin staining was detected, with the higher
concentrations surrounding the chromatin clusters (Fig.
2k). At metaphase I, g-tubulin was detected along spindle
microtubules (Fig. 2l). At early anaphase I, g-tubulin became aggregated around the spindle poles (Fig. 1m). In
late anaphase I and telophase I (Fig. 1, n and o), g-tubulin
became progressively more intensely localized around the
spindle midzone, but was completely excluded from the
midbody itself. At metaphase II (Fig. 1p), g-tubulin was
again localized along spindle microtubules in a pattern
similar to that of metaphase I. In addition to its spindle-
associated localization, g-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).
During meiotic maturation of pig oocytes, NuMA was
g-TUBULIN AND NuMA IN MAMMALIAN OOCYTES
FIG. 4. Disappearance of NuMA foci around spindle poles of pig oocytes after nocodazole treatment. Pig metaphase II oocytes were treated
with 10 mg/ml nocodazole for 3 h, then fixed and subjected to immunofluorescent confocal microscopy with anti-NuMA antibody. Correlative
DNA (a) and NuMA (b) staining is shown. A chromatin cluster in the first
polar body is indicated by an arrowhead. Arrows indicate the position of
aligned chromosomes. Bar 5 10 mm.
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 Dynamics
on the Metaphase II Spindle in Pig Oocytes
When pig metaphase II oocytes were cultured for 3 h in
the medium containing 10 mg/ml nocodazole, spindle mi-
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FIG. 6. Immunofluorescence localization of g-tubulin (a) and NuMA (b)
in mouse metaphase I oocytes. g-Tubulin and NuMA appears in green in
a and b, respectively, and DNA appears in red. An arrow indicates a
cytoplasmic g-tubulin aggregation. Bar 5 10 mm.
crotubules 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). g-Tubulin along spindle microtubules
also disappeared (data not shown).
When pig metaphase II oocytes were cultured for 3 h in
the medium containing 10 mg/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 g-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).
Species-Specific Localization Patterns of g-Tubulin
and NuMA During Meiosis
For comparative purposes, mouse metaphase I oocytes
were fixed and stained with anti-g-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. g-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 g-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 g-tubulin and NuMA during meiotic maturation, even
between different mammalian species (see Figs. 2l and 3l).
DISCUSSION
FIG. 5. Appearance of additional NuMA foci and concentrated g-tubulin
staining around spindle poles of pig oocytes after taxol treatment. Pig
metaphase II oocytes were treated with 10 mg/ml taxol for 3 h, then fixed
and subjected to immunofluorescent confocal microscopy with antiNuMA (a and b) and anti-g-tubulin (c and d) antibodies. Correlative DNA
(a, c) and NuMA or g-tubulin (b, d) staining is shown. A chromatin cluster
in the first polar body is indicated by an arrowhead. Arrows indicate the
position of aligned chromosomes. Bar 5 10 mm.
In fully grown G2-arrested mouse oocytes, several electron-dense condensations from which microtubules radiate
have been observed at the periphery of the GV [3]. These
structures have been identified as MTOCs using the 5051
antibody directed against human PCM [29]. The PCM [4]
and g-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
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FIG. 7. The postulated reorganization of
microtubule polarity during meiotic spindle elongation. In mitosis in theoretical
considerations, the minus ends of microtubules are kept attached to centrosomes
that are localized at spindle poles throughout chromosome segregation and spindle
elongation. In contrast, during segregation
in meiosis, chromosomes go past the positions occupied previously by the spindle
poles because of the absence of definitive
centrosomes to act as a block to chromosome passage. As a result, chromosomes
occupy the ends of the elongated spindle,
and microtubules are repositioned such
that the plus ends of the kinetochore microtubules grow outward and the minus
ends of microtubule are located inside the
elongated spindle. In this figure, the microtubules nucleated from the spindle
midbody are omitted for the sake of the
simplification.
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. g-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 g-tubulin was detected after GVBD
around condensed chromosomes where microtubule polymerization was detected. g-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 g-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, g-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 g-tubulin is localized not only at the centrosome but also on the surface
of spindle microtubules in mitotic cells, suggesting that gtubulin might act as a microtubule-stabilizing protein.
Therefore, g-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 enddirected 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 g-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 gtubulin 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 g-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 spin-
g-TUBULIN AND NuMA IN MAMMALIAN OOCYTES
dle 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 animalvegetal 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 g-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, g-tubulin
and NuMA consistently localized at the spindle poles before g-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. g-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 gtubulin 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.
1191
ACKNOWLEDGMENT
The authors thank Dr. Duane A. Compton (Dartmouth Medical School,
New Hampshire) for his generous contribution of anti-NuMA antibody.
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