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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02193
1 Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo,
Tokyo 112-8610, Japan.
2 Tateyama Marine Laboratory, Marine and Coastal Research Center, Ochanomizu
University, Koh-yatsu, Umi-no-Hoshi, Tateyama, Chiba 294-0301, Japan.
3 Takiyama 5-7-7, Higashikurume, Tokyo 203-0033, Japan.
Author for correspondence (e-mail:
snemo{at}cc.ocha.ac.jp)
Accepted 1 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODS...RESULTSDISCUSSIONREFERENCES |
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Key words: Centrioles, Centrosomes, Centrosome regulation, Centrosome duplication, Electrofusion, Maturation division, Meiosis, Starfish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODS...RESULTSDISCUSSIONREFERENCES |
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) and later, Washitani-Nemoto et al.
(Washitani-Nemoto et al.,
1994) found that suppression of polar body (PB) extrusions in
artificially activated oocytes induces parthenogenetic development, whereas
eggs that matured normally did not develop, even with artificial activation.
They suggested that the meiotic centrosomes retained in the eggs by the
failure of PB extrusion are diverted to mitosis-organizing centers in the
mitotic spindle, resulting in parthenogenetic development.
Washitani-Nemoto et al.
(Washitani-Nemoto et al.,
1994), Uetake et al. (Uetake
et al., 2002) and Zhang et al.
(Zhang et al., 2004) utilized
the suppression of PB extrusion as a useful tool for analysing the mechanism
of the `paternal inheritance of the centrosomes in development', first noted
by Boveri (Boveri, 1887). As we
know, control of the centrosome inheritance is an issue of fundamental
importance for all sexually reproductive organisms.
Centrosomal behaviour during normal meiosis in starfish oocytes is shown in
Fig. 1. According to Sluder et
al. (Sluder et al., 1989) and
Kato et al. (Kato et al.,
1990), each pole of a meiosis-I spindle in starfish oocytes has a
pair of centrioles (Fig. 1B),
but only one centriole is found in each pole of the meiosis-II spindle
(Fig. 1D). In other words, the
centrioles are not duplicated during meiosis II. Of the four centrioles in
meiosis I, two of them are inherited by the first PB (PB1), another one by the
second PB (PB2), and the remaining one by the mature egg during meiosis
(Fig. 1E).
Uetake et al. (Uetake et al.,
2002) used starfish oocytes that had formation of their PB
suppressed to investigate the behaviour of all the maternal
centrosomes/centrioles throughout meiosis. When the two pairs of meiosis-I
centrioles were retained in the oocyte by suppression of both PB1 and PB2
extrusion (`0pb egg'), they separated into four single centrioles in meiosis
II, but after completion of the meiotic process, only two were found with the
pronucleus in the mature egg. When the two centrioles of a meiosis-II spindle
were retained in the oocyte by suppression of PB2 extrusion alone (`1pb egg'),
only one was found after meiosis. When these PB-suppressed eggs (0pb and 1pb
eggs) were artificially activated, all the surviving centrioles duplicated to
form pairs, eventually organizing into mitotic spindles. Those findings
demonstrated that there is heterogeneity in the survival and reproductive
capacity of the maternal centrioles and that the centrosomes with the
reproductive centrioles are selectively cast off into the PB (PB1 and PB2),
resulting in a mature egg inheriting a non-reproductive centriole that would
degrade after the completion of meiosis
(Fig. 1E). Uetake et al.
(Uetake et al., 2002) thus
introduced the concept of `nonequivalence' of maternal centrioles.
Tamura and Nemoto (Tamura and Nemoto,
2001) had earlier examined the reproductive capacity of the
centrosomes in PB1 or PB2 by transplanting them into artificially activated
eggs, which revealed that one of the two centrioles in PB1 and the sole
centriole in PB2 are reproductive and able to form bipolar spindles leading to
cleavage and subsequent parthenogenetic development. Based on their results,
they also suggested that the four maternal centrioles are heterogeneous in
their reproductive capacity.
Such `nonequivalence' or `heterogeneity' among the maternal centrioles, however, does not become apparent until the completion of meiosis and an exploration of the mechanisms regulating the centrioles in meiosis, has to address two questions: (1) At what stage of meiosis are the fates of the centrioles determined? (2) What conditions are needed for the loss of function (`degradation') of half of the centrioles?
Our hypothesis was that the fate of the centrioles is determined before the resumption of meiosis, and that some factor in the cytoplasm of mature eggs is responsible for inducing the degradation of the centrioles. In order to test our theory, we developed a new technique for investigating the reproductive capacity of the centrioles. (In this paper we use the term `to degrade', to mean that centrioles lose their capacity to function as the mitotic division poles.)
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MATERIALS AND METHODS Experimental |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODS...RESULTSDISCUSSIONREFERENCES |
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; Picard et al.,
1988; Miyazaki et al.,
2000). The basis of our new experimental procedure is that the
centrosomes are in the loci of the respective premeiotic asters
(Uetake et al., 2002). First, we bisected an immature oocyte (Fig. 2A,B) and removed the GV in the nucleated half with a micropipette (Fig. 2C). The resultant enucleated fragment should retain the pair of premeiotic asters, each with a centrosome at the center. The fragment was then subjected to electric fusion with a mature egg (Fig. 2E), so that the premeiotic centrosomes were suddenly transferred into the mature cytoplasm, without experiencing meiotic divisions. These `heteroplasmic conjugates' (Fig. 2F) were the material for the present study. In one experiment, they were artificially activated without sperm, and then continuously observed by light microscopy for the emergence of single asters or mitotic figures and the occurrence of nuclear divisions. In another experiment, non-activated conjugates were examined by transmission electron microscopy for the number of surviving centrioles.
Oocyte preparation
Oocytes of the starfish Asterina pectinifera during the breeding
season in spring-summer were used. To obtain follicle-free immature oocytes
arrested at prophase of meiosis I, isolated ovaries were treated with
Ca2+-free artificial seawater and then transferred into filtered
natural seawater to induce spawning of oocytes
(Nemoto et al., 1980).
Preparation of the `centrosome-bearing fragments'
The oocytes in seawater were placed in a dish coated with 1% agar and each
one was manually bisected with a fine glass needle into an animal
(GV-containing) and vegetal (non-nucleated) fragment
(Kiyomoto and Shirai, 1993).
The GV-containing fragment was kept as small as possible
(Fig. 2B). A micropipette
connected to a microinjector (IM-5B; Narishige, Tokyo, Japan) on a
micromanipulator (NO-202, Narishige) was then inserted into the GV-containing
fragment, opposite the animal pole (Fig.
2C), and the GV was very slowly and continuously aspirated out
into the micropipette according to the procedure of Miyazaki et al.
(Miyazaki et al., 2000). The
size of the fragments was further reduced to about 100 µm in diameter by
enucleation, resulting in a volume that was about 25% that of an intact oocyte
(160 µm in diameter). An essential feature of our technique is the transfer
of the two premeiotic centrosomes into the cytoplasm of a mature egg, with
minimal transfer of immature cytoplasm, which is the reason for reducing the
size of the non-nucleated fragment. To remove both the jelly layer and the
vitelline coat, the fragments were treated with 0.01% actinase (Kaken
Pharmaceutical, Tokyo, Japan) in seawater for 10-15 minutes and rinsed several
times in seawater before their use as centrosome donors.
Determining the presence of meiotic centrosomes in the fragments
Miyazaki et al. (Miyazaki et al.,
2000) showed that oocytes retain a pair of premeiotic asters even
after aspiration of the GV. To confirm that the two centrosomes in the loci of
the premeiotic asters were retained in our fragments, we carried out indirect
immunofluorescence staining using an anti-
-tubulin antibody, the
specific probe for centrosomes, according to the methods of Uetake et al.
(Uetake et al., 2002). The
fragments deprived of the vitelline coat after treatment with 0.01% actinase
were immersed in an extraction medium, plated onto glass slides, fixed with 6%
paraformaldehyde and incubated overnight with the rabbit anti--tubulin
polyclonal antibody (T3559, Sigma-Aldrich Co., St Louis, MO, USA). Next, the
samples were stained with a Texas Red-labelled goat anti-rabbit IgG antibody
(Biosource International, Camarillo, CA, USA) and examined with a fluorescence
microscope (OPTIPHOT, Nikon, Tokyo, Japan). The two centrosomes appeared as
two spots in the fragment (Fig.
3).
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). Mature eggs with both PB1 and PB2 (cf.
Fig. 2E) were used for the
electric fusion process. It is known that a normally matured egg with both PB1
and PB2 will not cleave even after activation without sperm, indicating the
absence of reproductive centrioles (Obata
and Nemoto, 1984;
Washitani-Nemoto et al., 1994;
Tamura and Nemoto, 2001;
Uetake et al., 2002).
Electric fusion of fragments and mature eggs
A chamber for electric fusion designed by Yoneda
(Yoneda, 2000) was filled with
a 0.88 M solution of mannitol with 0.4 mM CaCl2 and 0.1 mM
MgSO4 (Yoneda,
1997). One fragment and one mature egg were transferred into the
chamber and placed side by side in the center along the line of the electric
field between the two planar electrodes. Each round of electric pulses was
routinely four repetitions of a pulse sequence comprising a high frequency AC
field and a brief rectangular DC pulse
(Yoneda, 1997). The frequency
of the AC field was fixed at 2.5 MHz. The peak-to-peak amplitude was 200
Vp-p/cm. The duration of each sequence was 10-15 seconds. The duration of the
brief rectangular DC pulse was fixed at 50 µseconds. The voltage of the DC
pulse was 250-290 V/cm.
To date, two fusion procedures have been reported, one for fusing two
immature oocytes and another for fusing two maturing oocytes
(Yoneda, 1997;
Yoneda, 2000;
Masui et al., 2001). In the
present study, we developed a new technique for fusing an immature oocyte and
a mature egg, as explained in the Results.
Artificial activation of conjugates
The fusion product, or `conjugate', was activated with 10 µM calcium
ionophore A 23187 (Calbiochem-Novabiochem, La Jolla, CA, USA) for 10 minutes,
rinsed several times in seawater and then allowed to develop.
Light microscopy
A microscope equipped with both differential interference-contrast and
polarization optics (HPD; Nikon, Tokyo, Japan) was used. Microphotographs were
taken with Neopan 400 Presto film (Fuji Photo Film, Tokyo, Japan).
Transmission electron microscopy
Following the procedure of Kato et al.
(Kato et al., 1990), each
conjugate was washed briefly with 0.53 M NaCl solution and fixed with
glutaraldehyde-OsO4 mixture [1% glutaraldehyde, 1% OsO4
and 0.45 M sodium acetate in 0.05 M sodium phosphate buffer (pH 6.4)] for 20
minutes at room temperature. After dehydration in an ethanol series, the
conjugates were stained en bloc with uranyl nitrate and lead acetate, and then
embedded in Poly/Bed 812 (Polyscience Inc., Warrington, UK) on a flat plate of
silicone rubber. The blocks were trimmed to an area of approximately 5 mm and
serially sectioned at 0.15 µm thickness with an ultramicrotome (Ultracut
UCT, Leica, Wien, Austria). However, because the present conjugates were very
large (up to 200 µm in diameter) and there was not a natural marker of the
loci of the asters, we began making serial 1 µm-thick sections until we
found a very faint radial structure, or trace of the aster, and then began
thin sectioning. The thin sections were examined in an electron microscope
(JEM-1230, JEOL, Tokyo, Japan) to determine the number of centrioles in each
of the asters.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODS...RESULTSDISCUSSIONREFERENCES |
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) until a bulge formed on the surface of the mature egg where
it was in contact with the bulge on the fragment surface. The bulges then
fused, which lead to fusion of the entire fragment and mature egg. It took
3-10 minutes to create the heteroplasmic conjugate and up to three conjugates
were often obtained at a time. Immediately after the fusion, the conjugates
were removed from the fusion chamber for a brief rinse with fresh seawater,
and then activated by a 10-minute treatment with calcium ionophore. The narrow
neck joining the pair gradually broadened and they eventually formed a single
sphere.
It is known that the chain of electric pulses for fusion may activate some
mature eggs, as evidenced by the breakdown of the pronuclear envelope, which
takes place about 1 hour later (Yoneda,
1997; Yoneda,
2000). In the case of our conjugates, incidental activation by the
fusion pulse alone may cause the cleavage of the first mitotic cycle. In our
experimental protocol the purpose of starting the ionophore treatment
immediately (within 10 minutes) after the fusion was to cancel any effect of
precocious activation by the fusion pulses.
Development of activated conjugates
On activation with calcium ionophore, the conjugates underwent a cycle of
cleavages (Fig. 4), the first
cleavage furrow appearing about 60 minutes after activation. Often the furrow
regressed (Fig. 4A) and the egg
remained as a single cell (Fig.
4B). At the time of the next cycle such eggs directly divided into
four blastomeres (Fig. 4C,D)
and the third cleavage formed eight blastomeres
(Fig. 4E). The cleavage
interval was about 40 minutes, which is similar to normal embryos.
We consider that it was the fusion with the centrosome-bearing fragment that enabled the activated egg to begin the cycle of cleavages, indicating that the premeiotic centrosomes in the fragment were diverted into the mitosis-organizing centers of the conjugates.
Nuclear events and formation of mitotic asters in the conjugates
For detailed observation of nuclear events and the formation of mitotic
asters, activated conjugates in 80% seawater were compressed to 60 µm
thickness between a glass slide and cover slip. The compression enabled
precise timing of the nuclear changes, although it inhibited the formation of
the cleavage furrow. When microscopic observation started about 5 minutes
after activation at 20°C, the female pronucleus was usually retained. If
it was not, we discontinued observing these conjugates, because they must have
been activated spontaneously long before the ionophore activation.
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). Within 7±3 (mean ± s.d.) minutes of NEBD, two asters suddenly became visible and their loci varied among the conjugates: in some, both asters formed near where the pronucleus had been located, in others, only one aster formed near the site of the pronucleus and the other aster formed at a distance from the nuclear site, and in still other conjugates both asters were located apart from the nuclear site. We designated these three patterns of the location of the formed asters as Patterns 1, 2, and 3 (Figs 5, 6, 7). A common feature of all three patterns so far observed was that the number of asters forming at the first mitosis was always two (Fig. 8).
Pattern 1 (8 conjugates)
As shown in Fig. 5,
polarization microscopy revealed that each of the asters formed a spindle
aster, and a bipolar spindle formed at first mitosis. Two nuclei then emerged.
After the breakdown of the two nuclei in the next round of mitosis, two
bipolar spindles were assembled and four nuclei formed. In the third round,
the four nuclei broke down and four bipolar spindles appeared, resulting in
formation of eight nuclei. Thus in each of the cycles, the number of division
poles and nuclei doubled (Fig.
8).
Pattern 2 (4 conjugates)
At first mitosis, a monopolar (half) spindle formed at the nuclear site
(Fig. 6) with the other aster
remaining at a distance. A nucleus then formed at the site of the monopolar
spindle and following its breakdown in the next round of mitosis, a bipolar
spindle formed and two nuclei then formed. The isolated aster had now doubled.
In the third round, the two nuclei broke down and two bipolar spindles formed,
resulting in formation of four nuclei. The number of isolated asters was now
four. Thus in each of the cycles, the number of asters doubled
(Fig. 8).
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We have thus classified 24 conjugates into Patterns 1, 2 and 3. The remaining 4 of the 28 conjugates failed to undergo the second round of mitosis and were excluded from the analysis.
A feature specific to Pattern 3 conjugates is the appearance of `aster-like structure'. We are confident that this structure is unrelated to the premeiotic centrosomes. A brief notes on the aster-like structure is given later, in the Discussion.
Number of asters and centrioles in the conjugates before ionophore activation
For further analysis of the heterogeneity among meiotic centrioles, we
needed to know the number of surviving centrioles in our conjugates. We kept
formed conjugates in seawater without calcium-ionophore activation. If the
conjugates are incidentally activated by fusion pulses alone, they would
undergo the first cleavage about 1 hour later (cf.
Fig. 4A). Therefore, to avoid
using those conjugates that had been incidentally activated by fusion pulses,
we routinely waited more than 120 minutes and selected those conjugates that
were undivided and retained their spherical profile. They were then subjected
to fixation for transmission electron microscopy.
It was very difficult to detect the faint trace of a single aster on the thick sections, but with practice, we succeeded in locating two asters in one conjugate. They were about 40 µm apart (Fig. 9) and in the center of each aster we found a single centriole, which indicates that, of the four centrioles derived from the immature oocyte, two survived in the mature cytoplasm of the conjugates, and the remaining two `degraded', i.e. they lost the ability to organize the mitotic asters.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODS...RESULTSDISCUSSIONREFERENCES |
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For simplicity of discussion, we will take Pattern 1 as a `typical' case in which the two asters emerged near the site where the egg pronucleus had been located, each aster forming one pole of the bipolar spindle in the first nuclear division. As shown in Fig. 5, the number of bipolar spindles increased in a 2-4-8 fashion in each of the subsequent cycles.
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) confirmed that the maternal centrosomes/centrioles form a
bipolar mitotic spindle at the same stage during the first mitosis as in
normally fertilized eggs. In this respect the behaviour of the mitotic asters
in our Pattern 1 conjugates replicated that of the asters in 0pb eggs. Uetake
et al. (Uetake et al., 2002)
found that two centrioles survive after the completion of meiosis and that
each of the two surviving centrioles in 0pb eggs reproduces during the first S
phase, and in fact they noted a pair of centrioles with an orthogonal
configuration in each of the two centrosomes forming the bipolar spindle at
the first mitosis. It is thus reasonable to suppose that the poles of the
bipolar spindle at the first mitosis in the present conjugates also had paired
centrioles. The regular doubling of the bipolar spindles in the second and
third cell cycles (Fig. 5)
infers the presence of paired centrioles in each pole of the spindle at first
mitosis and therefore the number of centrioles in our conjugates at first
mitosis would be four. We have demonstrated one centriole in each of the two
asters in a non-activated conjugate at the pronuclear stage
(Fig. 9), so there is no doubt
that the two centrioles replicated once, probably during the first S phase
initiated by activation of the conjugates.
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However, the number of asters that formed at the first mitosis was invariably two, common to all three patterns. Moreover, we observed, in all three patterns, that each of the asters doubled at each of the subsequent mitotic cycles. We consider that the results obtained in the Pattern 2 and Pattern 3 conjugates also support our conclusion that only two of four centrioles survive.
What is remarkable about the maternal centrioles in our conjugates is that they had not undergone meiotic divisions and had not contributed to the formation of the meiotic spindles and yet, of the four centrioles only two survived with the capacity to replicate. Hence we conclude that their fate was determined while they were in the fully-grown immature oocyte, well before the resumption of meiosis.
Possible structural heterogeneity of the centrioles
Recent studies by Tamura and Nemoto
(Tamura and Nemoto, 2001) and
Uetake et al. (Uetake et al.,
2002) on artificial parthenogenesis in starfish introduced the
concept of `heterogeneity' or `nonequivalence' of the reproductive capacity of
the maternal centrioles. A typical example is the meiosis-II spindle: one pole
of the spindle positioned beneath the cell surface is inherited by the forming
PB2, and the other pole, located in the deeper cytoplasm, is left in the
mature egg. Each pole contains a single centriole. The studies showed that the
PB2 centriole has reproductive capacity, whereas the egg centriole is lost
after the completion of meiosis. How the pole of the reproductive centriole
selectively locates itself beneath the cell surface to be cast off into the
PB2 is a newly raised question. Tamura and Nemoto
(Tamura and Nemoto, 2001) and
Uetake et al. (Uetake et al.,
2002) consider that it has a device for anchoring itself to the
cell surface, a structure unique to the reproductive centriole. Such
`structural heterogeneity' must be linked to the heterogeneity in reproductive
capacity.
Thus, in order for the pole of the meiosis-II spindle containing the reproductive centriole to be correctly positioned beneath the cell surface, the fate of the centriole has to have been determined by the time of meiosis II. This was confirmed in the present study. Actually we found that the fate of the centriole was already fixed at the stage of the fully-grown immature oocyte. Whether the time its fate is determined can be traced back further to an even earlier stage of oogenesis is a subject for future study.
Process of degradation of the maternal centrioles
In the case of 0pb/1pb eggs, the `nonreproductive centrioles' are lost
shortly after the completion of meiosis
(Uetake et al., 2002). Nuclear
events, such as the formation of the pronucleus or cell-cycle arrest at the G1
phase, arising just after the completion of meiosis, suggest changes in the
egg cytoplasm that trigger these events. Uetake et al.
(Uetake et al., 2002) argue
that the supposed changes in the cytoplasm `may be related to the suppression
of some maternal centrosomes/centrioles'. A similar suppression was also
observed in our conjugates. The maternal centrioles transferred directly into
the mature cytoplasm had not undergone meiotic divisions, yet two centrioles
degraded. We anticipate that the cytoplasmic environment of the mature egg is
the necessary condition for inducing the destined centrioles to decay.
Notes on the `aster-like structure'
Tamura and Nemoto (Tamura and Nemoto,
2001) described a similar structure (`monaster') that formed at
the site of the pronucleus in intact eggs artificially activated without
sperm. It emerged at each mitotic cycle, never duplicated and remained single.
Sluder et al. (Sluder et al.,
1989) also observed the formation of such a monaster in fertilized
starfish eggs when syngamy of the sperm and egg pronuclei was artificially
prevented. Based on the common morphology of our `aster-like structure' and
the monaster, their site of appearance, and the timing of their formation, we
regard them as identical. Uetake et al.
(Uetake et al., 2002)
demonstrated that the monaster in activated intact eggs does not have a region
recognized by anti--tubulin antibody, indicating the absence of a
centrosome. We have also recently observed (unpublished data) that injection
of the antibody into fertilized eggs inhibits aster formation by the sperm
centrosome, but does not inhibit the formation of the monaster. Zhang et al.
(Zhang et al., 2004) argue
that, in the monaster, chromosomes locate to its center
(Tamura and Nemoto, 2001;
Uetake et al., 2002),
differently from the monasters formed by centrosomes, where chromosomes locate
on the periphery of the asters (Glover et
al., 1995; Gonzalez et al.,
1998). We believe that the consideration on the nature of the
monaster stated above should apply to our `aster-like structure' as well, i.e.
it is unrelated to the premeiotic centrosomes.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODS...RESULTSDISCUSSIONREFERENCES |
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Uetake, Y., Kato, K. H., Washitani-Nemoto, S. and Nemoto, S. (2002). Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies. Dev. Biol. 247,149 -164.[CrossRef][Medline]
Washitani-Nemoto, S., Saitoh, C. and Nemoto, S. (1994). Artificial parthenogenesis in starfish eggs: behavior of nuclei and chromosomes resulting in tetraploidy of parthenogenotes produced by the suppression of polar body extrusion. Dev. Biol. 163,293 -301.[CrossRef][Medline]
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Yoneda, M. (2000). Heteroplasmic conjugates formed by the fusion of starfish oocyte pairs with a 12 minute time difference in the maturation phase. Dev. Growth Differ. 42,121 -128.[Medline]
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