Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH
Kaori Harada,
Eiko Oita and
Kazuyoshi Chiba*
Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku,
Tokyo 112-8610, Japan
*
Author for correspondence (e-mail:
kchiba{at}cc.ocha.ac.jp)
Accepted 5 June 2003
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SUMMARY
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Reinitiation of meiosis in oocytes usually occurs as a two-step process
during which release from the prophase block is followed by an arrest in
metaphase of the first or second meiotic division [metaphase I (MI) or
metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly
understood, although it is a widely observed phenomenon in invertebrates. The
blockage of fully grown starfish oocytes in prophase of meiosis I is released
by the hormone 1-methyladenine. It has been believed that meiosis of starfish
oocytes proceeds completely without MI or MII arrest, even when fertilization
does not occur. Here we show that MI arrest of starfish oocytes occurs in the
ovary after germinal vesicle breakdown. This arrest is maintained both by the
Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH
in the ovary before spawning. Immediately after spawning into seawater,
activation of Na+/H+ antiporters via a heterotrimeric G
protein coupling to a 1-methyladenine receptor in the oocyte leads to an
intracellular pH increase that can overcome the MI arrest even in the presence
of active MAP kinase.
Key words: MAP kinase, Meiosis, Metaphase arrest, Na+/H+ antiporter, Starfish
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Introduction
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In most animals, fully grown oocytes are arrested at prophase of meiosis I
and resume meiosis when triggered by external stimuli, such as hormones
(Masui and Clarke, 1979
). In
invertebrates, oocytes of insects and ascidians are arrested at metaphase I
(MI) and meiosis reinitiation is triggered by fertilization, although the
mechanism of MI arrest is unknown. In vertebrates, maturing oocytes progress
through meiosis I, and then arrest in metaphase II (MII) until fertilization
(Colas and Guerrier, 1995
). The
cytoplasmic activity responsible for MII arrest is termed cytostatic factor
(CSF) (Masui, 1996
). CSF is
generated by the activated Mos/MEK/MAP kinase (MAPK) cascade
(Gotoh and Nishida, 1995
;
Singh and Arlinghaus, 1997
;
Gebauer and Richter, 1997
;
Sagata, 1997
). The protein
kinase p90Rsk is a target of MAPK causing CSF arrest
(Bhatt and Ferrell, 1999
;
Gross et al., 1999
), and is
involved in the inhibition of the anaphase-promoting complex (APC) which is an
E3 ubiquitin ligase targeting cyclin B
(Maller et al., 2002
;
Sagata, 1997
). Also, the APC
inhibitor Emi1 blocks the APC activation and prevents mitotic exit in
CSF-arrested oocytes (Reimann and Jackson,
2002
). Fertilization causes a transient increase in cytoplasmic
calcium concentration, leading to the activation of calmodulin-dependent
protein kinase II (Lorca et al.,
1993
), followed by APC activation, cyclin B destruction and
completion of meiosis.
Meiosis reinitiation in starfish oocytes is induced by the hormonal
stimulation of 1-methyladenine (1-MA)
(Kanatani et al., 1969
). The
receptor of 1-MA coupling to the hetero trimeric G protein mediates the
activation of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt, which
results in the activation of Cdc2 kinase and cyclin B complex, inducing
germinal vesicle breakdown (GVBD) (Chiba et
al., 1993
; Jaffe et al.,
1993
; Nakano et al.,
1999
; Sadler and Ruderman,
1998
; Okumura et al.,
2002
). MAPK in starfish oocytes is activated after GVBD by a newly
synthesized starfish homolog of Mos functioning as a MAPK kinase kinase (MEK
kinase) (Tachibana et al.,
2000
). MAPK activity decreases after the second polar body
formation, when fertilization occurs during meiosis
(Tachibana et al., 1997
).
The standard procedure in experiments involves starfish oocytes being
isolated from the ovary, placed in seawater (SW), and then treated with 1-MA.
These oocytes proceed completely through meiosis I and II without metaphase
arrest. However, this situation is rather artificial, since oocytes are
naturally stimulated by 1-MA in the ovary. In this study, to induce natural
spawning, we injected 1-MA into the body cavity of female starfish and found
that a MI arrest, which was maintained by the MAPK pathway, occurred in the
ovary. Release of the MI arrest was induced by an intracellular pH (pHi)
increase when the oocyte was spawned into SW.
 |
Materials and methods
|
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Animals and oocytes
Starfish (Asterina pectinifera) were collected on the Pacific
coast of Honshu, Japan, and kept in laboratory aquaria supplied with
circulating SW at 15°C. To induce spawning, 6 ml of SW containing 0.1 mM
1-MA was injected into the body cavity of a female animal using a 22 gauge
needle. In some experiments, 8 mg radial nerve/ml (as a source of gonad
stimulating substance; GSS) (Kanatani et
al., 1969
) was used to induce spawning. To induce synchronous GVBD
in the ovary, 1-MA or GSS was injected into several points of the body
cavity.
Microinjection into starfish oocytes and quantification of injection
volumes were done as previously described
(Chiba et al., 1993
;
Chiba et al., 1999a
). All
experiments were done at 20°C unless otherwise indicated.
Immunofluorescence microscopy
Oocytes were washed several times with cold calcium-free SW. The oocytes
were then treated with extraction buffer containing detergent
(Shirai et al., 1990
) and
fixed with 100% methanol for 1 hour at-20°C. After fixation, they were
transferred to PBS-T (phosphate-buffered saline/0.05% Tween 20) and left to
stand for 5 minutes. They were then incubated with a mouse monoclonal antibody
against
-tubulin (Amersham Corp., Buckinghamshire, England) for 50
minutes, washed with PBS-T, then stained with a FITC-conjugated goat
anti-mouse IgG antibody (Tago, Burlingame, CA) for 40 minutes. DNA was stained
with DAPI (Sigma) for 30 minutes, and washed with PBS-T.
Determination of pHi with BCECF-dextran
A dextran (10 kDa) conjugate of 2',
7'-bis[2-carboxyethyl]-5-[and- 6]-carboxyfluorescein (BCECF) (Molecular
Probes) was dissolved at 2 mM in 100 mM potassium aspartate and 20 mM Hepes at
pH 7.2. The volume injected was 2% of the total oocyte volume. To estimate
pHi, an inverted light microscope (DMIRB; Leica) was connected by an adapter
tube to a HiSCA CCD camera (C6790) of the ARGUS/HiSCA image processing system
(Hamamatsu Photonics K. K.). Excitation light from a xenon lamp was alternated
between 450 and 490 nm under computer control (C6789; Hamamatsu Photonics K.
K.). The emitted light passed through a dichroic beam splitter at 510 nm and
through a 515- to 560-nm emission filter (Leica). The ratios of the emission
intensities at 490/450 nm were calculated using the ARGUS/HiSCA image
processing system. Model intracellular medium containing 300 mM glycine, 175
mM KCl, 185 mM mannitol, 20 mM NaCl, 5 mM MgCl2, 25 mM Hepes, and
25 mM Pipes, adjusted to the indicated pH with KOH and 100 µM digitonin to
permeabilize the oocytes, was used for calibration.
Preparation of the oocyte homogenate and supernatant
The cell-free preparation (oocyte supernatant) was made as previously
described (Chiba et al.,
1999b
). Briefly, oocytes (1 ml) just undergoing GVBD were washed
twice in 10 ml of ice-cold buffer P (150 mM glycine, 100 mM EGTA, 200 mM Hepes
buffer, pH 7.0). After the oocytes were sedimented by gravity, buffer P was
removed as completely as possible. Then the oocytes were transferred to a net
of 60 µm mesh in the neck of a microtube (1.5 ml) and pressed onto the net
with the cap of the tube. When the tube was centrifuged at 1400
g for 3 seconds, oocytes were homogenized by passage through
the net. The homogenate was centrifuged at 20,000 g for 15
minutes at 0°C. The supernatant was transferred to a microtube and frozen
by immersion in liquid nitrogen. Before use, the frozen supernatant was thawed
at 15°C, and kept on ice.
SDS-PAGE and immunoblot analysis
The cell-free preparation was boiled for 5 minutes in sample buffer, and
separated by electrophoresis on a 10% SDS-polyacrylamide gel, and the proteins
were transferred to a PVDF transfer membrane (Millipore, Bedford, MA). The
membrane was blocked with PBS-T containing 5% skim milk, and incubated with an
anti-starfish cyclin B antibody at 1:1000 for 1 hour at room temperature.
After the membrane was washed with PBS-T, it was incubated with a
horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:1000) for 1
hour. After the membrane was washed, the bound antibody was detected using a
chemiluminescent substrate (ECL; Amersham Pharmacia Biotech, Piscataway, NJ)
and a LAS-1000 Luminescent image analyzer (Fuji Photo Film Co., Ltd., Tokyo,
Japan).
 |
Results and discussion
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First polar body formation is blocked in the ovary
Spawning started 30-50 minutes after the injection of 1-MA or GSS into the
starfish body cavity, and continued for over 3 hours. Released oocytes did not
have germinal vesicles, indicating that GVBD had occurred in the ovary before
spawning. Indeed, we could see that all oocytes were undergoing GVBD in the
ovary when we dissected the animals and isolated oocytes from them 40 minutes
after the injection of 1-MA into the body cavity.
When 1-MA is applied to oocytes isolated from animals without 1-MA
injection, GVBD and first polar body formation usually occur 20 and 70 minutes
after 1-MA treatment, respectively. We expected that the timing of GVBD of
oocytes in the ovary of 1-MA-injected animals would be similar. However, to
our surprise, oocytes spawned from animals that had been injected with 1-MA
158 minutes previously had not formed the first polar bodies when they were
observed immediately after spawning. These oocytes formed first polar bodies
183 minutes after 1-MA injection (about 30 minutes after spawning:
Fig. 1, bottom panels). Similar
results were obtained whenever oocytes were examined just after spawning
(Fig. 1, 38 or 98 minutes after
1-MA injection). Thus, the first polar body formation was blocked in the
ovary, while GVBD proceeded normally.

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Fig. 1. Extrusion of polar bodies after spawning. Photographs were taken at the
indicated times (minutes) after the injection of 1-MA into the body cavity of
the recipient animal. (Top panels) Immediately after spawning (35 minutes
after the injection of 1-MA into the body cavity), the oocyte was inseminated.
The first and second polar bodies (arrowheads: 1stPB and 2ndPB) were formed
after the elevation of the fertilization envelope, indicating that they were
extruded after fertilization or spawning. (Middle and bottom panels) Oocytes
spawned into SW at 95 or 155 minutes after the injection of 1-MA were
inseminated. Similarly, polar bodies were extruded after spawning. Scale bar:
50 µm.
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MI arrest occurs in the ovaries of stimulated animals
To determine the stage of oocytes arrested in the ovary after GVBD, the
oocytes were stained with DAPI and an anti-microtubule antibody. As shown in
Fig. 2, all oocytes were at
prometaphase 45 minutes after the injection of 1-MA into the body cavity. At
105 minutes, 57±20% (mean ± s.e.m., n=4) of the oocytes
were at metaphase (Fig. 2,
105), and the remaining oocytes were at prometaphase. At 165 minutes,
72±11% (mean ± s.e.m., n=3) of the oocytes were at
metaphase (Fig. 2, 165), and
the rest were at prometaphase. Thus, MI arrest occurred in the ovaries of
stimulated animals. Since some oocytes were at prometaphase even at 165
minutes, the progression from prometaphase to metaphase appears to be retarded
in the ovary. After spawning, MI arrest was released
(Fig. 2, 75, 135 and 195 minute
samples). Extrusion of the first and second polar bodies then occurred,
without additional arrest (Fig.
1). It is well known that optimal development of starfish embryos
occurs when oocytes are fertilized between GVBD and the first polar body
formation, since protection against polyspermy is gradually lost after the
first polar body formation (Meijer and
Guerrier, 1984
). Thus, the occurrence of MI arrest before spawning
ensures that all maturing oocytes can be fertilized before the first polar
body extrusion, even if the spawning period is relatively long.

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Fig. 2. MI arrest in the ovaries of stimulated animals. (Left column) Oocytes
spawned into SW at 45, 105 and 165 minutes after the injection of 1-MA into
the body cavity of the animal were immediately collected and treated with
extraction buffer, followed by fixation and staining with DAPI and an
anti-tubulin antibody to visualize chromosomes (blue) and tubulin (green).
(Right column) Oocytes incubated in normal SW for 30 minutes after spawning
were similarly treated and stained at 75, 135, and 195 minutes. Representative
figures are shown. (Top row) Prometaphase at 45 minutes and anaphase at 75
minutes. (Middle row) Metaphase at 105 minutes and anaphase at 135 minutes.
(Bottom row) Metaphase at 165 minutes and anaphase at 195 minutes. Scale bar:
2 µm.
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Na+/H+antiporter is activated by 1-MA
Na+/H+ antiporters (Na+/H+
exchangers: NHEs) are a family of plasma membrane proteins that catalyze the
electroneutral exchange of intracellular H+ for extracellular
Na+ (Counillon and Pouyssegur,
2000
). They play a central role in a variety of cell functions,
including regulation of intracellular pH, cell volume and cell growth
(Dibrov and Fliegel, 1998
;
Fliegel et al., 1998
). They are
activated by stimuli such as hormones, growth factors and osmotic shrinkage.
In starfish, there are contradictory reports showing that 1-MA induces a pHi
increase (Johnson and Epel,
1982
) or not (Peaucellier et
al., 1988
). These discrepancies may be due to differences of the
species of the animals or the methods used to measure pHi. In the present
study using a dextran (10 kDa)-conjugate of BCECF, we clearly found that 1-MA
treatment in normal SW resulted in an increase in pHi of about 0.4 pH units
(Fig. 3A). It is well known
that the absence of extracellular Na+ inhibits the
Na+/H+ antiporter-induced pHi increase. As expected, in
zero-Na+ artificial SW, the pHi increase of 1-MA-treated oocytes
was blocked (Fig. 3A). Also,
5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of
Na+/H+ antiporters, blocked the pHi increase induced by
1-MA in SW (Fig. 3A).

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Fig. 3. An increase of pHi during oocyte maturation induced by
Na+/H+ antiporters coupling to 1-MA signal transduction.
(A) Representative traces of pHi before and after 1-MA treatments.
Fluorescence ratios of BCECF-dextran were measured every 30 seconds using a
single oocyte before and after 1-MA treatments. Symbols represent oocytes
treated with 1 µM 1-MA in normal SW (open circles, n=40), or in
zero-Na+ artificial SW (0 NaSW) (closed diamonds, n=25)
containing 480 mM choline chloride, 55 mM MgCl2, 10 mM
CaCl2, 5 mM KCl, 2.5 mM KHCO3, pH 8.0 adjusted with KOH,
or in normal SW with 0.6 mM EIPA (open diamonds, n=22). Open
triangles represent oocytes in normal seawater without 1-MA treatment
(n=11). (B) Representative traces of pHi after Gß (0.6
µM) microinjection. Gß purified from bovine brain was stored in
0.6% Na+ cholate, 100 mM NaCl, 20 mM Tris-HCl, pH 8.0, and
microinjected as previously described
(Chiba et al., 1993 ).
Fluorescence ratios of a single oocyte before and after Gß
microinjection were measured. Symbols represent oocytes microinjected with
Gß in normal SW (open circles, n=22) or in
zero-Na+ artificial SW (closed diamonds, n=6), or
microinjected in normal SW with the buffer used for Gß (open
triangles, n=8). (C) The fluorescence ratios of a single oocyte in
artificial SW with (open diamonds, n=14) or without (open circles,
n=40) 0.1 mM LY294002 were measured before and after 1-MA
treatment.
|
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When G protein ß
subunits (Gß
) were injected into
oocytes without 1-MA, the pHi increase as well as GVBD occurred
(Fig. 3B), indicating that G
protein coupling to the 1-MA receptor
(Tadenuma et al., 1992
) is
involved in the activation of Na+/H+ antiporters. An
effector of Gß
is PI 3-kinase
(Nakano et al., 1999
;
Sadler and Ruderman, 1998
),
and a specific inhibitor of PI 3-kinase, LY294002, inhibited the 1-MA-induced
pHi elevation (Fig. 3C),
supporting the hypothesis that the Na+/H+ antiporter
functions downstream of PI 3-kinase in the signaling pathway of 1-MA-induced
starfish oocyte maturation.
The pHi increase induced by 1-MA is not required for the induction of
GVBD
While GVBD of maturing oocytes in zero-Na+ artificial SW
occurred 20 minutes after 1-MA addition, polar body formation was blocked
(Fig. 4). Thus, the pHi
increase induced by 1-MA was not required for the induction of GVBD, but
appeared to be required for the extrusion of polar bodies. As mentioned above,
polar body formation was blocked in the ovaries of stimulated animals,
suggesting that the pHi increase of oocytes in the ovary is also blocked.

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Fig. 4. Inhibition of polar body formation in zero-Na+ artificial SW
after 1-MA addition. GVBD occurred 20 minutes after 1-MA addition both in
normal SW and zero-Na+ artificial SW. In normal SW, the first and
second polar body (arrowheads) were extruded 70-90 minutes and 100-120 minutes
after 1-MA addition, respectively. In zero-Na+ artificial SW, polar
bodies were not extruded.
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The pHi increase of oocytes is blocked in the ovary, causing MI
arrest
To test whether the MI arrest during starfish oocyte maturation in the
ovary is dependent on the inhibition of a rise in pHi, BCECF was microinjected
into the oocytes in the ovaries of animals stimulated by 1-MA. This
experiment, however, did not succeed, since the packed oocytes in the ovary
were very fragile. Therefore, we isolated the ovaries from stimulated animals
and transferred them to zero-Na+ artificial SW without 1-MA. Then,
the oocytes arrested at MI in the ovaries were isolated in zero-Na+
artificial SW to block a possible pHi increase, and injected with BCECF. As
shown in Fig. 5A, when the
zero-Na+ artificial SW was replaced by normal SW containing
Na+ ions, a significant rise of pHi occurred, suggesting that pHi
of maturing oocytes in the ovaries is kept at low level.

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Fig. 5. Blockage of pHi increase of oocytes in the ovary of stimulated animals. (A)
Ovaries were isolated from the stimulated animals 15 minutes before
measurement of fluorescence ratios. They were immersed in zero-Na+
artificial SW and dissociated oocytes were obtained. Oocytes were continuously
treated with zero-Na+ artificial SW for 15 minutes, during which
time they were microinjected with BCECF. The zero-Na+ artificial SW
was replaced by normal SW 60 minutes or 180 minutes after injection of 1-MA
into the body cavity of the animal, followed by measurement of fluorescence
ratios. (B) Ovaries were isolated from non-stimulated animals and immersed in
zero-Na+ artificial SW. Then, oocytes in the ovaries were isolated
and microinjected with BCECF. The zero-Na+ artificial SW was
replaced by normal SW at 10 minutes, and again replaced by zero-Na+
artificial SW at 60 minutes. A small rise of pHi occurred when the
zero-Na+ artificial SW was replaced by normal SW. (C) The pHi
increase of oocytes from non-stimulated animals was induced by 1-MA in normal
SW, which was replaced by zero-Na+ artificial SW without 1-MA at 65
minutes, and again replaced by normal SW at 93 minutes. High pHi induced by
normal SW containing 1-MA was not affected by zero-Na+ artificial
SW. (D) Ovaries were isolated from the stimulated animals and immersed in
normal SW. Immediately, oocytes in the ovaries were isolated and microinjected
with BCECF, and the pHi of oocytes was measured. pHi increased with time after
removal of the ovary from the animal.
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Interestingly, the pHi of oocytes arrested at MI (approx. pH 6.7;
Fig. 5A, time 0) was lower than
that of oocytes from non-stimulated animals in normal SW (approx. pH 7.1;
Fig. 3A, time 0). Similarly,
low pHi was observed when immature oocytes were isolated from ovaries treated
with zero-Na+ artificial SW (approx. pH 6.6;
Fig. 5B, time 0). Since
zero-Na+ artificial SW treatment by itself did not cause a
significant decrease of the pHi in either immature
(Fig. 5B, time 60) or maturing
oocytes (Fig. 5C, time 65), the
low pHi of oocytes in the ovaries is not artificially induced by
zero-Na+ artificial SW, but is related to the physiological
condition. Instead, pHi of oocytes from non-stimulated animals in normal SW
(approx. pH 7.1; Fig. 3A, time
0) seems to be artificially induced, since a small rise of pHi occurred when
the zero-Na+ artificial SW was replaced by normal SW
(Fig. 5B, time 10). Although we
do not know the mechanism of the pHi increase of ovarian oocytes induced by
normal SW, such pHi elevation may prevent detection of the pHi rise induced by
1-MA in some species of starfish.
In another series of experiments, we isolated the ovaries from stimulated
animals 180 minutes after 1-MA injection and transferred them directly to
normal SW without 1-MA, followed by a rapid injection of BCECF. As shown in
Fig. 5D, the pHi increased with
time after removal of the ovary from the animal. Thus, in the ovary, the pHi
elevation of oocytes undergoing GVBD is blocked, which causes MI arrest. Also,
it is likely that the Na+/H+ antiporters in the ovaries
of stimulated animals are maintained in an activated state even after
isolation of the oocytes in zero-Na+ artificial SW without 1-MA.
When Na+ is added, the pre-activated Na+/H+
antiporters start working. A similar situation may occur in vivo; in the
ovary, the concentration of Na+ may be low, and the MI arrest may
be released by the Na+ in SW immediately after spawning. Another
possibility is that the Na+/H+ antiporters of oocytes in
the ovary may be inhibited by CO2 as shown in sea urchin sperm in
semen (Johnson et al.,
1983
).
MI arrest is maintained by the MAPK pathway and low pHi
Hormonal stimulation of starfish oocytes by 1-MA leads to the activation of
the cdc2/cyclin B complex in the cytoplasm without the requirement for new
protein synthesis (Kishimoto,
1999
; Doree and Hunt,
2002
). During metaphase, the cdc2/cyclin B complex is stable, but
50-60 minutes after 1-MA addition, cyclin B is suddenly degraded by the
proteasome, which results in exit from metaphase. To determine whether MI
arrest in the ovary is caused by the pHi-dependent inhibition of cyclin B
degradation, we adjusted the pH of a cell-free preparation from oocytes
undergoing GVBD to 7.0 or 7.3, and incubated the extract. As shown in
Fig. 6A, cyclin B in the
cell-free preparation was completely degraded after 60 minutes incubation at
pH 7.3, while no degradation was observed at pH 7.0. Also, when the cell-free
preparation at pH 7.0 was incubated with the MEK inhibitor U0126 to inhibit
the MAPK pathway, cyclin B was degraded after 60 minutes incubation.
Activation of MAPK at pH 7.0 and 7.3, and inactivation of MAPK by U0126 at pH
7.0 were confirmed as shown in Fig.
6B. Thus, the MAPK pathway is required for establishing MI arrest
at lower pH (<pH 7.0). At higher pH (>pH 7.3), MI arrest does not occur,
although the MAPK is still activated. These results clearly show that MAPK
cannot inhibit cyclin B degradation at higher pH.

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Fig. 6. Effects of pH and the MAPK pathway on the destruction of cyclin B in a
cell-free preparation. (A) The cell-free preparation at pH 7.0 or 7.3 with or
without U0126 (0.1 mM) was incubated for 0, 20, 40, 60 and 85 minutes. After
incubation, sample buffer for SDS-PAGE was added to stop the reaction. Then,
the samples were analyzed by 10% SDS-PAGE, followed by immunoblotting with an
anti-cyclin B antibody. (B) The MAPK activity of the cell-free preparation at
pH 7.0 or 7.3 with or without U0126 (0.1 mM). Activation of MAPK was inhibited
by U0126. MAPK was activated at pH 7.0 and 7.3 (arrow), and inactivated at pH
7.0 with U0126 (arrowhead). Each sample was analyzed by 12.5% SDS-PAGE,
followed by immunoblotting with an anti-MAPK (ERK1) antibody.
|
|
MI arrest of starfish oocytes has not been reported previously, since
starfish oocytes from non-stimulated animals are usually treated with normal
SW containing 1-MA for induction of GVBD, which causes an increase of pHi
before GVBD. At the lower pHi of maturing oocytes in the ovary, the MAPK
pathway may inhibit the APC-dependent degradation of cyclin B. After spawning,
cyclin B degradation is induced by the pHi increase while the MAPK pathway is
still active, and is involved in the blockage of DNA synthesis during meiosis
(Tachibana et al., 2000
).
Identification of the target of MAPK regulating the MI arrest in a
pHi-dependent manner will be the next aim of our studies.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr T. Kishimoto for providing an anti-cyclin B antibody, and Dr L.
A. Jaffe for reading the manuscript. This study was supported by a grant from
the Human Frontier Science Program and grants from the Ministry of Education,
Culture, Sports and Sciences of Japan.
 |
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