1 Department of Biology, Miyagi University of Education, Aoba-ku, Sendai, Miyagi
980-0845, Japan
2 Misaki Marine Biological Station, the University of Tokyo, Miura, Kanagawa
238-0225, Japan
* Author for correspondence (e-mail: deguchi{at}staff.miyakyo-u.ac.jp)
Accepted 15 October 2002
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Summary |
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Key words: Intracellular Ca2+, Fertilization, Ca2+ channels, Serotonin, Ins(1,4,5)P3
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Introduction |
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As for MI-type bivalves, temporal patterns of Ca2+ increases at
fertilization have been analyzed in five different species: Mytilus,
Crassostrea, Ruditapes, Limaria and Hiatella. When the
Ca2+ indicator Fluo-3 is introduced as AM ester, only a single
blunt Ca2+ increase, which persists for several minutes, is
observed in fertilized oocytes of Mytilus
(Abdelmajid et al., 1993) and
Ruditapes (Leclerc et al.,
2000
). In the oocytes injected with Ca2+ indicators
such as Fura-2 and Calcium Green-1, however, Ca2+ response at
fertilization comprises an initial sharp Ca2+ transient and
subsequent repetitive Ca2+ spikes (Ca2+ oscillations) in
all of the five species, including Mytilus and Ruditapes
(Deguchi and Osanai, 1994a
;
Deguchi and Morisawa, 1997
).
In Mytilus, the initial Ca2+ transient at fertilization
arises almost synchronously in the oocyte without forming a point-source
Ca2+ wave (Deguchi and Osanai,
1994a
). A more recent analysis revealed that the increased
Ca2+ starts from the entire oocyte cortex and spreads inwardly to
the center, taking the form of a `cortical flash' pattern
(Stricker, 1999
), during the
rising phase of the initial Ca2+ transient
(Deguchi and Morisawa, 1997
).
This initial transient is not affected by heparin, an antagonist of inositol
1,4,5-trisphosphate [Ins(1,4,5)P3] receptors, but is
suppressed by blockers of voltage-gated Ca2+ channels such as
methoxyverapamil (D-600) (Deguchi et al.,
1996
). Pharmacological experiments in another species,
Ruditapes, suggest that voltage-gated Ca2+ channels are
progressively situated on the plasma membrane of oocytes during the oocyte
maturation from PI to MI (Leclerc et al.,
2000
). These data collectively suggest that the initial
Ca2+ transient at fertilization in MI-type bivalves is mainly due
to the influx of external Ca2+ through voltage-gated
Ca2+ channels distributed over the plasma membrane. However, the
phase of Ca2+ oscillations, which occurs after the initial
Ca2+ transient, persists even after the removal of external
Ca2+ in all MI-type bivalves tested
(Deguchi and Osanai, 1994a
). In
Mytilus, the phase of Ca2+ oscillations is completely
blocked by heparin but not by D-600
(Deguchi et al., 1996
), and
each Ca2+ spike during this phase takes the form of a point-source
Ca2+ wave (Deguchi and
Morisawa, 1997
), which seems to be a common pattern of
Ca2+ release from internal stores in fertilized oocytes or eggs of
many other animals (Stricker,
1999
). These results suggest that the phase of Ca2+
oscillations, unlike an initial Ca2+ transient, is chiefly
regulated by Ca2+ release from
Ins(1,4,5)P3-sensitive stores in MI-type bivalves.
Therefore, MI-arrested oocytes of MI-type bivalves seem to possess at least
two pathways to produce intracellular Ca2+ increases:
Ca2+ influx via voltage-gated Ca2+ channels and
Ca2+ release from internal stores via
Ins(1,4,5)P3 receptors.
The contribution of Ca2+ influx to intracellular Ca2+
increases at fertilization has been suggested in several PI-type bivalves. In
Spisula, fertilization causes depolarization of the plasma membrane
lasting for several minutes (Finkel and
Wolf, 1980), which may activate voltage-dependent Ca2+
channels. In Barnea, long-term 45Ca uptake, which is
inhibited by the addition of D-600, takes place at fertilization
(Dubé and Guerrier,
1982
). Among PI-type bivalves, Mactra is the only species
in which a temporal pattern of Ca2+ changes at fertilization is
known: sperm-induced Ca2+ increases comprise an initial large
Ca2+ transient and a subsequent submaximal plateau phase of
Ca2+ elevation, which persists up to the time of germinal vesicle
breakdown (GVBD) (Deguchi and Osanai,
1994b
). The plateau phase seems to be maintained by the continuous
influx of external Ca2+, since the elevated Ca2+
immediately returns to the resting level following the removal of external
Ca2+ during this phase (Deguchi
and Osanai, 1994b
). These results suggest that external
Ca2+ is the main source of the sperm-induced intracellular
Ca2+ increases in PI-type bivalves. In accordance with this view,
it has been shown in the PI-type bivalves that inhibition of Ca2+
influx at fertilization precludes GVBD
(Allen, 1953
;
Dubé and Guerrier,
1982
; Deguchi and Osanai,
1994b
), and that stimulation of this pathway with high
K+ seawater conversely triggers GVBD without insemination
(Guerrier et al., 1981
;
Dubé and Guerrier,
1982
; Deguchi and Osanai,
1994b
). In contrast to the accumulated evidence for the
contribution of Ca2+ influx, however, Bloom et al. found in
Spisula that GVBD can be induced by injection of
Ins(1,4,5)P3 into unfertilized oocytes, and that
intracellular concentrations of precursors of Ins(1,4,5)P3
become higher following fertilization
(Bloom et al., 1988
). Their
results raise the possibility that not only Ca2+ influx but also
Ca2+ release from Ins(1,4,5)P3-sensitive stores
might be involved in sperm-induced Ca2+ increases and responsible
for meiosis reinitiation from PI in PI-type bivalves.
The aim of the present study was to understand the mechanisms underlying the sperm-induced Ca2+ changes at fertilization in the PI-type bivalve Mactra chinensis. First, we investigated the spatiotemporal Ca2+ dynamics not only in the whole oocyte but also in more restricted regions, in the cytoplasm and inside the nucleus, at normal fertilization. Second, we clarified the main Ca2+ source and pathway for the sperm-induced Ca2+ changes. Finally, we examined whether unfertilized oocytes have the potential ability to use other Ca2+-mobilizing mechanisms that are quiescent at fertilization. Our results demonstrate that Mactra oocytes possess at least two pathways for producing cytoplasmic and nuclear Ca2+ increases. One is the Ca2+ influx mechanism via voltage-dependent Ca2+ channels, which is responsible for the Ca2+ increases at fertilization. The other is the Ca2+ release mechanism via Ins(1,4,5)P3 receptors, which may be a latent system and not play a central role, at least by the time of GVBD, in fertilized oocytes. This situation is quite different from that observed in the MI-type bivalves, where both the external and the internal Ca2+ sources are used in fertilized oocytes.
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Materials and Methods |
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Solutions
Unless otherwise specified, FSW was used as bathing medium for oocytes.
Ca2+-free seawater (CaFSW; 462 mM NaCl, 9.4 mM KCl, 48 mM MgCl and
10 mM EGTA) and low Ca2+ seawater (LCaSW; 449 mM NaCl, 9.4 mM KCl,
48 mM MgCl, 12 mM CaCl and 10 mM EGTA; 2 mM of free Ca2+) were
generally supplemented with 10 mM Tris and adjusted to pH 8.3. Stock solutions
of methoxyverapamil (D-600; Sigma, St Louis, MO) and serotonin
(5-hydroxytryptamine, 5-HT; Sigma) were prepared at 20 mM in DMSO:ethanol
(1:3) and at 10 mM in distilled water, respectively, and diluted just before
use. The former vehicle (DMSO + ethanol) alone had no inhibitory or
stimulatory effect on intracellular Ca2+ changes.
Microinjection
The method of microinjection was essentially equivalent to that described
previously (Deguchi and Osanai,
1994a). Ca2+ indicators, Calcium Green-1 10 kDa dextran
(10 kDa CGD) and 70 kDa dextran (70 kDa CGD), were purchased from Molecular
Probes (Eugene, OR) and prepared at 1.0 and 0.5 mM, respectively, in an
injection buffer containing 100 mM K aspartate and 10 mM Hepes (pH 7.0). In
some experiments, 10 kDa CGD was further supplemented with 20 mg/ml 3-kDa
heparin (Sigma) and/or 600 µM myo-inositol 1,4,5-trisphosphate
P4(5)-1-(2-nitrophenyl) ethyl ester [caged
Ins(1,4,5)P3; Calbiochem, San Diego, CA]. The tip of a
micropipette was inserted into the cytoplasm (or into the nucleus in some
experiments) of PI-arrested oocyte, and the injection buffer containing the
chemicals was ejected by water pressure. Estimated concentrations of the
injected chemicals in the cytoplasm or nucleus ranged from 2 to 4% of the
original concentrations in a micropipette. The dye-injected oocytes were
incubated in FSW for at least 30 minutes, and those oocytes that underwent
GVBD during the period were discarded.
Ca2+ imaging
All fluorescence measurements were carried out at 20-24°C. One to three
dye-injected oocytes were introduced into a measurement chamber, where they
were slightly compressed by two coverslips adhered with a strip of
double-sided adhesive tape as a spacer. These oocytes were observed with a
DIAPHOT-TMD inverted microscope (Nikon, Tokyo, Japan) equipped with
epifluorescence apparatus (TMD-EF2) with an excitation filter (450-490 nm), a
dichroic mirror (510 nm), and an emission filter (520-560 nm). Fluorescence
images of the oocytes were captured with a silicon-intensified target tube
(SIT) camera (C-2400; Hamamatsu Photonics, Hamamatsu, Japan) and continuously
recorded on videotape. For each targeted oocyte, changes in fluorescence
intensity within a circle (two-thirds of the oocyte diameter) calculated by an
image processor (ARGUS 50/CA, Hamamatsu Photonics) were continuously displayed
on a screen during the recording period.
The measuring oocytes in the chamber were inseminated, exposed to various
agents, or irradiated with UV light when steady levels of fluorescence
intensities were confirmed. For insemination, sperm suspension diluted with
FSW was added to the chamber. The final sperm concentrations were
104-105 sperm/ml in most experiments, whereas much
denser suspension was used for the oocytes incubated with D-600, since
fertilization was not easily established in the presence of the drug probably
due to its inhibitory effect on the sperm acrosome reaction. To exchange
external medium during the measurement, new medium (1.5 ml) was added
after withdrawal of the original medium (residue:
100 µl), and this
procedure was repeated at least twice. For the liberation of
Ins(1,4,5)P3, caged
Ins(1,4,5)P3-injected oocytes were globally irradiated
with UV light (at 380 nm) for 10-15 seconds, during which the blue light for
CGD excitation was withdrawn. The gaps during the fluorescence recording (see
Fig. 5C) correspond to the UV
irradiation periods.
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During or after fluorescence measurements, oocytes were checked for the presence or absence of GVBD. To obtain the spatiotemporal Ca2+ pattern at `normal' fertilization, each oocyte that had undergone GVBD was withdrawn from the chamber and further cultured individually in a hole of a 96-well culture plate for observation of the subsequent mitotic process. In this case, data analysis was restricted to those oocytes that developed to early trochophores. In some experiments, measured oocytes were fixed in methanol:acetic acid (3:1), washed with distilled water 20-30 hours later, and then stained with 10 µM 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) for 30 minutes to visualize sperm nuclei in them.
Data analysis
Fluorescence images on videotape were converted into digital images and
processed with NIH Image (a public domain image processing software for the
Macintosh computer). The sequential digitized images, each of which was
constituted by averaging four successive images, were captured at the interval
of 3 seconds for temporal analysis of long-term Ca2+ changes or of
0.4 seconds for detailed spatiotemporal analysis of each Ca2+
transient. To investigate temporal Ca2+ patterns in the whole
oocyte, cytoplasm or nucleus, the values of average fluorescence intensities
calculated in each region (F) were normalized by dividing them by the resting
value (F0). In most cases, the F0 value was obtained
from the image just before the first detectable Ca2+ increase (=
zero time in each Ca2+ trace), which is considered as the time of
fertilization for inseminated oocytes
(Deguchi and Osanai, 1994a).
Although F0 levels were somewhat different in respective oocytes,
temporal changes in the normalized value (F/F0) after the same
treatment were almost constant, suggesting that the initial fluorescence
intensities were mainly affected by intracellular dye concentrations rather
than resting Ca2+ levels. In some experiments, the exact onset time
of nuclear envelope breakdown was determined by detecting leakage of 70 kDa
CGD through the nuclear envelope; F values in the dye-free region (e.g.
nuclear region of the oocyte where 70 kDa CGD was injected into the cytoplasm)
were initially lower and mainly came from the fluorescence of the surrounding
dye, but prominently increased as the nuclear envelope breakdown progressed.
In this case, the F0 value was obtained from the image at
2
minutes before the beginning of this event (see
Fig. 1B,C, Fig. 4B). For analyzing the
detailed spatiotemporal property of Ca2+ increase, sequential
fluorescence images were normalized by dividing them by the resting image just
before each Ca2+ increase in a pixel-to-pixel manner and expressed
as pseudocolor images. The zero time in each montage (Figs
3,
6) indicates the initiation
time of the Ca2+ increase.
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Results |
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Since fluorescence images in this study were not obtained with confocal microscopy, there was a possibility that fluorescence signals from the cytoplasm and nucleus might be mixed together, giving inaccurate information about Ca2+ changes. To distinguish between these two signals and confirm the respective Ca2+ patterns, 70 kDa CGD was injected into the cytoplasm or nucleus; unlike 10 kDa CGD, 70 kDa CGD could not diffuse across the nuclear envelope (Fig. 1B,C, top-right panels). The cytoplasm-restricted CGD showed a transient increase in F/F0 at fertilization (Fig. 1B), and the first detectable increase in F/F0 was also seen throughout the oocyte cortex (data not shown). Similarly, a Ca2+ transient at fertilization was observed with 70 kDa CGD in the nucleus (n=5; Fig. 1C); an increased F/F0 signal was initially distributed just beneath the whole nuclear envelope and spread to the center of the nucleus (Fig. 3B). These data are fully consistent with the above observation with 10 kDa CGD.
In 10 kDa CGD-injected oocytes, the total duration of Ca2+
increases from the onset of initial Ca2+ transient to the end of
plateau phase was 615±32 seconds (mean±s.e.m., n=7).
Under bright-field observation, GVBD can be detected shortly after the
cessation of the plateau (Deguchi and
Osanai, 1994b). However, the fluorescence of 70 kDa CGD began to
leak from cytoplasm to nucleus at 473±20 seconds (n=8;
arrowhead in Fig. 1B) or from
nucleus to cytoplasm at 506±52 seconds (n=5; arrowhead in
Fig. 1C). These results
indicate that the nuclear envelope begins to break down shortly before, rather
than after, the end of plateau in fertilized oocytes.
Mechanism of Ca2+ changes at fertilization
When external Ca2+ around fertilized Mactra oocytes is
removed, a plateau phase of Ca2+ elevation is abolished immediately
(Deguchi and Osanai, 1994b).
This fact and the spatial pattern of the initial Ca2+ transient
(Fig. 3A) imply that the
Ca2+ changes at fertilization are totally dependent on the
Ca2+ influx pathway. The following experiments were done to verify
this scenario.
To reduce Ca2+ entry into fertilized oocytes, artificial
seawater with low Ca2+ concentration was first tested. Since the
presence of 1 mM or less external Ca2+ precluded fertilization
itself, artificial seawater containing 2 mM free Ca2+ was used
as low Ca2+ seawater (LCaSW). In the oocytes fertilized in LCaSW,
an initial Ca2+ transient was not suppressed (n=5;
Fig. 2A); a cortical flash
pattern similar to that observed at normal fertilization occurred during its
rising phase (data not shown). However, the cytoplasmic and nuclear
Ca2+ levels during the subsequent plateau phase were considerably
lower and the duration of this phase was much longer in LCaSW (compare
Fig. 1A and
Fig. 2A). The total duration
including the initial Ca2+ transient and the following plateau
under these conditions was 1237±92 seconds (n=5). GVBD was
visualized shortly after the end of plateau under bright-field
observation.
As a next attempt to suppress Ca2+ influx, the effect of D-600,
an effective inhibitor of voltagedependent Ca2+ channels in bivalve
oocytes (Dubé and Guerrier,
1982; Deguchi et al.,
1996
), was examined. In the oocytes incubated with 100 µM D-600
in FSW, an initial Ca2+ transient at fertilization was not
abolished (n=8; Fig.
2B). However, a cortical flash pattern of its rising phase became
asymmetrical or incomplete (Fig.
3C), compared with that observed at normal fertilization
(Fig. 3A). Following the
initial transient, the increased Ca2+ returned to the resting level
without being maintained at the submaximal level (8/8). There was no further
Ca2+ increase after the resting Ca2+ level had been
attained in 4 of the 8 oocytes (Fig.
2B). In the remaining 4 oocytes, the resting Ca2+ state
was maintained for several minutes, and then additional small Ca2+
transients appeared (data not shown). Most of the D-600-treated oocytes (7/8)
failed to undergo GVBD. The exceptional one oocyte (1/8) showed a plateau
phase of Ca2+ elevation following the additional small
Ca2+ transients and resulted in delayed GVBD (occurring at
20
minutes after fertilization, data not shown).
Combined applications of LCaSW and D-600 almost totally abolished
sperm-induced Ca2+ changes. One (n=3;
Fig. 2C) or multiple
(n=3; data not shown) Ca2+ increases of barely detectable
size appeared in 6 of 7 oocytes and no Ca2+ change occurred in the
remaining one. Each of the slight Ca2+ increases exhibited either a
localized Ca2+ elevation restricted to one cortical region or a
rather uniform increase throughout the entire oocyte (data not shown). None of
the 7 oocytes underwent GVBD. After the experiments, 5 out of the 7 oocytes
were washed with FSW, activated by excess K+ seawater
(Deguchi and Osanai, 1994b),
and then fixed for the staining with DAPI. In all cases, a few decondensed
sperm nuclei were detected (data not shown), suggesting successful sperm
entries under conditions where LCaSW and D-600 were simultaneously
applied.
In contrast to the inhibitory effects of LCaSW and D-600, heparin had no serious influence on sperm-induced Ca2+ changes. Following insemination, the oocyte injected with 20 mg/ml heparin displayed an initial Ca2+ transient and a subsequent plateau phase (n=9; Fig. 2D), resulting in GVBD (9/9). The initial Ca2+ transient showed a cortical flash pattern similar to that observed at normal fertilization (data not shown). The only unusual point in the heparin-injected oocytes was that some additional Ca2+ transients, each of which took the form of a cortical flash rather than a point-source propagating wave, appeared between the initial transient and the plateau phase in 4 of 9 oocytes (data not shown). It should be noted that the same concentration of heparin completely blocked an Ins(1,4,5)P3-induced Ca2+ increase (see the next section). The above data collectively suggest that Ca2+ influx through Ca2+ channels on the plasma membrane, but not Ca2+ release from Ins(1,4,5)P3-sensitive stores, contributes to Ca2+ changes at fertilization.
In all cases described above, the F/F0 level in the nucleus became obviously greater than that in the cytoplasm toward the end of plateau phase when the oocytes were advancing to GVBD (Fig. 1A, Fig. 2A,D). The following experiments were performed to examine whether this situation is necessary for GVBD. When FSW was replaced by Ca2+-free seawater (CaFSW) at 4 minutes after fertilization in 10 kDa CGD-injected oocytes, both cytoplasmic and nuclear Ca2+ elevations were terminated prematurely (total duration of Ca2+ increases: 320±11 seconds, n=6) and higher F/F0 level in the nucleus was not produced subsequently (Fig. 4A). However, GVBD was not inhibited in these oocytes. To determine the precise time required for the onset of nuclear envelope breakdown, 70 kDa CGD was used under these conditions (Fig. 4B); leakage of 70 kDa CGD from cytoplasm to nucleus occurred at 461±17 seconds (n=6) after fertilization, with a similar timing for normally fertilized oocytes continuously bathed in FSW (see above). These results indicate that the higher nuclear F/F0 level around the final part of plateau phase is not necessarily required for the progression of GVBD.
Potential ability of oocytes to release Ca2+ from internal
stores
It is known that 5-HT can stimulate Ca2+ release from internal,
probably Ins(1,4,5)P3-sensitive Ca2+ stores in
the bivalves such as Ruditapes
(Guerrier et al., 1993) and
Hiatella (Deguchi and Osanai,
1995
). In the next series of experiments, the effect of 5-HT on
Ca2+ changes in Mactra oocytes was investigated. When
unfertilized oocytes were exposed to 100 nM 5-HT in FSW, a large
Ca2+ transient was immediately caused (n=7;
Fig. 5A). During its rising
phase, an increased F/F0 signal first took place at one cortical
region and propagated across the oocyte in a wave-like fashion
(Fig. 6A). The initial
Ca2+ wave always started from the restricted point of the oocyte
cortex which was situated around the edge of the space between two coverslips
(the left side of each fluorescence image) in a chamber, the site where
effective concentration of 5-HT must be first attained. In most cases, the
first Ca2+ transient comprised an initial peak and following
smaller but oscillatory Ca2+ spikes
(Fig. 5A, inset); each
Ca2+ spike took the form of a point-source Ca2+ wave
which began to propagate before the increased Ca2+ in the preceding
Ca2+ spike completely returned to the resting level (e.g. the
second Ca2+ wave starting at 5.2 seconds in
Fig. 6A). A similar
Ca2+ transient, a set of an initial peak and following smaller
Ca2+ spikes, repeatedly appeared when 5-HT was continuously present
(Fig. 5A). The number of
Ca2+ transients during a period of 20 minutes was 5.3±0.9
(n=7). The rising phase of the later Ca2+ transients also
exhibited a propagating Ca2+ wave pattern, although the wave
starting point sometimes changed even in the same oocyte
(Fig. 6A,B). In the
5-HT-treated oocytes, GVBD was only induced when the first Ca2+
transient just after the 5-HT stimulation was maintained for a relatively long
time (2/7; data not shown).
Repetitive Ca2+ transients induced by 5-HT were also detected when nuclear Ca2+ changes were monitored with 70 kDa CGD injected into the nucleus (n=6; Fig. 5B). The number of Ca2+ transients during a period of 20 minutes was 4.7±1.1 (n=6). In these oocytes, each Ca2+ transient was initiated as a Ca2+ wave propagating across the nucleus (Fig. 6C). GVBD took place in 1 of the 6 cases.
Finally, 5-HT was applied in the absence of external Ca2+ to determine whether 5-HT-induced Ca2+ oscillations are dependent on Ca2+ influx. When oocytes were exposed to 100 nM 5-HT in CaFSW, an initial large Ca2+ transient and subsequent repetitive Ca2+ transients, each of which was typically accompanied by oscillatory Ca2+ spikes, were similarly produced (data not shown). The number of Ca2+ transients during a period of 20 minutes was 4.0±0.9 (n=4), which was not significantly different from the values in FSW. The spatiotemporal property of each Ca2+ transient was also not different from that detected in FSW (data not shown). Under conditions where 5-HT was applied in CaFSW, 1 of the 4 oocytes resulted in GVBD.
To confirm the existence of an Ins(1,4,5)P3-induced Ca2+ release mechanism in Mactra oocytes, the effect of `caged' derivative of Ins(1,4,5)P3 was examined. In the oocytes injected with 600 µM caged Ins(1,4,5)P3, a single UV irradiation caused a single Ca2+ transient lasting for 1-3 minutes during the incubation in FSW (7/7; data not shown), in CaFSW (4/5; data not shown), and in LCaSW with 100 µM D-600 (6/8; data not shown). Among the oocytes showing such a short-lived Ca2+ transient (n=17), GVBD was induced in only three cases. In contrast, repeated UV irradiations generally produced a long-lived Ca2+ increase even in CaFSW (n=6; Fig. 5C) and triggered GVBD more frequently (4/6). The UV-induced Ca2+ transient in caged Ins(1,4,5)P3-injected oocytes was completely blocked by simultaneous injection of 20 mg/ml heparin; none of 9 examined oocytes displayed any Ca2+ change after UV irradiation (data not shown). When 5 out of the 9 oocytes were inseminated subsequently, Ca2+ increases and resultant GVBD were invariably induced (5/5; data not shown), indicating that they had never lost sensitivities to Ca2+ change itself. These results demonstrate that Mactra oocytes are equipped with an Ins(1,4,5)P3 receptor-mediated Ca2+ release mechanism, which can produce, if forcibly stimulated, a considerable Ca2+ increase enough to trigger meiosis reinitiation from PI.
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Discussion |
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In addition to the measurement of cytoplasm- or nucleus-restricted
Ca2+ changes, the precise start time of GVBD was determined by the
experiments with 70 kDa CGD; the nuclear envelope began to break down shortly
before, rather than after, the end of plateau phase in normally fertilized
Mactra oocytes. In parallel experiments with 10 kDa CGD, an
interesting phenomenon of higher F/F0 level in the nucleus than in
the cytoplasm became noticeable around the final part of plateau phase [a
similar phenomenon is also reported in Urechis and analyzed in detail
(Stephano and Gould, 1997)].
This result suggests the selective accumulation of Ca2+ in the
nucleus just before the nuclear envelope breakdown in fertilized
Mactra oocytes, although there is a possibility that the phenomenon
is partly due to artifacts, such as the different behavior of fluorescent
Ca2+ indicators in the cytoplasmic and nuclear environments
(Thomas et al., 2000
). The
finding led us to conceive that such a situation might play a role in
regulating the progression of GVBD. However, GVBD was neither inhibited nor
delayed by application of CaFSW at 4 minutes after fertilization, which
abolished the plateau phase of cytoplasmic and nuclear Ca2+
elevations prematurely and prevented the subsequent appearance of higher
F/F0 level in the nucleus. By contrast, GVBD is completely blocked
when the same treatment with CaFSW is carried our within 3 minutes of
fertilization (Deguchi and Osanai,
1994b
). Therefore, it is likely that the initial period of
cytoplasmic and/or nuclear Ca2+ elevations at fertilization serves
as a prerequisite trigger for meiosis reinitiation from PI, but that the later
part of plateau phase, during which heterogeneous F/F0 levels in
the cytoplasm and nucleus are established, is no longer required for the
progression of subsequent meiotic events, including the disassembly of the
nuclear envelope. A similar scheme might be applied to other PI-type
protostomes including Spisula
(Dubé and Guerrier,
1982
) and Urechis
(Gould and Stephano, 1989
;
Stephano and Gould, 1997
). It
is known that the cell cycle transition from PI to MI can be triggered without
an intracellular Ca2+ increase in a variety of MI-type protostomes
such as bivalves (e.g. Kyozuka et al.,
1997
), limpets (Gould et al.,
2001
) and nemertean worms
(Stricker and Smythe,
2000
).
Stimulation with 5-HT caused repetitive Ca2+ transients in
Mactra oocytes, although the drug had a weak effect on triggering
GVBD (see also Fong et al.,
1996). The 5-HT-induced Ca2+ oscillations proceeded
without external Ca2+ in contrast to the situation observed at
fertilization. Spatiotemporal analysis of the 5-HT-induced Ca2+
oscillations in this study, which is the first demonstration in protostome
oocytes, revealed that the rising phase of each Ca2+ transient
takes the form of a point-source Ca2+ wave propagating across the
whole oocyte, including the cytoplasm and nucleus. The ability for substantial
Ca2+ release from internal stores in Mactra oocytes was
also confirmed by the experiments with caged Ins(1,4,5)P3;
continuous application of Ins(1,4,5)P3 not only produced a
long-lived Ca2+ increase but also triggered GVBD without a
contribution of external Ca2+. These results indicate that
Mactra oocytes have the potentiality not only to release internally
stored Ca2+ through the interaction between
Ins(1,4,5)P3 and its receptors but also to produce
repetitive Ca2+ waves, as observed at fertilization in MI-type
bivalves (Deguchi and Morisawa,
1997
) and many other non-PI-type animals (see
Stricker, 1999
). The existence
of Ins(1,4,5)P3-induced Ca2+ release mechanism
is also reported in Urechis
(Stephano and Gould, 1997
),
although it is unknown whether this release alone can produce a sufficient
amount of Ca2+ to provoke GVBD in this species. These results,
together with the study on Spisula oocytes showing that GVBD is
induced by Ins(1,4,5)P3 injection
(Bloom et al., 1988
), imply
that PI-arrested oocytes in PI-type protostomes are equipped with this
universal Ca2+-mobilizing system.
It remains unknown why the Ins(1,4,5)P3-dependent
Ca2+ release pathway is activated at fertilization in non-PI-type
animals, but not in PI-type species such as Mactra. There are
essentially two different possibilities to account for the differences between
PI and non-PI-type animals. One possibility is that different factors exist in
sperm, which stimulate different pathways in oocytes or eggs at fertilization.
In various animals, including nemertean worms
(Stricker, 1997), ascidians
(Kyozuka et al., 1998
;
McDougall et al., 2000
;
Runft and Jaffe, 2000
) and
vertebrates (Swann, 1990
;
Yamamoto et al., 2001
),
injection of sperm extract (SE) into unfertilized oocytes or eggs of the same
species has been shown to produce intracellular Ca2+ changes
similar to those seen at fertilization. It seems likely that the SE-induced
Ca2+ changes are mainly regulated by Ca2+ release
through an Ins(1,4,5)P3 receptor-mediated mechanism
(Oda et al., 1999
;
Runft and Jaffe, 2000
). The
active components of SE, which are recognized as soluble proteins in all
animals described above (Stricker,
1999
), are effective beyond species, even in heterologous
combinations of gametes obtained from distantly related animals
(Stricker et al., 2000
).
However, the existence of SE and its effect on Ca2+ changes have
not yet been confirmed in PI-type animals. However, it has been reported in
the PI-type Urechis that a sperm acrosomal protein, which externally
acts on the oocyte plasma membrane, causes Ca2+ influx via
voltage-gated Ca2+ channels and resultant intracellular
Ca2+ changes similar to those seen at fertilization
(Gould and Stephano, 1989
;
Stephano and Gould, 1997
).
The other possibility is based on differences in the ability of oocytes or
eggs to generate intracellular Ca2+ increases. There are
considerable structural changes in the endoplasmic reticulum (ER), which is
the most likely candidate for internal Ca2+ stores, during the
transition from PI to MI in starfish
(Jaffe and Terasaki, 1994) and
nemertean worm oocytes (Stricker et al.,
1998
). An Ins(1,4,5)P3-induced Ca2+
release mechanism develops as oocyte maturation advances in starfish
(Chiba et al., 1990
) and
hamster oocytes (Fujiwara et al.,
1993
). Intracellular stocks of polyphosphoinositides, precursors
of Ins(1,4,5)P3, increase between PI and MI in MI-type
limpet oocytes (Borg et al.,
1992
). Such circumstances observed in the non-PI-type animals,
which all indicate the incomplete establishment of an
Ins(1,4,5)P3-mediated Ca2+ release mechanism at
PI stage, may be responsible in part for the inability of Mactra
oocytes to use internally stored Ca2+ at fertilization. However,
additional reasons may also be required, considering the fact that substantial
Ca2+ release can be induced in Mactra oocytes stimulated
with 5-HT or Ins(1,4,5)P3 instead of sperm. Recently, it
has been demonstrated that some intermediate molecules such as Src family
kinases and phospholipase C
play an essential role in the signal
transduction between sperm (or SE) and the production of
Ins(1,4,5)P3 in several non-PI-type animals
(Carroll et al., 1997
;
Runft and Jaffe, 2000
;
Abassi et al., 2000
;
Sato et al., 2000
). It might
be possible that such molecules are lacking (or their activities are
suppressed by other molecules) in PI-arrested oocytes. In MI-type ascidian
oocytes, it is also proposed that sperm- or SE-induced Ca2+
oscillations are regulated by cyclin B1-dependent kinase activity
(Levasseur and McDougall,
2000
; McDougall et al.,
2000
), which is low at PI stage. In fact, injection of SE causes
Ca2+ oscillations in MI-arrested ascidian oocytes but the same
procedure does not produce any Ca2+ change in PI-arrested oocytes,
in spite of the fact that injection of Ins(1,4,5)P3 is
effective in inducing a considerable Ca2+ increase even in the
immature stage (McDougall et al.,
2000
).
The present study clearly demonstrated that oocytes of the PI-type bivalve Mactra predominantly use a Ca2+ influx pathway at fertilization, in spite of the potential ability to release internally stored Ca2+. Moreover, possible differences in the mechanism underlying intracellular Ca2+ increases at fertilization between PI-type and other types of animals were pointed out and discussed. Further studies, including the identification of sperm-derived factors and their downstream pathways leading to intracellular Ca2+ increases in oocytes or eggs, are required to explain the differences between PI- and non-PI-type animals. Bivalves will be suitable materials for such a comparison, since there are PI- and MI-type species where spatiotemporal Ca2+ patterns are both clarified.
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