External Ca2+ is predominantly used for cytoplasmic and nuclear Ca2+ increases in fertilized oocytes of the marine bivalve Mactra chinensis

Ryusaku Deguchi1,* and Masaaki Morisawa2

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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Oocytes of the marine bivalve Mactra chinensis are spawned and arrested at the germinal vesicle stage (first meiotic prophase) until fertilization, without undergoing a process called oocyte maturation. As is the case of other animals, a fertilized oocyte of the bivalve displays increases in intracellular free Ca2+. We have clarified here the spatiotemporal patterns and sources of the intracellular Ca2+ changes at fertilization. Shortly after insemination, increased Ca2+ simultaneously appeared at the whole cortical region of the oocyte and spread inwardly to the center, attaining the maximal Ca2+ levels throughout the oocyte, including the cytoplasm and nucleus. The initial maximal Ca2+ peak was followed by a submaximal plateau phase of cytoplasmic and nuclear Ca2+ elevations, which persisted for several minutes. The nuclear envelope began to break down shortly before the termination of the plateau phase. These sperm-induced Ca2+ changes were inhibited by suppression of the influx of external Ca2+ from seawater but not by disturbance of the release of internal Ca2+ from inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-sensitive stores, suggesting that the increased Ca2+ is from an external source. In contrast to the situation observed at fertilization, an oocyte artificially stimulated with serotonin (5-hydroxytryptamine, 5-HT) displayed repetitive Ca2+ transients, each of which started from one cortical region and propagated across the oocyte as a Ca2+ wave. The 5-HT-induced Ca2+ transients persisted even in the absence of external Ca2+. Experiments with caged Ins(1,4,5)P3 revealed that Ca2+ release from Ins(1,4,5)P3-sensitive stores is another pathway that is sufficient to trigger meiosis reinitiation from the first prophase. These results demonstrate that Mactra oocytes can potentially use two different Ca2+-mobilizing pathways: Ca2+ influx producing a centripetal Ca2+ wave from the whole cortex and Ca2+ release from Ins(1,4,5)P3-sensitive stores producing a point-source propagating Ca2+ wave. However, it seems likely that the Ca2+ influx pathway is predominantly activated at fertilization.

Key words: Intracellular Ca2+, Fertilization, Ca2+ channels, Serotonin, Ins(1,4,5)P3


    Introduction
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 Introduction
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Fully-grown oocytes arrested at the first meiotic prophase (prophase I, PI) in ovaries progress oocyte maturation, when exposed to hormones or released from inhibitory substances, to acquire the ability for fertilization in most animal species. These oocytes are again arrested at species-specific stages including the first metaphase (metaphase I, MI), second metaphase (metaphase II, MII), and pronuclear stage (PN), until fertilization (Masui, 1985Go). In the bivalves such as Mytilus and Ruditapes, the second arrest of meiosis occurs at MI prior to fertilization (MI-type) (Masui, 1985Go; Osanai and Kuraishi, 1988Go). In contrast, there are some bivalve species (e.g. Spisula and Mactra) in which meiosis reinitiation from PI is physiologically triggered concomitantly with fertilization without a process of oocyte maturation (PI-type) (Masui, 1985Go; Deguchi and Osanai, 1994bGo). Regardless of the stages of fertilization, single or multiple increases in intracellular Ca2+ in fertilized oocytes or eggs have been detected in all animal species investigated so far (reviewed by Jaffe, 1985Go; Miyazaki et al., 1993Go; Stricker, 1999Go). The Ca2+ increases are recognized as essential for the oocytes or eggs to be released from the cell cycle arrest (for a review, see Whitaker and Patel, 1990Go).

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., 1993Go) and Ruditapes (Leclerc et al., 2000Go). 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, 1994aGo; Deguchi and Morisawa, 1997Go). In Mytilus, the initial Ca2+ transient at fertilization arises almost synchronously in the oocyte without forming a point-source Ca2+ wave (Deguchi and Osanai, 1994aGo). 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, 1999Go), during the rising phase of the initial Ca2+ transient (Deguchi and Morisawa, 1997Go). 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., 1996Go). 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., 2000Go). 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, 1994aGo). In Mytilus, the phase of Ca2+ oscillations is completely blocked by heparin but not by D-600 (Deguchi et al., 1996Go), and each Ca2+ spike during this phase takes the form of a point-source Ca2+ wave (Deguchi and Morisawa, 1997Go), which seems to be a common pattern of Ca2+ release from internal stores in fertilized oocytes or eggs of many other animals (Stricker, 1999Go). 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, 1980Go), 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, 1982Go). 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, 1994bGo). 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, 1994bGo). 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, 1953Go; Dubé and Guerrier, 1982Go; Deguchi and Osanai, 1994bGo), and that stimulation of this pathway with high K+ seawater conversely triggers GVBD without insemination (Guerrier et al., 1981Go; Dubé and Guerrier, 1982Go; Deguchi and Osanai, 1994bGo). 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., 1988Go). 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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Gametes
Adult specimens of the marine clam Mactra chinensis were collected in Tokyo Bay from July to August and kept in an aquarium with running seawater at 12-18°C. PI-arrested oocytes were obtained by dissecting and agitating the ovaries and then washed two or three times with filtered seawater (FSW). The oocytes were incubated in FSW for at least 60 minutes, and only the batches showing less than 10% spontaneous meiosis reinitiation, which was judged by the presence of GVBD, were used. Sperm was collected in the same manner, stored in a refrigerator, and properly diluted with FSW prior to insemination.

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, 1994aGo). 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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Fig. 5. Temporal patterns of Ca2+ changes induced by 5-HT and Ins(1,4,5)P3. The oocyte injected with 10 kDa (A, into the cytoplasm) or 70 kDa CGD (B, into the nucleus) was exposed to 5-HT (final concentration: 100 nM) in FSW. In C, the oocyte injected with 10 kDa CGD plus 600 µM caged Ins(1,4,5)P3 was irradiated with UV light six times (arrows) in CaFSW. The F/F0 values were calculated in the whole oocyte (A,C) or in the nuclear region alone (B). An inset in A depicts the expanded Ca2+ pattern of the first transient just after the addition of 5-HT. GVBD did not occur in A or B. In contrast, GVBD occurred in C just after the recording period.

 

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, 1994aGo). 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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Fig. 1. Temporal patterns of Ca2+ changes in the cytoplasm and nucleus in normally fertilized oocytes. To monitor the Ca2+ changes, 10 kDa CGD (A) or 70 kDa CGD (B) was injected into the cytoplasm, or 70 kDa CGD was injected into the nucleus (C). The relative fluorescence levels of CGD (F/F0 values) were calculated in the cytoplasmic and nuclear regions separately. The steep increases in F/F0 (arrowheads in B and C) indicate the leakage of 70 kDa CGD through the nuclear envelope. A pair of right panels shows fluorescence images before (top) and after (bottom) each fluorescence measurement. All of the oocytes underwent GVBD on schedule (at ~10 minutes) and then developed to early trochophores.

 


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Fig. 4. Effect of Ca2+-free seawater (CaFSW) on sperm-induced Ca2+ changes. The oocyte where 10 kDa (A) or 70 kDa CGD (B) was injected into the cytoplasm was inseminated in FSW, which was replaced by CaFSW at 4 minutes after fertilization. The F/F0 values were calculated in the cytoplasmic and nuclear regions separately. The steep increase in F/F0 (arrowhead in B) indicates the leakage of 70 kDa CGD through the nuclear envelope. The two oocytes underwent GVBD on schedule.

 


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Fig. 3. Ca2+ dynamics during the rising phase of the initial Ca2+ transient at fertilization. The data in A, B and C were obtained from the oocytes for Fig. 1A, 1C and 2B, respectively. Sequential fluorescence images were acquired every 0.4 seconds, 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 was defined as the time of the first visible Ca2+ increase at fertilization.

 


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Fig. 6. Ca2+ dynamics during the rising phase of 5-HT-induced Ca2+ transients. The data in A and B were obtained from the first Ca2+ transient (initiated at 0) and the fifth transient (initiated at 1278 seconds), respectively, in the same oocyte for Fig. 5A. In C, a rising pattern of the third transient (initiated at 537 seconds) in the oocyte for Fig. 5B was investigated. Sequential fluorescence images were acquired every 0.4 seconds, 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 was defined as the initiation time of each Ca2+ transient, which is independent of the time of 5-HT addition.

 



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Fig. 2. Effects of low Ca2+ seawater (LCaSW), D-600, and heparin on sperm-induced Ca2+ changes. The oocyte where 10 kDa CGD was injected into the cytoplasm was preincubated for 6-8 minutes in LCaSW (A), in FSW containing 100 µM D-600 (B), or in LCaSW containing 100 µM D-600 (C), and then inseminated. In D, the oocyte injected with 10 kDa CGD plus 20 mg/ml heparin was inseminated in FSW. The F/F0 values were calculated in the cytoplasmic and nuclear regions separately (A,D) or in the whole oocyte (B,C). GVBD was prominently delayed (occurred at ~25 minutes after fertilization) in A, or prevented in B and C. In contrast, GVBD took place on schedule in D.

 


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Spatiotemporal Ca2+ changes at normal fertilization
When 10 kDa CGD was injected into the cytoplasm of PI-arrested oocytes, its fluorescence signals were distributed not only in the cytoplasm but also inside the nucleus (Fig. 1A, topright panel), indicating that the dye can diffuse across the nuclear envelope. A temporal pattern of intracellular Ca2+ changes at normal fertilization, expressed as F/F0, was basically identical to that monitored with a ratiometric Ca2+ dye, Fura-2 (Deguchi and Osanai, 1994bGo); an initial large Ca2+ transient was followed by a lower plateau phase lasting for several minutes (n=7; Fig. 1A). The rising phase of the initial Ca2+ transient exhibited a cortical flash pattern; an increased F/F0 signal first took place around the whole cortical region of the oocyte and then spread inwardly, attaining a peak level throughout the oocyte within several seconds (Fig. 3A). The peak F/F0 values calculated in the cytoplasmic and nuclear regions were almost the same (Fig. 1A). These results suggest that external Ca2+ enters the oocyte through the whole plasma membrane at the time of fertilization, and that the increased Ca2+ rapidly spreads over the cytoplasm and even into the nucleus.

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, 1994bGo). 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, 1994bGo). 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, 1982Go; Deguchi et al., 1996Go), 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, 1994bGo), 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., 1993Go) and Hiatella (Deguchi and Osanai, 1995Go). 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.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present analysis using 10 kDa CGD revealed that an initial large Ca2+ transient just after fertilization clearly exhibits a cortical flash pattern in Mactra oocytes; a Ca2+ increase first took place at the whole oocyte cortex and then spread throughout the cytoplasm and even inside the nucleus. Such a pattern of centripetal Ca2+ increase in the cytoplasm or nucleus was also confirmed by 70 kDa CGD injected into either of the two regions. The initial transient was almost completely blocked by simultaneous treatments with LCaSW and D-600, although either treatment alone had an insufficient effect. In contrast, this transient was not suppressed by heparin at all. These results strongly suggest that the initial Ca2+ transient just after fertilization is mainly due to Ca2+ influx via voltage-gated Ca2+ channels on the plasma membrane. The initial transient was followed by a plateau phase, during which submaximal levels of cytoplasmic and nuclear Ca2+ were maintained. The plateau phase was also suppressed by LCaSW and D-600, but not by heparin. These results, together with the fact that the plateau phase is immediately abolished by the addition of CaFSW [Deguchi and Osanai, 1994bGo) and this study], suggest that this phase is regulated by continuous Ca2+ influx via voltage-gated Ca2+ channels. It is, therefore, likely that the Ca2+ influx mechanism is totally responsible for the cytoplasmic and nuclear Ca2+ increases in fertilized Mactra oocytes. A similar spatiotemporal Ca2+ pattern at fertilization and its dependence on Ca2+ influx have also been demonstrated in the PI-type echiuran worm Urechis (Jaffe et al., 1979Go; Stephano and Gould, 1997Go).

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, 1997Go)]. 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., 2000Go). 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, 1994bGo). 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, 1982Go) and Urechis (Gould and Stephano, 1989Go; Stephano and Gould, 1997Go). 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., 1997Go), limpets (Gould et al., 2001Go) and nemertean worms (Stricker and Smythe, 2000Go).

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., 1996Go). 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, 1997Go) and many other non-PI-type animals (see Stricker, 1999Go). The existence of Ins(1,4,5)P3-induced Ca2+ release mechanism is also reported in Urechis (Stephano and Gould, 1997Go), 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., 1988Go), 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, 1997Go), ascidians (Kyozuka et al., 1998Go; McDougall et al., 2000Go; Runft and Jaffe, 2000Go) and vertebrates (Swann, 1990Go; Yamamoto et al., 2001Go), 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., 1999Go; Runft and Jaffe, 2000Go). The active components of SE, which are recognized as soluble proteins in all animals described above (Stricker, 1999Go), are effective beyond species, even in heterologous combinations of gametes obtained from distantly related animals (Stricker et al., 2000Go). 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, 1989Go; Stephano and Gould, 1997Go).

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, 1994Go) and nemertean worm oocytes (Stricker et al., 1998Go). An Ins(1,4,5)P3-induced Ca2+ release mechanism develops as oocyte maturation advances in starfish (Chiba et al., 1990Go) and hamster oocytes (Fujiwara et al., 1993Go). Intracellular stocks of polyphosphoinositides, precursors of Ins(1,4,5)P3, increase between PI and MI in MI-type limpet oocytes (Borg et al., 1992Go). 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{gamma} 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., 1997Go; Runft and Jaffe, 2000Go; Abassi et al., 2000Go; Sato et al., 2000Go). 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, 2000Go; McDougall et al., 2000Go), 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., 2000Go).

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.


    Acknowledgments
 
We thank H. Shirakawa (Department of Physiology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan) for providing us with the technique of image processing using NIH Image. This work was supported by a Research Fellowship and Grant from the Japan Society for Promotion of Science for Young Scientists to R.D. (no. 4141).


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 Results
 Discussion
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