External Ca2+ Is Predominantly Used for Cytoplasmic and Nuclear Ca2+ Increases in Fertilized Oocytes of the Marine Bivalve Mactra Chinensis

Research Article 367 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 1Department of Biology, Miyagi University of Education, Aoba-ku, Sendai, Miyagi 980-0845, Japan 2Misaki Marine Biological Station, the University of Tokyo, Miura, Kanagawa 238-0225, Japan *Author for correspondence (e-mail: [email protected])

Summary Oocytes of the marine bivalve Mactra chinensis are source. In contrast to the situation observed at fertilization, spawned and arrested at the germinal vesicle stage (first an oocyte artificially stimulated with serotonin (5- meiotic prophase) until fertilization, without undergoing a hydroxytryptamine, 5-HT) displayed repetitive Ca2+ process called oocyte maturation. As is the case of other transients, each of which started from one cortical region animals, a fertilized oocyte of the bivalve displays increases and propagated across the oocyte as a Ca2+ wave. The 5- in intracellular free Ca2+. We have clarified here the HT-induced Ca2+ transients persisted even in the absence 2+ spatiotemporal patterns and sources of the intracellular of external Ca . Experiments with caged Ins(1,4,5)P3 2+ 2+ Ca changes at fertilization. Shortly after insemination, revealed that Ca release from Ins(1,4,5)P3-sensitive stores increased Ca2+ simultaneously appeared at the whole is another pathway that is sufficient to trigger meiosis cortical region of the oocyte and spread inwardly to the reinitiation from the first prophase. These results center, attaining the maximal Ca2+ levels throughout the demonstrate that Mactra oocytes can potentially use two oocyte, including the cytoplasm and nucleus. The initial different Ca2+-mobilizing pathways: Ca2+ influx producing maximal Ca2+ peak was followed by a submaximal plateau a centripetal Ca2+ wave from the whole cortex and Ca2+ 2+ phase of cytoplasmic and nuclear Ca elevations, which release from Ins(1,4,5)P3-sensitive stores producing a point- persisted for several minutes. The nuclear envelope began source propagating Ca2+ wave. However, it seems likely to break down shortly before the termination of the plateau that the Ca2+ influx pathway is predominantly activated at phase. These sperm-induced Ca2+ changes were inhibited fertilization. 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, Key words: Intracellular Ca2+, Fertilization, Ca2+ channels, 2+ suggesting that the increased Ca is from an external Serotonin, Ins(1,4,5)P3

Introduction et al., 1993; Stricker, 1999). The Ca2+ increases are recognized Fully-grown oocytes arrested at the first meiotic prophase as essential for the oocytes or eggs to be released from the cell (prophase I, PI) in ovaries progress oocyte maturation, when cycle arrest (for a review, see Whitaker and Patel, 1990). exposed to hormones or released from inhibitory substances, As for MI-type bivalves, temporal patterns of Ca2+ increases to acquire the ability for fertilization in most animal species. at fertilization have been analyzed in five different species: These oocytes are again arrested at species-specific stages Mytilus, Crassostrea, Ruditapes, Limaria and Hiatella. When including the first metaphase (metaphase I, MI), second the Ca2+ indicator Fluo-3 is introduced as AM ester, only a metaphase (metaphase II, MII), and pronuclear stage (PN), single blunt Ca2+ increase, which persists for several minutes, until fertilization (Masui, 1985). In the bivalves such as Mytilus is observed in fertilized oocytes of Mytilus (Abdelmajid et al., and Ruditapes, the second arrest of meiosis occurs at MI prior 1993) and Ruditapes (Leclerc et al., 2000). In the oocytes to fertilization (MI-type) (Masui, 1985; Osanai and Kuraishi, injected with Ca2+ indicators such as Fura-2 and Calcium 1988). In contrast, there are some bivalve species (e.g. Spisula Green-1, however, Ca2+ response at fertilization comprises an and Mactra) in which meiosis reinitiation from PI is initial sharp Ca2+ transient and subsequent repetitive Ca2+ physiologically triggered concomitantly with fertilization spikes (Ca2+ oscillations) in all of the five species, including without a process of oocyte maturation (PI-type) (Masui, 1985; Mytilus and Ruditapes (Deguchi and Osanai, 1994a; Deguchi Deguchi and Osanai, 1994b). Regardless of the stages of and Morisawa, 1997). In Mytilus, the initial Ca2+ transient at fertilization, single or multiple increases in intracellular Ca2+ fertilization arises almost synchronously in the oocyte without in fertilized oocytes or eggs have been detected in all animal forming a point-source Ca2+ wave (Deguchi and Osanai, species investigated so far (reviewed by Jaffe, 1985; Miyazaki 1994a). A more recent analysis revealed that the increased Ca2+ 368 Journal of Cell Science 116 (2) starts from the entire oocyte cortex and spreads inwardly to the and that intracellular concentrations of precursors of center, taking the form of a ‘cortical flash’ pattern (Stricker, Ins(1,4,5)P3 become higher following fertilization (Bloom et 1999), during the rising phase of the initial Ca2+ transient al., 1988). Their results raise the possibility that not only Ca2+ 2+ (Deguchi and Morisawa, 1997). This initial transient is not influx but also Ca release from Ins(1,4,5)P3-sensitive stores affected by heparin, an antagonist of inositol 1,4,5- might be involved in sperm-induced Ca2+ increases and trisphosphate [Ins(1,4,5)P3] receptors, but is suppressed responsible for meiosis reinitiation from PI in PI-type bivalves. by blockers of voltage-gated Ca2+ channels such as The aim of the present study was to understand the methoxyverapamil (D-600) (Deguchi et al., 1996). mechanisms underlying the sperm-induced Ca2+ changes at Pharmacological experiments in another species, Ruditapes, fertilization in the PI-type bivalve Mactra chinensis. First, we suggest that voltage-gated Ca2+ channels are progressively investigated the spatiotemporal Ca2+ dynamics not only in the situated on the plasma membrane of oocytes during the oocyte whole oocyte but also in more restricted regions, in the maturation from PI to MI (Leclerc et al., 2000). These data cytoplasm and inside the nucleus, at normal fertilization. collectively suggest that the initial Ca2+ transient at fertilization Second, we clarified the main Ca2+ source and pathway for the in MI-type bivalves is mainly due to the influx of external Ca2+ sperm-induced Ca2+ changes. Finally, we examined whether through voltage-gated Ca2+ channels distributed over the unfertilized oocytes have the potential ability to use other Ca2+- plasma membrane. However, the phase of Ca2+ oscillations, mobilizing mechanisms that are quiescent at fertilization. Our which occurs after the initial Ca2+ transient, persists even after results demonstrate that Mactra oocytes possess at least two the removal of external Ca2+ in all MI-type bivalves tested pathways for producing cytoplasmic and nuclear Ca2+ (Deguchi and Osanai, 1994a). In Mytilus, the phase of Ca2+ increases. One is the Ca2+ influx mechanism via voltage- oscillations is completely blocked by heparin but not by D-600 dependent Ca2+ channels, which is responsible for the Ca2+ (Deguchi et al., 1996), and each Ca2+ spike during this phase increases at fertilization. The other is the Ca2+ release 2+ takes the form of a point-source Ca wave (Deguchi and mechanism via Ins(1,4,5)P3 receptors, which may be a latent Morisawa, 1997), which seems to be a common pattern of Ca2+ system and not play a central role, at least by the time of release from internal stores in fertilized oocytes or eggs of GVBD, in fertilized oocytes. This situation is quite different many other animals (Stricker, 1999). These results suggest that from that observed in the MI-type bivalves, where both the the phase of Ca2+ oscillations, unlike an initial Ca2+ transient, external and the internal Ca2+ sources are used in fertilized 2+ is chiefly regulated by Ca release from Ins(1,4,5)P3-sensitive oocytes. 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- Materials and Methods gated Ca2+ channels and Ca2+ release from internal stores via Gametes Ins(1,4,5)P3 receptors. Adult specimens of the marine clam Mactra chinensis were collected The contribution of Ca2+ influx to intracellular Ca2+ in Tokyo Bay from July to August and kept in an aquarium with increases at fertilization has been suggested in several PI-type running seawater at 12-18°C. PI-arrested oocytes were obtained by bivalves. In Spisula, fertilization causes depolarization of the dissecting and agitating the ovaries and then washed two or three plasma membrane lasting for several minutes (Finkel and Wolf, times with filtered seawater (FSW). The oocytes were incubated in FSW for at least 60 minutes, and only the batches showing less than 1980), which may activate voltage-dependent Ca2+ channels. 45 10% spontaneous meiosis reinitiation, which was judged by the In Barnea, long-term Ca uptake, which is inhibited by the presence of GVBD, were used. Sperm was collected in the same addition of D-600, takes place at fertilization (Dubé and manner, stored in a refrigerator, and properly diluted with FSW prior Guerrier, 1982). Among PI-type bivalves, Mactra is the only to insemination. 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 Solutions plateau phase of Ca2+ elevation, which persists up to the time Unless otherwise specified, FSW was used as bathing medium for oocytes. Ca2+-free seawater (CaFSW; 462 mM NaCl, 9.4 mM KCl, of germinal vesicle breakdown (GVBD) (Deguchi and Osanai, 2+ 1994b). The plateau phase seems to be maintained by the 48 mM MgCl and 10 mM EGTA) and low Ca seawater (LCaSW; 2+ 2+ 449 mM NaCl, 9.4 mM KCl, 48 mM MgCl, 12 mM CaCl and 10 mM continuous influx of external Ca , since the elevated Ca EGTA; ~2 mM of free Ca2+) were generally supplemented with 10 immediately returns to the resting level following the removal mM Tris and adjusted to pH 8.3. Stock solutions of methoxyverapamil 2+ of external Ca during this phase (Deguchi and Osanai, (D-600; Sigma, St Louis, MO) and serotonin (5-hydroxytryptamine, 1994b). These results suggest that external Ca2+ is the main 5-HT; Sigma) were prepared at 20 mM in DMSO:ethanol (1:3) and source of the sperm-induced intracellular Ca2+ increases in PI- at 10 mM in distilled water, respectively, and diluted just before use. type bivalves. In accordance with this view, it has been shown The former vehicle (DMSO + ethanol) alone had no inhibitory or in the PI-type bivalves that inhibition of Ca2+ influx at stimulatory effect on intracellular Ca2+ changes. fertilization precludes GVBD (Allen, 1953; Dubé and Guerrier, 1982; Deguchi and Osanai, 1994b), and that stimulation of this Microinjection pathway with high K+ seawater conversely triggers GVBD The method of microinjection was essentially equivalent to that without insemination (Guerrier et al., 1981; Dubé and Guerrier, described previously (Deguchi and Osanai, 1994a). Ca2+ indicators, 1982; Deguchi and Osanai, 1994b). In contrast to the Calcium Green-1 10 kDa dextran (10 kDa CGD) and 70 kDa dextran 2+ accumulated evidence for the contribution of Ca influx, (70 kDa CGD), were purchased from Molecular Probes (Eugene, OR) however, Bloom et al. found in Spisula that GVBD can be and prepared at 1.0 and 0.5 mM, respectively, in an injection buffer induced by injection of Ins(1,4,5)P3 into unfertilized oocytes, containing 100 mM K aspartate and 10 mM Hepes (pH 7.0). In some Ca2+ influx in fertilized bivalve oocytes 369 experiments, 10 kDa CGD was further supplemented with 20 mg/ml values of average fluorescence intensities calculated in each region (F) 3-kDa heparin (Sigma) and/or 600 µM myo-inositol 1,4,5- were normalized by dividing them by the resting value (F0). In most 4(5) trisphosphate P -1-(2-nitrophenyl) ethyl ester [caged Ins(1,4,5)P3; cases, the F0 value was obtained from the image just before the first Calbiochem, San Diego, CA]. The tip of a micropipette was inserted detectable Ca2+ increase (= zero time in each Ca2+ trace), which is into the cytoplasm (or into the nucleus in some experiments) of PI- considered as the time of fertilization for inseminated oocytes arrested oocyte, and the injection buffer containing the chemicals was (Deguchi and Osanai, 1994a). Although F0 levels were somewhat ejected by water pressure. Estimated concentrations of the injected different in respective oocytes, temporal changes in the normalized chemicals in the cytoplasm or nucleus ranged from 2 to 4% of the value (F/F0) after the same treatment were almost constant, suggesting original concentrations in a micropipette. The dye-injected oocytes that the initial fluorescence intensities were mainly affected by were incubated in FSW for at least 30 minutes, and those oocytes that intracellular dye concentrations rather than resting Ca2+ levels. In underwent GVBD during the period were discarded. 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. Ca2+ imaging nuclear region of the oocyte where 70 kDa CGD was injected into the All fluorescence measurements were carried out at 20-24°C. One to cytoplasm) were initially lower and mainly came from the three dye-injected oocytes were introduced into a measurement fluorescence of the surrounding dye, but prominently increased as the chamber, where they were slightly compressed by two coverslips nuclear envelope breakdown progressed. In this case, the F0 value was adhered with a strip of double-sided adhesive tape as a spacer. These obtained from the image at ~2 minutes before the beginning of this oocytes were observed with a DIAPHOT-TMD inverted microscope event (see Fig. 1B,C, Fig. 4B). For analyzing the detailed (Nikon, Tokyo, Japan) equipped with epifluorescence apparatus spatiotemporal property of Ca2+ increase, sequential fluorescence (TMD-EF2) with an excitation filter (450-490 nm), a dichroic mirror images were normalized by dividing them by the resting image just (510 nm), and an emission filter (520-560 nm). Fluorescence images before each Ca2+ increase in a pixel-to-pixel manner and expressed as of the oocytes were captured with a silicon-intensified target tube pseudocolor images. The zero time in each montage (Figs 3, 6) (SIT) camera (C-2400; Hamamatsu Photonics, Hamamatsu, Japan) indicates the initiation time of the Ca2+ increase. 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, Results Hamamatsu Photonics) were continuously displayed on a screen Spatiotemporal Ca2+ changes at normal fertilization during the recording period. The measuring oocytes in the chamber were inseminated, exposed When 10 kDa CGD was injected into the cytoplasm of PI- to various agents, or irradiated with UV light when steady levels of arrested oocytes, its fluorescence signals were distributed not fluorescence intensities were confirmed. For insemination, sperm only in the cytoplasm but also inside the nucleus (Fig. 1A, top- suspension diluted with FSW was added to the chamber. The final right panel), indicating that the dye can diffuse across the sperm concentrations were 104-105 sperm/ml in most experiments, nuclear envelope. A temporal pattern of intracellular Ca2+ whereas much denser suspension was used for the oocytes incubated changes at normal fertilization, expressed as F/F0, was with D-600, since fertilization was not easily established in the basically identical to that monitored with a ratiometric Ca2+ presence of the drug probably due to its inhibitory effect on the sperm dye, Fura-2 (Deguchi and Osanai, 1994b); an initial large Ca2+ acrosome reaction. To exchange external medium during the transient was followed by a lower plateau phase lasting for measurement, new medium (~1.5 ml) was added after withdrawal of several minutes (n=7; Fig. 1A). The rising phase of the initial the original medium (residue: ~100 µl), and this procedure was Ca2+ transient exhibited a cortical flash pattern; an increased 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 F/F0 signal first took place around the whole cortical region of (at 380 nm) for 10-15 seconds, during which the blue light for CGD the oocyte and then spread inwardly, attaining a peak level excitation was withdrawn. The gaps during the fluorescence recording throughout the oocyte within several seconds (Fig. 3A). The (see Fig. 5C) correspond to the UV irradiation periods. peak F/F0 values calculated in the cytoplasmic and nuclear During or after fluorescence measurements, oocytes were checked regions were almost the same (Fig. 1A). These results suggest for the presence or absence of GVBD. To obtain the spatiotemporal that external Ca2+ enters the oocyte through the whole plasma Ca2+ pattern at ‘normal’ fertilization, each oocyte that had undergone membrane at the time of fertilization, and that the increased GVBD was withdrawn from the chamber and further cultured Ca2+ rapidly spreads over the cytoplasm and even into the individually in a hole of a 96-well culture plate for observation of the nucleus. subsequent mitotic process. In this case, data analysis was restricted to those oocytes that developed to early trochophores. In some Since fluorescence images in this study were not obtained experiments, measured oocytes were fixed in methanol:acetic acid with confocal microscopy, there was a possibility that (3:1), washed with distilled water 20-30 hours later, and then stained fluorescence signals from the cytoplasm and nucleus might be with 10 µM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; mixed together, giving inaccurate information about Ca2+ Sigma) for 30 minutes to visualize sperm nuclei in them. 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 Data analysis not diffuse across the nuclear envelope (Fig. 1B,C, top-right Fluorescence images on videotape were converted into digital images panels). The cytoplasm-restricted CGD showed a transient and processed with NIH Image (a public domain image processing increase in F/F at fertilization (Fig. 1B), and the first software for the Macintosh computer). The sequential digitized 0 detectable increase in F/F0 was also seen throughout the oocyte images, each of which was constituted by averaging four successive 2+ images, were captured at the interval of 3 seconds for temporal cortex (data not shown). Similarly, a Ca transient at analysis of long-term Ca2+ changes or of 0.4 seconds for detailed fertilization was observed with 70 kDa CGD in the nucleus spatiotemporal analysis of each Ca2+ transient. To investigate (n=5; Fig. 1C); an increased F/F0 signal was initially temporal Ca2+ patterns in the whole oocyte, cytoplasm or nucleus, the distributed just beneath the whole nuclear envelope and spread 370 Journal of Cell Science 116 (2) to the center of the nucleus (Fig. 3B). These data are fully arrowhead in Fig. 1C). These results indicate that the nuclear consistent with the above observation with 10 kDa CGD. envelope begins to break down shortly before, rather than after, In 10 kDa CGD-injected oocytes, the total duration of Ca2+ the end of plateau in fertilized oocytes. 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 Mechanism of Ca2+ changes at fertilization the cessation of the plateau (Deguchi and Osanai, 1994b). When external Ca2+ around fertilized Mactra oocytes is However, the fluorescence of 70 kDa CGD began to leak from removed, a plateau phase of Ca2+ elevation is abolished cytoplasm to nucleus at 473±20 seconds (n=8; arrowhead in immediately (Deguchi and Osanai, 1994b). This fact and the Fig. 1B) or from nucleus to cytoplasm at 506±52 seconds (n=5; 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 voltage- dependent 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) Fig. 1. Temporal patterns of Ca2+ changes in the cytoplasm and nucleus in 2+ 2+ showed a plateau phase of Ca elevation following normally fertilized oocytes. To monitor the Ca changes, 10 kDa CGD (A) or 70 the additional small Ca2+ transients and resulted in kDa CGD (B) was injected into the cytoplasm, or 70 kDa CGD was injected into delayed GVBD (occurring at ~20 minutes after the nucleus (C). The relative fluorescence levels of CGD (F/F0 values) were calculated in the cytoplasmic and nuclear regions separately. The steep increases fertilization, data not shown). in F/F0 (arrowheads in B and C) indicate the leakage of 70 kDa CGD through the Combined applications of LCaSW and D-600 nuclear envelope. A pair of right panels shows fluorescence images before (top) almost totally abolished sperm-induced Ca2+ and after (bottom) each fluorescence measurement. All of the oocytes underwent changes. One (n=3; Fig. 2C) or multiple (n=3; data GVBD on schedule (at ~10 minutes) and then developed to early trochophores. not shown) Ca2+ increases of barely detectable size Ca2+ influx in fertilized bivalve oocytes 371

A 10-kDa CGD B 10-kDa CGD 3.2 2.0 ) LCaSW ) D-600 0 cytoplasm 0 whole 2.8 nucleus oocyte F/F F/F ( (

1.6 e 2.4 e c c n n e e c 2.0 c s s e e r r 1.2 o o u 1.6 u l l f f

e e v 1.2 v i i t t a a 0.8 l l sperm e 0.8 sperm e R R 0 300 600 900 1200 1500 1800 0 300 600 900 Time (s) Time (s) C 10-kDa CGD D 10-kDa CGD + heparin 2.0 ) ) LCaSW + D-600 3.2 0 0 whole cytoplasm oocyte nucleus F/F F/F 2.8 ( (

1.6 e e c c 2.4 n n e e c c s s 2.0 e e r r 1.2 o o u u l l 1.6 f f

e e v v i i 1.2 t t a a 0.8 sperm l l e e 0.8 sperm R R 0 300 600 900 0 300 600 900 Time (s) Time (s)

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.

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. 372 Journal of Cell Science 116 (2)

2+ appeared in 6 of 7 oocytes and no Ca change occurred in the A 10-kDa CGD remaining one. Each of the slight Ca2+ increases exhibited

2+ ) either a localized Ca elevation restricted to one cortical 0 2.0 cytoplasm nucleus region or a rather uniform increase throughout the entire oocyte

(data not shown). None of the 7 oocytes underwent GVBD. 1.6 CaFSW After the experiments, 5 out of the 7 oocytes were washed with FSW, activated by excess K+ seawater (Deguchi and Osanai, 1.2 1994b), and then ﬁxed for the staining with DAPI. In all cases, a few decondensed sperm nuclei were detected (data not 0.8 sperm shown), suggesting successful sperm entries under conditions Relative fluorescence (F/F where LCaSW and D-600 were simultaneously applied. 0 300 600 900 In contrast to the inhibitory effects of LCaSW and D-600, Time (s) heparin had no serious inﬂuence on sperm-induced Ca2+ B 70-kDa CGD (cytoplasm) changes. Following insemination, the oocyte injected with 20 2.8 )

2+ 0 cytoplasm mg/ml heparin displayed an initial Ca transient and a 2.4 nucleus subsequent plateau phase (n=9; Fig. 2D), resulting in GVBD 2+ 2.0 (9/9). The initial Ca transient showed a cortical flash pattern CaFSW similar to that observed at normal fertilization (data not 1.6 shown). The only unusual point in the heparin-injected oocytes 1.2 was that some additional Ca2+ transients, each of which took 0.8 the form of a cortical flash rather than a point-source sperm propagating wave, appeared between the initial transient and Relative fluorescence (F/F 0.4 the plateau phase in 4 of 9 oocytes (data not shown). It should 0 300 600 900 be noted that the same concentration of heparin completely Time (s) 2+ blocked an Ins(1,4,5)P3-induced Ca increase (see the next Fig. 4. Effect of Ca2+-free seawater (CaFSW) on sperm-induced 2+ section). The above data collectively suggest that Ca influx Ca2+ changes. The oocyte where 10 kDa (A) or 70 kDa CGD (B) was through Ca2+ channels on the plasma membrane, but not Ca2+ injected into the cytoplasm was inseminated in FSW, which was 2+ release from Ins(1,4,5)P3-sensitive stores, contributes to Ca replaced by CaFSW at 4 minutes after fertilization. The F/F0 values changes at fertilization. were calculated in the cytoplasmic and nuclear regions separately. In all cases described above, the F/F0 level in the nucleus The steep increase in F/F0 (arrowhead in B) indicates the leakage of became obviously greater than that in the cytoplasm toward the 70 kDa CGD through the nuclear envelope. The two oocytes end of plateau phase when the oocytes were advancing to underwent GVBD on schedule. 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 place at one cortical region and propagated across the oocyte (CaFSW) at 4 minutes after fertilization in 10 kDa CGD- in a wave-like fashion (Fig. 6A). The initial Ca2+ wave always injected oocytes, both cytoplasmic and nuclear Ca2+ elevations started from the restricted point of the oocyte cortex which was were terminated prematurely (total duration of Ca2+ increases: situated around the edge of the space between two coverslips 320±11 seconds, n=6) and higher F/F0 level in the nucleus was (the left side of each fluorescence image) in a chamber, the site not produced subsequently (Fig. 4A). However, GVBD was not where effective concentration of 5-HT must be first attained. inhibited in these oocytes. To determine the precise time In most cases, the first Ca2+ transient comprised an initial peak required for the onset of nuclear envelope breakdown, 70 kDa and following smaller but oscillatory Ca2+ spikes (Fig. 5A, CGD was used under these conditions (Fig. 4B); leakage of 70 inset); each Ca2+ spike took the form of a point-source Ca2+ kDa CGD from cytoplasm to nucleus occurred at 461±17 wave which began to propagate before the increased Ca2+ in seconds (n=6) after fertilization, with a similar timing for the preceding Ca2+ spike completely returned to the resting normally fertilized oocytes continuously bathed in FSW (see level (e.g. the second Ca2+ wave starting at 5.2 seconds in Fig. 2+ above). These results indicate that the higher nuclear F/F0 level 6A). A similar Ca transient, a set of an initial peak and around the final part of plateau phase is not necessarily following smaller Ca2+ spikes, repeatedly appeared when 5-HT required for the progression of GVBD. 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 Potential ability of oocytes to release Ca2+ from internal propagating Ca2+ wave pattern, although the wave starting stores point sometimes changed even in the same oocyte (Fig. 6A,B). It is known that 5-HT can stimulate Ca2+ release from internal, In the 5-HT-treated oocytes, GVBD was only induced when 2+ 2+ probably Ins(1,4,5)P3-sensitive Ca stores in the bivalves such the first Ca transient just after the 5-HT stimulation was as Ruditapes (Guerrier et al., 1993) and Hiatella (Deguchi and maintained for a relatively long time (2/7; data not shown). Osanai, 1995). In the next series of experiments, the effect of Repetitive Ca2+ transients induced by 5-HT were also 5-HT on Ca2+ changes in Mactra oocytes was investigated. detected when nuclear Ca2+ changes were monitored with 70 When unfertilized oocytes were exposed to 100 nM 5-HT in kDa CGD injected into the nucleus (n=6; Fig. 5B). The number FSW, a large Ca2+ transient was immediately caused (n=7; Fig. of Ca2+ transients during a period of 20 minutes was 4.7±1.1 2+ 5A). During its rising phase, an increased F/F0 signal first took (n=6). In these oocytes, each Ca transient was initiated as a Ca2+ influx in fertilized bivalve oocytes 373

2+ 3.0 The spatiotemporal property of each Ca transient was also not different from that detected in FSW (data not shown). 2.0 Under conditions where 5-HT was applied in CaFSW, 1 of the A 10-kDa CGD 3.0 4 oocytes resulted in GVBD. 1.0 2+ ) whole To conﬁrm the existence of an Ins(1,4,5)P3-induced Ca 0 oocyte 2.5 0 20 40

F/F release mechanism in Mactra oocytes, the effect of ‘caged’

e ( derivative of Ins(1,4,5)P3 was examined. In the oocytes nc 2.0 e µ c injected with 600 M caged Ins(1,4,5)P3, a single UV

es 2+ 1.5 irradiation caused a single Ca transient lasting for 1-3

fluor minutes during the incubation in FSW (7/7; data not shown), e v i 1.0 in CaFSW (4/5; data not shown), and in LCaSW with 100 µM lat

e 5-HT

R D-600 (6/8; data not shown). Among the oocytes showing such 0.5 2+ 0 300 600 900 1200 1500 a short-lived Ca transient (n=17), GVBD was induced in only Time (s) three cases. In contrast, repeated UV irradiations generally 2+ B 70-kDa CGD (nucleus) produced a long-lived Ca increase even in CaFSW (n=6; Fig. 5C) and triggered GVBD more frequently (4/6). The UV-

) 2+ 0 2.0 nucleus induced Ca transient in caged Ins(1,4,5)P3-injected oocytes

F/F was completely blocked by simultaneous injection of 20 mg/ml e ( heparin; none of 9 examined oocytes displayed any Ca2+ nc 1.6 e c change after UV irradiation (data not shown). When 5 out of es 2+ 1.2 the 9 oocytes were inseminated subsequently, Ca increases fluor

e and resultant GVBD were invariably induced (5/5; data not v i 5-HT shown), indicating that they had never lost sensitivities to Ca2+ lat 0.8 e

R change itself. These results demonstrate that Mactra oocytes 2+ 0 300 600 900 1200 1500 are equipped with an Ins(1,4,5)P3 receptor-mediated Ca Time (s) release mechanism, which can produce, if forcibly stimulated, 2+ C 10-kDa CGD + caged IP3 a considerable Ca increase enough to trigger meiosis 2.4 reinitiation from PI.

) whole CaFSW 0 oocyte

F/F 2.0

e ( Discussion nc e

c 1.6 UV UV The present analysis using 10 kDa CGD revealed that an initial es UV 2+ UV large Ca transient just after fertilization clearly exhibits a

fluor 1.2 UV 2+ e cortical ﬂash pattern in Mactra oocytes; a Ca increase ﬁrst v i

lat 0.8 took place at the whole oocyte cortex and then spread e UV R throughout the cytoplasm and even inside the nucleus. Such a 0 150 300 450 600 pattern of centripetal Ca2+ increase in the cytoplasm or nucleus Time (s) was also confirmed by 70 kDa CGD injected into either of the two regions. The initial transient was almost completely Fig. 5. Temporal patterns of Ca2+ changes induced by 5-HT and blocked by simultaneous treatments with LCaSW and D-600, 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- although either treatment alone had an insufficient effect. In HT (final concentration: 100 nM) in FSW. In C, the oocyte injected contrast, this transient was not suppressed by heparin at all. 2+ with 10 kDa CGD plus 600 µM caged Ins(1,4,5)P3 was irradiated These results strongly suggest that the initial Ca transient just 2+ with UV light six times (arrows) in CaFSW. The F/F0 values were after fertilization is mainly due to Ca influx via voltage-gated calculated in the whole oocyte (A,C) or in the nuclear region alone Ca2+ channels on the plasma membrane. The initial transient (B). An inset in A depicts the expanded Ca2+ pattern of the first was followed by a plateau phase, during which submaximal transient just after the addition of 5-HT. GVBD did not occur in A or levels of cytoplasmic and nuclear Ca2+ were maintained. The B. In contrast, GVBD occurred in C just after the recording period. 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 Ca2+ wave propagating across the nucleus (Fig. 6C). GVBD CaFSW [(Deguchi and Osanai, 1994b) and this study], suggest took place in 1 of the 6 cases. that this phase is regulated by continuous Ca2+ influx via Finally, 5-HT was applied in the absence of external Ca2+ voltage-gated Ca2+ channels. It is, therefore, likely that the to determine whether 5-HT-induced Ca2+ oscillations are Ca2+ influx mechanism is totally responsible for the dependent on Ca2+ influx. When oocytes were exposed to cytoplasmic and nuclear Ca2+ increases in fertilized Mactra 100 nM 5-HT in CaFSW, an initial large Ca2+ transient and oocytes. A similar spatiotemporal Ca2+ pattern at fertilization subsequent repetitive Ca2+ transients, each of which was and its dependence on Ca2+ influx have also been demonstrated typically accompanied by oscillatory Ca2+ spikes, were in the PI-type echiuran worm Urechis (Jaffe et al., 1979; similarly produced (data not shown). The number of Ca2+ Stephano and Gould, 1997). transients during a period of 20 minutes was 4.0±0.9 (n=4), In addition to the measurement of cytoplasm- or nucleus- which was not significantly different from the values in FSW. restricted Ca2+ changes, the precise start time of GVBD was 374 Journal of Cell Science 116 (2)

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. determined by the experiments with 70 kDa CGD; the nuclear meiosis reinitiation from PI, but that the later part of plateau envelope began to break down shortly before, rather than after, phase, during which heterogeneous F/F0 levels in the the end of plateau phase in normally fertilized Mactra oocytes. cytoplasm and nucleus are established, is no longer required In parallel experiments with 10 kDa CGD, an interesting for the progression of subsequent meiotic events, including the phenomenon of higher F/F0 level in the nucleus than in the disassembly of the nuclear envelope. A similar scheme might cytoplasm became noticeable around the final part of plateau be applied to other PI-type protostomes including Spisula phase [a similar phenomenon is also reported in Urechis and (Dubé and Guerrier, 1982) and Urechis (Gould and Stephano, analyzed in detail (Stephano and Gould, 1997)]. This result 1989; Stephano and Gould, 1997). It is known that the cell suggests the selective accumulation of Ca2+ in the nucleus just cycle transition from PI to MI can be triggered without an before the nuclear envelope breakdown in fertilized Mactra intracellular Ca2+ increase in a variety of MI-type protostomes oocytes, although there is a possibility that the phenomenon is such as bivalves (e.g. Kyozuka et al., 1997), limpets (Gould et partly due to artifacts, such as the different behavior of al., 2001) and nemertean worms (Stricker and Smythe, 2000). fluorescent Ca2+ indicators in the cytoplasmic and nuclear Stimulation with 5-HT caused repetitive Ca2+ transients in environments (Thomas et al., 2000). The finding led us to Mactra oocytes, although the drug had a weak effect on conceive that such a situation might play a role in regulating triggering GVBD (see also Fong et al., 1996). The 5-HT- the progression of GVBD. However, GVBD was neither induced Ca2+ oscillations proceeded without external Ca2+ inhibited nor delayed by application of CaFSW at 4 minutes in contrast to the situation observed at fertilization. after fertilization, which abolished the plateau phase of Spatiotemporal analysis of the 5-HT-induced Ca2+ oscillations cytoplasmic and nuclear Ca2+ elevations prematurely and in this study, which is the first demonstration in protostome 2+ prevented the subsequent appearance of higher F/F0 level in the oocytes, revealed that the rising phase of each Ca transient nucleus. By contrast, GVBD is completely blocked when the takes the form of a point-source Ca2+ wave propagating across same treatment with CaFSW is carried our within 3 minutes of the whole oocyte, including the cytoplasm and nucleus. The fertilization (Deguchi and Osanai, 1994b). Therefore, it is ability for substantial Ca2+ release from internal stores in likely that the initial period of cytoplasmic and/or nuclear Ca2+ Mactra oocytes was also confirmed by the experiments with elevations at fertilization serves as a prerequisite trigger for caged Ins(1,4,5)P3; continuous application of Ins(1,4,5)P3 not Ca2+ influx in fertilized bivalve oocytes 375 only produced a long-lived Ca2+ increase but also triggered considering the fact that substantial Ca2+ release can be GVBD without a contribution of external Ca2+. These results induced in Mactra oocytes stimulated with 5-HT or indicate that Mactra oocytes have the potentiality not only to Ins(1,4,5)P3 instead of sperm. Recently, it has been release internally stored Ca2+ through the interaction between demonstrated that some intermediate molecules such as Src Ins(1,4,5)P3 and its receptors but also to produce repetitive family kinases and phospholipase Cγ play an essential role in Ca2+ waves, as observed at fertilization in MI-type bivalves the signal transduction between sperm (or SE) and the (Deguchi and Morisawa, 1997) and many other non-PI-type production of Ins(1,4,5)P3 in several non-PI-type animals animals (see Stricker, 1999). The existence of Ins(1,4,5)P3- (Carroll et al., 1997; Runft and Jaffe, 2000; Abassi et al., 2000; induced Ca2+ release mechanism is also reported in Urechis Sato et al., 2000). It might be possible that such molecules are (Stephano and Gould, 1997), although it is unknown whether lacking (or their activities are suppressed by other molecules) this release alone can produce a sufficient amount of Ca2+ to in PI-arrested oocytes. In MI-type ascidian oocytes, it is also provoke GVBD in this species. These results, together with the proposed that sperm- or SE-induced Ca2+ oscillations are study on Spisula oocytes showing that GVBD is induced by regulated by cyclin B1-dependent kinase activity (Levasseur Ins(1,4,5)P3 injection (Bloom et al., 1988), imply that PI- and McDougall, 2000; McDougall et al., 2000), which is low arrested oocytes in PI-type protostomes are equipped with this at PI stage. In fact, injection of SE causes Ca2+ oscillations in universal Ca2+-mobilizing system. MI-arrested ascidian oocytes but the same procedure does not 2+ 2+ It remains unknown why the Ins(1,4,5)P3-dependent Ca produce any Ca change in PI-arrested oocytes, in spite of the release pathway is activated at fertilization in non-PI-type fact that injection of Ins(1,4,5)P3 is effective in inducing a animals, but not in PI-type species such as Mactra. There are considerable Ca2+ increase even in the immature stage essentially two different possibilities to account for the (McDougall et al., 2000). differences between PI and non-PI-type animals. One The present study clearly demonstrated that oocytes of the possibility is that different factors exist in sperm, which PI-type bivalve Mactra predominantly use a Ca2+ influx stimulate different pathways in oocytes or eggs at fertilization. pathway at fertilization, in spite of the potential ability to In various animals, including nemertean worms (Stricker, release internally stored Ca2+. Moreover, possible differences 1997), ascidians (Kyozuka et al., 1998; McDougall et al., 2000; in the mechanism underlying intracellular Ca2+ increases at Runft and Jaffe, 2000) and vertebrates (Swann, 1990; fertilization between PI-type and other types of animals were Yamamoto et al., 2001), injection of sperm extract (SE) into pointed out and discussed. Further studies, including the unfertilized oocytes or eggs of the same species has been identification of sperm-derived factors and their downstream shown to produce intracellular Ca2+ changes similar to those pathways leading to intracellular Ca2+ increases in oocytes or seen at fertilization. It seems likely that the SE-induced Ca2+ eggs, are required to explain the differences between PI- and changes are mainly regulated by Ca2+ release through an non-PI-type animals. Bivalves will be suitable materials for Ins(1,4,5)P3 receptor-mediated mechanism (Oda et al., 1999; such a comparison, since there are PI- and MI-type species Runft and Jaffe, 2000). The active components of SE, which where spatiotemporal Ca2+ patterns are both clarified. are recognized as soluble proteins in all animals described above (Stricker, 1999), are effective beyond species, even in We thank H. Shirakawa (Department of Physiology, Tokyo heterologous combinations of gametes obtained from distantly Women’s Medical University School of Medicine, Shinjuku-ku, related animals (Stricker et al., 2000). However, the existence Tokyo, Japan) for providing us with the technique of image processing of SE and its effect on Ca2+ changes have not yet been using NIH Image. This work was supported by a Research Fellowship and Grant from the Japan Society for Promotion of Science for Young confirmed in PI-type animals. However, it has been reported in Scientists to R.D. (no. 4141). 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 References intracellular Ca2+ changes similar to those seen at fertilization Abassi, Y. A., Carroll, D. J., Giusti, A. F., Robert, J., Belton, R. J., Jr and (Gould and Stephano, 1989; Stephano and Gould, 1997). Foltz, K. R. (2000). 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R. (1988). Inositol trisphosphate, mechanism develops as oocyte maturation advances in starfish inositol phospholipid metabolism, and germinal vesicle breakdown in surf (Chiba et al., 1990) and hamster oocytes (Fujiwara et clam oocytes. Dev. Biol. 129, 532-540. al., 1993). Intracellular stocks of polyphosphoinositides, Borg, B., Renzis, G. D., Payan, P. and Ciapa, B. (1992). Activation of polyphosphoinositide metabolism at artificial maturation of Patella vulgata precursors of Ins(1,4,5)P3, increase between PI and MI in MI- oocytes. Dev. Biol. 149, 206-212. type limpet oocytes (Borg et al., 1992). Such circumstances Carroll, D. J., Ramarao, C. S., Mehlmann, L. M., Roche, S., Terasaki, M. observed in the non-PI-type animals, which all indicate the and Jaffe, L. A. (1997). Calcium release at fertilization in starfish eggs is 2+ mediated by phospholipase Cγ. J. Cell Biol. 138, 1303-1311. incomplete establishment of an Ins(1,4,5)P3-mediated Ca Chiba, K., Kado, R. T. and Jaffe, L. A. (1990). Development of calcium release mechanism at PI stage, may be responsible in part for release mechanisms during starfish oocyte maturation. Dev. Biol. 140, 300- 2+ the inability of Mactra oocytes to use internally stored Ca at 306. fertilization. However, additional reasons may also be required, Deguchi, R. and Osanai, K. (1994a). Repetitive intracellular Ca2+ increases 376 Journal of Cell Science 116 (2)

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