FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LIM, D.
Right arrow Articles by SANTELLA, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LIM, D.
Right arrow Articles by SANTELLA, L.
(The FASEB Journal. 2002;16:1050-1056.)
© 2002 FASEB

Activation of oocytes by latrunculin A

DMITRI LIM, KLAUS LANGE* and LUIGIA SANTELLA1

Laboratory of Cell Biology, Stazione Zoologica ‘A. Dohrn’ Villa Comunale I-80121, Naples, Italy; and
* Kladower Damn 25b, 14089 Berlin, Germany

1Correspondence: Laboratory of Cell Biology, Stazione Zoologica ‘A. Dohrn’, Villa Comunale I-80121, Naples, Italy. E-mail: santella{at}alpha.szn.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Actin depolymerization by latrunculin A (LAT-A) in mature starfish oocytes induces a massive calcium mobilization that results in the discharge of the cortical granules and in the elevation of the fertilization envelope. The Ca2+ liberation starts as a circumscribed subplasma membrane hotspot, which is followed by a flash of Ca2+ increase restricted to the cortical layer. Ca2+ propagates rapidly from these peripheral regions to the center of the oocyte, initiating calcium oscillations. Blockade of the inositol 1,4,5-trisphosphate receptors with heparin does not affect the liberation of Ca2+ at the initial hotspot or the cortical flash, but abolishes the centripetal spreading of the wave and the Ca2+ oscillations. In Ca2+-free medium, LAT-A also initiates Ca2+ release at a discrete cortical point, but then propagates throughout the cell without first forming the uniform cortical flash. The latter is thus linked to the influx of external Ca2+, somehow promoted by the depolymerization of cortical (microvillar) actin. The Ca2+ response to spermatozoa (i.e., peripheral hotspot, cortical flash, globalization of the signal) closely mimics that promoted by LAT-A. Thus, the initial cortical release of Ca2+ promoted by the sperm may be due to the depolymerization of actin.—Lim, D., Lange, K., Santella, L. Activation of oocytes by latrunculin A.


Key Words: intracellular calcium • fertilization • actin depolymerization • starfish oocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CHANGES IN INTRACELLULAR calcium are essential to egg activation. Although a single Ca2+ wave or repetitive Ca2+ spikings are routinely detected just after fertilization in a spectrum of species ranging from marine invertebrate to mammals, the mechanism by which the sperm triggers the calcium signal is obscure (see ref 1 for a recent discussion of the problem). In echinoderms, the increase in intracellular calcium has been linked to the activation of inositol 1,4,5-trisphosphate (InsP3) and ryanodine/cyclicADP-ribose receptors (2 , 3) since the injection of either InsP3 or cyclic ADPr-ribose (cADPr) induces a massive increase in intracellular Ca2+, leading to cortical granules exocytosis and elevation of the fertilization envelope (4) . It has been suggested that InsP3 would initiate Ca2+ release at fertilization of sea urchin eggs whereas cADPr would be the endogenous regulator of the Ca2+-induced Ca2+ release (CICR) (5 , 6) . However, a recent report indicates that cyclic GMP and cADPr stimulate InsP3 production (7) . Sea urchin eggs and starfish oocytes also respond to the newly discovered Ca2+-mobilizing messenger nicotinic acid adenine nucleotide phosphate (NAADP) (8 , 9) . One peculiar aspect of NAADP first documented in starfish oocytes is its linkage to the influx of external Ca2+, which produces a cortical flash when the injected caged messenger is liberated (10 , 11) . This confirms the suggestion that the Ca2+ store sensitive to NAADP is independent of those that respond to cADPr and InsP3 (12) . The activation of the putative cortical NAADP receptors is preliminary to the propagation of the Ca2+ wave by activation of the InsP3 receptors (13) . The type of Ca2+ response and the nature of the Ca2+ stores active in starfish oocytes at fertilization are thus the subject of this contribution. Given the importance of the actin cytoskeleton in the cortical region of the oocytes (14) , we decided to explore its role in the initial Ca2+ mobilization event in starfish oocytes. Here we show that actin depolymerization by latrunculin A (LAT-A), which is more effective than cytochalasin B (15) , induces an intracellular Ca2+ mobilization that initiates at a circumscribed subplasma membrane hotspot in mature oocytes (16) suspended in sea water. This initial liberation is followed by a sudden flash of Ca2+ increase restricted to the cortical layer of the oocytes. Ca2+ then propagates rapidly from these peripheral regions to the center of the oocyte. Globalization of the Ca2+ signal results in rapid discharge of the cortical granules and in the elevation of the fertilization envelope. The Ca2+ response after the interaction of spermatozoa with the oocytes suspended in sea water closely mimics that promoted by LAT-A: it begins with an initial circumscribed peripheral release, followed by a cortical flash and by the InsP3-mediated globalization of the signal. We propose that the initial cortical release of Ca2+ promoted by the sperm may be due to the depolymerization of actin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of gametes
Starfish (Astropecten auranciacus) were collected during the breeding season in February-June in the gulf of Naples and kept in running natural sea water (16°C). Immature oocytes (containing the germinal vesicle, nucleus) were dissected from the ovaries and kept in artificial sea water (ASW: 460 mM NaCl, 10.1 mM KCl, 9.2 mM CaCl2, 35.9 mM MgCl2, 17.5 mM MgSO4, 2.5 mM NaHCO3, pH 8.0) for 30 min before use. Oocytes in which the breakdown of the germinal vesicle occurred spontaneously were discarded. Maturation was promoted by adding the hormone 1-methyladenine (Sigma Chemical Co., St. Louis, MO) at a final concentration of 5 µM. For fertilization experiments, 1 µL of dry sperm was suspended in 2 mL of artificial sea water, and 40 µL of this suspension was added to 1 mL of the oocyte suspension to obtain a final sperm dilution of 1:50,000.

Microinjections and calcium measurements
The calcium fluorescent dye OR Green 488 BAPTA-1 coupled to a 10 kDa dextran (OGBD; Molecular Probes, Eugene, OR) was injected into the cytoplasm of mature oocytes. The concentration of the dye in the pipette (diameter of the tip 1 µm) was adjusted to 5 mg/mL with injection buffer (IB; 450 mM potassium chloride, 10 mM HEPES, pH 7.0). The volume of injected dye corresponded to 1–2% of the total cell volume; thus, the final concentration of injected substances in the cellular environment was 50- to 100-fold lower than in the micropipette. Cytosolic Ca2+ changes were measured using either a cooled CCD camera (MicroMax, Princeton Instruments, Inc., Trenton, NJ) mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective or a confocal laser scanning microscope Olympus FVX-ZM-IL (Olympus Optical Co., Ltd., Japan), an UplanApo 20x/0.70 objective, laser power 20%, and confocal aperture no. 2. Fluorescence images were processed with a MetaMorph Imaging System software (Universal Imaging Corporation, West Chester, PA). To exclude variations of fluorescence intensity, the signals were corrected for variations in dye concentration by normalizing fluorescence (F) against baseline fluorescence (F0). The region of interest to measure the fluorescence level was positioned as shown in the scheme of the figures.

Chemicals
LAT-A was purchased from Molecular Probes. A 3 mM stock solution in dimethyl sulfoxide (DMSO) was prepared and kept frozen. ASW or Ca2+-free sea water (CaFSW) containing 6 µM LAT-A was prepared just before the experiment. Mature oocytes (70 min after 1-MA addition) were gently transferred to LAT-A containing ASW and kept in a free position during the Ca2+ measurement acquisition. The concentration of DMSO in the experimental bath did not exceed 0.2%. For experiments on oocytes injected with OGBD, cells were washed twice in a solution containing 470 mM NaCl, 10.1 mM KCl, 35.9 mM MgCl2, 17.5 mM MgSO4, 2.5 mM NaHCO3, pH 8.0, supplemented with 2 mM EGTA. The oocytes were transferred to CaFSW containing 1 mM EGTA. A 50 mg/mL solution of heparin (MW=6000) (Sigma) in the injection buffer was prepared just before the injection experiment. The 8NH2cADPr antagonist of the cADPr-sensitive channel (Sigma) was prepared as a 1 mM stock solution in IB and kept frozen. For microinjection experiments, the stock solution of 8NH2cADPr was dissolved at a final concentration of 250 µM.

Transmission and scanning electron microscopy
For transmission electron microscopy, control mature oocyte and oocytes treated with LAT-A were fixed first with 1% glutaraldeide-90% sea water for 1 h at room temperature. Oocytes were rinsed several times in ASW and postfixed in 1% osmium tetroxide for 0.5–1 h. The samples were dehydrated in a graded alcohol series and embedded in EPON 812. Sections were stained with 2% uranyl acetate and 0.2% lead citrate and examined with a Philips 400 transmission electron microscope. For scanning electron microscopy, the microvilli on the surface of the oocyte were visualized by removing the follicle cells and the vitelline envelope with 0.01% actinase-E (Kaken, Pharmaceutical CO., LTD, Japan) for 10 min. The treated oocytes were then washed several times in ASW. After fixation in glutaraldehyde, the samples were dehydrated in the ethanol series and critical point dried from carbon dioxide, cemented to specimen stubs, and spattered with gold. A Philips 505 scanning electron microscope was used for the observations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Latrunculin A experiments
Oocytes incubated in sea water were injected with the calcium fluorescent dye Oregon green 488 BAPTA-1 coupled to an OGBD 70 min after the addition of the maturing hormone 1-methyladenine (1-MA). Immediately afterward, LAT-A was added to the chamber and the Ca2+ response monitored by a cooled CCD camera. Figure 1 A shows that the response initiated 6 min and 14 s after the addition of LAT-A, which was the time evidently necessary for it to depolymerize actin, at a circumscribed cortical site (see the second fluorescent image). 24 s after the first local Ca2+ increase, a uniform fluorescent ring suddenly appeared in the entire cortical region of the oocyte (cortical flash) (see the fourth fluorescent image). The cortical flash failed to decay, but spread instead centripetally to the entire oocyte in ~60 s at a rate of 5 µm/s, strongly resembling the rate of spreading of the Ca2+ wave at fertilization calculated for the nemertean worm Cerebratulus lacteus (17) . The graph of the relative fluorescence of the Ca2+ indicator (Fig. 1B ) offers a numerical equivalent of the colors shown in the fluorescent images in the two sites analyzed in the cortical region (violet and green circles; blue circle for the central area). After the initial increase at a distinct point (time 0 on the graph), the relative fluorescence of the cortical flash reached an amplitude of 0.5 ± 0.15 arbitrary units. The Ca2+ increase in the center of the cell peaked at a relative fluorescence value of 0.7 ± 0.1 (n=35) 75 s after the initial Ca2+ elevation.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. Ca2+ release induced by the addition of LAT-A to mature oocytes suspended in sea water. A) The Ca2+ increase, monitored with a cooled CCD camera, initiated in one cortical site of the oocyte and yielded a uniform cortical flash (fourth fluorescent image, arrow). The Ca2+ wave then spread from the cortex in a centripetally directed wave. B) Graph of the relative fluorescence offers a numerical equivalent of the colors visualized in the fluorescent images of panel A. The regions of interest (ROIs) were positioned as shown.

The increase of Ca2+ induced by LAT-A in the cortical region of the cell could have been due to the influx of external Ca2+ through calcium channels in the oolemma. LAT-A was thus applied to oocytes kept in Ca2+-free sea water containing 1 mM EGTA (Fig. 2 A). As in the experiments in Ca2+-containing sea water, an initial circumscribed cortical site of fluorescence was observed 6 min and 19 s after LAT-A addition (see the second fluorescent image). However, after this initial increase, the Ca2+ wave spread throughout the cell without first generating a cortical flash (see the third fluorescent image), showing that the latter was generated by the influx of external Ca2+. The graph of the relative fluorescence 1.1 ± 0.15 (n=15) (Fig. 2B ) shows the Ca2+ increase at the initial point in the cortex (violet circle in the scheme) propagating at the same rate measured in the experiment in the presence of external Ca2+ (5 µm/s), and reaching the opposite pole of the cell in ~60 s.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 2. Ca2+ release induced by the addition of LAT-A to mature oocytes suspended in Ca2+-free sea water. A) The Ca2+ wave spread throughout the cell from the initial point without first generating a cortical flash. The cortical Ca2+ then propagated from one point over the entire cell surface. B) A graph of the relative fluorescence shows that the initial Ca2+ increase in one point of the cortex propagated throughout the oocyte, reaching the opposite pole.

The effect of LAT-A was also analyzed using confocal microscopy, monitoring two sites within the cortical region and in the center of an equatorial plane of the oocyte. The confocal images in Fig. 3 A confirmed the findings made with the cooled CCD camera (see Fig. 1A for comparison). The cortical Ca2+ flash seen in normal sea water with the cooled CCD camera was well visible, but the initial circumscribed site of the Ca2+ increase was visible only with the CCD camera (see second and third images of Fig. 1A ). This was apparently due to the difficulty of detecting a Ca2+ increase in a small region that could be out of the confocal plane of the oocyte. The Ca2+ increase shown in Fig. 3B was identical to that observed with the cooled CCD camera in oocytes kept in Ca2+-free sea water.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 3. Confocal laser scanning imaging of Ca2+ release induced by LAT-A. A) In sea water, the Ca2+ release induced by LAT-A treatment yielded a uniform cortical flash and then spread from the cortex in a centripetally directed wave. B) In Ca2+-free sea water, the cortical Ca2+ increased from one cortical point, then propagated over the entire cell surface without generating a cortical flash.

The cortical flash of Ca2+ induced by LAT-A and subsequent spreading of the wave were followed by the elevation of the fertilization envelope, as imaged by transmitted light microscopy (Fig. 4 ). Transmission electron microscopy of mature control oocytes (data not shown) showed the cortical granules in close association with the plasma membrane. Figure 4 shows the fertilization envelope of an oocyte 9 min after LAT-A addition. As with fertilization, the vitelline layer lifted from the oocyte’s surface after the fusion and the release of the cortical granule material to the perivitelline space.



View larger version (170K):
[in this window]
[in a new window]
  		Figure 4. Transmission electron micrograph of a mature oocyte treated with LAT-A for 9 min. The fertilization envelope elevated from the surface of the oocytes (arrow) after the extrusion of the cortical granules. Bar = 1 µm. The inset shows the elevation of the fertilization envelope (arrow) imaged by transmitted light microscopy.

The Ca2+ response to the addition of spermatozoa to mature oocytes observed with the cooled CCD camera strikingly resembled that induced by LAT-A. The sperm induced an initial small elevation of Ca2+ at the site of interaction, which expanded to a uniform cortical flash and then spread to the entire oocyte (Fig. 5 ). The cortical flash after exposure to spermatozoa had already been observed, for example, in the nemertean worm C. lacteus (17) , in whose eggs "the cortical flash became visible after the sperm attached to the oolemma and eventually yielded a rise in [Ca2+]i throughout the entire ooplasm in a manner suggestive of a centripetally directed wave". The cortical flash in C. lacteus has been claimed to involve calcium influx from outside based on experiments on the blockade of oolemmal calcium channels with cobalt (17) . Ca2+ influx at fertilization also occurs in sea urchin eggs due to rapid depolarization of the plasma membrane produced by the insertion of sperm cation channels (18) , amplified by the influx of calcium through voltage-dependent (L-type) channels (19) . However, no reports available in the literature had so far shown a distinct point source pattern of propagation. As mentioned above, this was probably due to the difficulty of detecting a Ca2+ increase restricted to a very limited region when using only a confocal plane of the oocyte instead of imaging the whole cell. Figure 5A shows that in all imaged oocytes (n=20), the cortical flash started 6 s after the first Ca2+ elevation at the initial cortical point (second fluorescent image), reaching a relative fluorescence amplitude of ~0.3 ± 0.1 and decaying afterward in ~7 s. The wave spread from the same point throughout the oocyte reaching an amplitude of 0.9 ± 0.1 arbitrary units in ~3 min at a rate of 1.65 µm/s. The graph of the relative fluorescence in Fig. 5B shows the Ca2+ response to the sperm in different regions of the oocytes (see the scheme). The cortical flash is well visible at the opposite oocyte pole (small spike of the green line in the graph), although it initiates at the point of sperm fusion. In this region (violet circle and line), the cortical ring was difficult to detect because of the superposition of the propagating wave.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 5. Intracellular Ca2+ increase after fertilization of a control mature oocyte. A) An overlay of the relative fluorescence of the intracellular Ca2+ increase visualized with OGBD in a mature control oocyte (upper panel) induced by the sperm. Relative fluorescence of the Ca2+ increase induced by the sperm (lower panel). B) The graph shows the Ca2+ increase in the cortical region and in the center of the oocyte (circles). The cortical flash is highly visible at the opposite oocyte pole (arrow).

Repetitive Ca2+ spikings induced by latrunculin A
After the first Ca2+ wave induced by LAT-A, the overall Ca2+ level declined toward the baseline in ~2 min, but was followed ~1 min later by a second wave that swept centripetally from the periphery of the cell. More cycles followed, repetitive waves occurring with decreasing frequency and amplitude for ~150 min, which may correspond to the time required to completely disassemble F-actin (Fig. 6 A).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 6. Repetitive Ca2+ spikings induced by LAT-A. A) The Ca2+ oscillations propagated across the entire ooplasm, the repetitive waves occurring with decreasing frequency and amplitude for ~150 min. B) Heparin preinjected into the oocytes completely inhibited the Ca2+ waves but failed to block the cortical Ca2+ elevation. C) Fluorescent image of the cortical Ca2+ flash in the inset of panel B sandwiched between images in which Ca2+ was at baseline level.

Experiments were performed to establish whether the increase in intracellular Ca2+ was linked to the increased metabolism of phosphoinositides, as suggested by the cytoskeletal dependence of InsP3-generating phospholipases (20) . Oocytes were incubated for 20 min with the phospholipase inhibitor U73122 (10 µM) before the addition of LAT-A. Although under the experimental conditions the height of the Ca2+ spikes was slightly lower, the elevation of the fertilization envelope and the oscillatory pattern occurred normally (data not shown). It was then decided to investigate whether the initial local Ca2+ liberation induced by LAT-A elicited the Ca2+ oscillations by activating InsP3-sensitive receptors, as would have been expected from the accepted ability of Ca2+ to promote InsP3 channel opening (21) . The InsP3 receptor antagonist heparin was preinjected into mature oocytes before treatment with LAT-A. Figure 6B shows that the Ca2+ response was radically altered in all experiments (n=15), but the long acquisition times used to follow the late-onset oscillations prevented the detection of the initial cortical Ca2+ liberation event. The uniform cortical Ca2+ flash occurred normally, reaching a relative fluorescence peak of 0.15 ± 0.1; n = 15 (Fig. 6B ). However, the centripetal spreading of the Ca2+ wave failed to follow it. The inset in Fig. 6C shows the fluorescent image of the cortical Ca2+ rise sandwiched between images in which Ca2+ was at baseline level. Thus, in line with previous observations (22 , 23) , the spreading of the Ca2+ wave from the cortex to the remainder of the oocyte was indeed mediated by the InsP3 receptors. The possible involvement of ryanodine/cADPr receptors was also probed using the specific antagonist 8NH2-cADPr (24) . Its preinjection failed to inhibit the LAT-A-induced Ca2+ oscillations, which actually reached significantly higher peak amplitudes 1.2 ± 0.08 (n=7) than in the controls 0.98 ± 0.1 (n=13).

The hypothesis was tested that LAT-A promoted the influx of extracellular Ca2+ by inducing the depolymerization of actin in the microvilli. Figure 7 A shows the surface of a mature oocyte examined with the scanning electron microscope 70 min after the addition of 1-MA. The oocytes were pretreated at the immature stage with actinase (10 min, 0.01%) to remove the follicle cells and the vitelline coat (14) . The elongated microvilli are distributed over the surface in regular form and length in a nondensely packed pattern. After 10 min in the presence of LAT-A, the length of the microvilli was clearly reduced and their shape became vesicular (Fig. 7B ), reminiscent of that prevailing in unfertilized sea urchin eggs treated with cytochalasin B (25) .



View larger version (107K):
[in this window]
[in a new window]
  		Figure 7. Scanning electron micrograph of the surface of a mature oocyte. A) Microvilli on the surface on a mature control oocyte 70 min after hormonal stimulation. B) Microvilli of a mature oocyte treated for 9 min with LAT-A. After the drug treatment, the microvilli collapsed onto the oocyte surface. Bar = 1 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has dissected the temporal sequence of Ca2+ responses that follow the interaction of oocytes with the sperm, documenting for the first time that a spatially restricted calcium increase at the point of sperm–egg interaction preceded the cortical flash previously observed by others. The initial Ca2+ increase at a restricted cortical point induced by the fertilizing sperm strikingly resembled that promoted by LAT-A, suggesting that the initial trigger of Ca2+ release beneath the plasma membrane could in both cases be linked to the depolymerization of actin. The uniform cortical flash of Ca2+ apparently was produced by Ca2+ penetration from the outside, as its absence was the only difference observed in the Ca2+ response to LAT-A in the absence of external Ca2+. Since the only known action of LAT-A is the depolymerization of F-actin, the results are compelling in suggesting that the actin cytoskeleton is involved in the regulation of Ca2+ channels. This would be in line with observations that cation exchangers and ion channels are modulated by actin effectors or by the interaction with G- or F-actin (26) . The problem, then, becomes that of understanding 1) why LAT-A, which evidently is available to the entire surface of the oocyte, only induced the initial Ca2+ liberation in a very restricted cortical area, and 2) the nature of the latrunculin-sensitive cortical Ca2+ store. One possibility is that polymerized actin itself was the Ca2+ store, as previously claimed in the literature (27 , 28) . The disassembly of the Ca2+-rich actin filament in a localized domain in the oocyte cortex (29) could set free a limited amount of Ca2+ and, at the same time, trigger the opening of plasma membrane channels, eventually leading to the globalization of the signal via the previously described canonical InsP3-dependent pathway (30) . Alternatively, disassembly of the actin cytoskeleton could promote the liberation of Ca2+ from conventional membrane-enclosed stores. One could mention previous findings of morphological changes during the maturation of starfish oocytes that are linked to the polymerization and depolymerization of cortical actin. 1-MA induces a rapid reorganization of the oocyte surface that culminates after 10 min in the formation of long spikes and in the elimination of microvilli as spikes form (14) . Long after microvilli are eliminated, the number of microvilli is restored, especially around the animal pole (31) .

In summary, it could be suggested that the initial Ca2+ response by LAT-A would be produced by acting on the microvilli at the animal hemisphere. Ca2+ could likely be directly liberated from depolymerized microvillar actin, or, indirectly, by neighboring membrane-enclosed deposits controlled by the actin cytoskeleton (27) . The initial local Ca2+ liberation would be followed by a series of Ca2+ release and reuptake events involving the InsP3-sensitive stores. Such a role for Ca2+ has been proposed, for example, in Xenopus laevis oocytes, where the close apposition of neighboring ‘puff sites’ is important for the generation of Ca2+ waves (30 , 32) . Results have appeared showing that cortical structures such as actin and the endoplasmic reticulum are involved in initiating the repetitive Ca2+ waves in fertilized ascidian oocytes (33) . Thus, in addition to having a role in the activation of oocytes, actin reorganization in the cortex may be the initial event that eventually leads to the sperm-induced globalization of the signal and the Ca2+ oscillations that may arise from the periodic opening of plasma membrane calcium channels (membrane oscillator) (34) . Repetitive Ca2+ waves originating at the site of sperm entry have been shown in different species. In ascidian oocytes, Ca2+ waves start from the site of injection of a sperm extract in the peripheral region and propagate across the ooplasm. If the injection of the sperm extract is performed in the central region of the oocyte (35) , the Ca2+ response is significantly delayed, indicating that the sensitivity to the extract is higher in the cortex of the cell (33 , 35 36 37) and dependent on the continuous Ca2+ entry by a capacitative mechanism (38 , 39) . Changes in the state of cortical actin induced by the sperm may play a pivotal role in the generation of Ca2+ waves at fertilization.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Ernesto Carafoli for helpful discussions and critical reading of the manuscript. We also wish to thank Mr. Gianni Gragnaniello for his help with the figures and the Marine Resources Service for maintaining the starfish.

Received for publication January 7, 2002. Revision received March 27, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Carafoli, E., Santella, L., Branca, D., Brini, M. (2001) Generation, control, and processing of cellular calcium signals. Crit. Rev. Biochem. Mol. Biol. 36,107-260[CrossRef][Medline]
  2. Swann, K., Whitaker, M. (1986) The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs. J. Cell. Biol. 103,2333-2342[Abstract/Free Full Text]
  3. Lee, H. C., Walseth, T. F., Bratt, G.T., Hayes, R. N., Clapper, D. L. (1989) Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J. Biol. Chem. 264,1608-1615[Abstract/Free Full Text]
  4. Abbott, A. L., Ducibella, T. (2001) Calcium and the control of mammalian cortical granule exocytosis. Front. Biosci. 6,D792-D806[Medline]
  5. Galione, A., Lee, H. C., Busa, W. B. (1991) Ca2+-induced Ca2+-release in sea urchin egg homogenates: modulation by cyclic ADP-ribose. Science 253,1143-1546[Abstract/Free Full Text]
  6. Lee, H. C. (1994) Cyclic ADP-ribose: a calcium mobilizing metabolite of NAD+. Mol. Cell. Biochem. 138,229-234[CrossRef][Medline]
  7. Kuroda, R., Kontani, K., Kanda, Y., Katada, T., Nakano, T., Satoh, Y., Suzuki, N., Kuroda, H. (2001) Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca2+ transients in fertilization of sea urchin eggs. Development 128,4405-4414[Abstract/Free Full Text]
  8. Lee, H. C., Aarhus, R. (1995) A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose. J. Biol. Chem. 270,2152-2157[Abstract/Free Full Text]
  9. Galione, A., Patel, S., Churchill, G. C. (2000) NAADP+-induced calcium release in sea urchin eggs. Biol. Cell 92,197-204[CrossRef][Medline]
  10. Santella, L., Kyozuka, K., Genazzani, A. A., De Riso, L., Carafoli, E. (2000) Nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release. Interactions among distinct Ca2+ mobilizing mechanisms in starfish oocytes. J. Biol. Chem. 275,8301-8306[Abstract/Free Full Text]
  11. Lim, D., Kyozuka, K., Gragnaniello, G., Carafoli, E., Santella, L. (2001) NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes. FASEB J. 15,2257-2267[Abstract/Free Full Text]
  12. Lee, H. C. (2001) Physiological functions of cyclicADP-ribose and NAADP as calcium messengers. Annu. Rev. Pharmacol. Toxicol. 41,317-345[CrossRef][Medline]
  13. Carroll, D. J., Ramarao, C. S., Melhmann, L. M., Roche, S., Terasaki, M., Jaffe, L. A. (1997) Calcium release at fertilization in starfish eggs is mediated by phospholipase C{gamma}. J. Cell Biol. 138,1303-1311[Abstract/Free Full Text]
  14. Schroeder, T. E., Stricker, S. A. (1983) Morphological changes during maturation of starfish oocytes: surface ultrastructure and cortical actin. Dev. Biol. 98,373-384[CrossRef][Medline]
  15. Wakatsuki, T., Schwab, B., Thompson, N. C., . Elson. E. L (2000) Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J. Cell Sci. 114,1025-1036[Abstract]
  16. Kanatani, H., Shirai, H., Nakanishi, K., Kurosawa, T. (1969) Isolation and identification on meiosis inducing substance in starfish Asterias amurensis. Nature (London) 221,273-274[CrossRef][Medline]
  17. Stricker, S. A. (1996) Repetitive calcium waves induced by fertilization in the nemertean worm Cerebratulus lacteus. Dev. Biol. 176,243-263[CrossRef][Medline]
  18. McCullow, D. H., Chambers, E. L. (1992) Fusion of membranes during fertilization. Increases of the sea urchin egg’s membrane capacitance and membrane conductance at the site of contact with the sperm. J. Gen. Physiol. 99,137-175[Abstract/Free Full Text]
  19. McDougall, A., Gillot, I., Whitaker, M. (1993) Thimerosal reveals calcium-induced calcium release in unfertilised sea urchin eggs. Zygote 1,35-42[Medline]
  20. Van Haelst, C., Rothstein, T. L. (1988) Cytochalasin stimulates phosphoinositides metabolism in murine B lymphocytes. J. Immunol. 140,1256-1258[Abstract]
  21. Taylor, C. W., Thorn, P. (2001) Calcium signalling: IP3 rises again and again. Curr. Biol. 11,R352-R355[CrossRef][Medline]
  22. Tovey, S. C., de Smet, P., Lipp, P., Thomas, D., Young, K. W., Missiaen, L., De Smedt, H., Parys, J. B., Berridge, M. J., Thuring, J., Holmes, A., Bootman, M. D. (2001) Calcium puffs are generic InsP(3)-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses. J. Cell Sci. 114,3979-3989[Abstract/Free Full Text]
  23. Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., Yukaki, M., Nakade, S., Mikoshiba, K. (1992) Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca2+-induced release and Ca2+ oscillations in hamster eggs. FEBS Lett. 309,180-184[CrossRef][Medline]
  24. Walseth, T. F., Lee, H. C. (1993) Synthesis and characterization of antagonists of cyclic-ADP-ribose-induced calcium release. Biochim. Biophys. Acta 1178,235-242[Medline]
  25. Eddy, E. M., Shapiro, B. M. (1976) Changes in the topography of the sea urchin egg after fertilization. J. Cell Biol. 71,35-48[Abstract/Free Full Text]
  26. Janmey, P. A. (1998) The cytoskeleton and cell signalling: component localization and mechanical coupling. Physiol. Rev. 78,763-781[Abstract/Free Full Text]
  27. Lange, K., Brandt, U. (1996) Calcium storage and release properties of F-actin: evidence for the involvement of F-actin in cellular calcium signaling. FEBS Lett. 395,137-142[CrossRef][Medline]
  28. Lange, J., Schlieps, K., Lange, K., Knoll-Köhler, E. (1997) Activation of calcium signaling in isolated rat hepatocytes is accompanied by shape changes of microvilli. Exp. Cell Res. 234,486-497[CrossRef][Medline]
  29. Lange, K. (1999) Microvillar Ca++ signaling: a new view of an old problem. J. Cell. Physiol. 180,19-35[CrossRef][Medline]
  30. Marchant, J., Callamaras, N., Parker, I. (1999) Initiation of IP3-mediated Ca2+ waves in Xenopus oocytes. EMBO J. 18,5285-5299[CrossRef][Medline]
  31. Hirai, H., Shida, H. (1979) Shortening of microvilli during the maturation of starfish oocyte from which vitelline coat was removed. Bull. Mar. Biol. Sta. Asamushi Tohoku Univ. 16,161-167
  32. Callamaras, N., Parker, I. (1999) Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal line scan imaging. J. Gen. Physiol. 113,199-213[Abstract/Free Full Text]
  33. Speksnijder, J. E. (1992) The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker. Dev. Biol. 153,259-271[CrossRef][Medline]
  34. Berridge, M. J., Dupont, G. (1994) Spatial and temporal signalling by calcium. Curr. Opin. Cell Biol. 6,267-274[CrossRef][Medline]
  35. Kyozuka, K., Deguchi, R., Mohri, T., Miyazaki, S. (1998) Injection of sperm extract mimics spatiotemporal dynamics of Ca2+ responses and progression of meiosis at fertilization of ascidian oocytes. Development 125,4099-4105[Abstract]
  36. Kume, S., Muto, A., Aruga, J., Nakagawa, T., Michikawa, T., Furuichi, T., Nakade, S., Okano, H., Mikoshiba, K. (1993) The Xenopus IP3 receptors: structure, function, and localization in oocytes and eggs. Cell 73,555-570[CrossRef][Medline]
  37. Mehlmann, L., Mikoshiba, K., Kline, D. (1996) Redistribution and increase in cortical inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse oocyte. Dev. Biol. 180,489-498[CrossRef][Medline]
  38. McGuinness, O. M., Moreton, R. B., Johnson, M. H., Berridge, M. J. (1996) A direct measurement of increased divalent cation influx in fertilized mouse oocytes. Development 122,2199-2206[Abstract]
  39. Mohri, T., Shirakawa, H., Oda, S., Sato, M. S., Mikoshiba, K., Miyazaki, S. (2001) Analysis of Mn2+/Ca2+ influx and release during Ca2+ oscillations in mouse eggs injected with sperm extract. Cell Calcium 29,311-325[CrossRef][Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
Q.-Y. Sun and H. Schatten
Regulation of dynamic events by microfilaments during oocyte maturation and fertilization
Reproduction, February 1, 2006; 131(2): 193 - 205.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. Whitaker
Calcium at Fertilization and in Early Development
Physiol Rev, January 1, 2006; 86(1): 25 - 88.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Lim, E. Ercolano, K. Kyozuka, G. A. Nusco, F. Moccia, K. Lange, and L. Santella
The M-phase-promoting Factor Modulates the Sensitivity of the Ca2+ Stores to Inositol 1,4,5-Trisphosphate via the Actin Cytoskeleton
J. Biol. Chem., October 24, 2003; 278(43): 42505 - 42514.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LIM, D.
Right arrow Articles by SANTELLA, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LIM, D.
Right arrow Articles by SANTELLA, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS