Keiichiro Kyozuka

EFFECTS OF 1-METHYLADENINE ON NUCLEAR CA2+ TRANSIENTS AND MEIOSIS RESUMPTION IN STARFISH OOCYTES ARE MIMICKED BY THE NUCLEAR INJECTION OF INOSITOL 1,4 ,5-TRISPHOSPHATE AND CADP-RIBOSE

The treatment of prophase-arrested starfish oocytes with the hormone 1-methyladenine (1-MA) induces the elevation of Ca2+ in both the cytoplasm and the germinal vesicle (nucleus), and is followed by the resumption of meiosis. The injection of the modulators of the intracellular Ca2+ channels inositol 1,4,5-trisphosphate (InsP3) or cyclic adenosine diphosphate ribose (cADPr) into the germinal vesicle promoted meiosis resumption in the absence of 1-MA in about 50% of the oocytes. Caged InsP3 or caged cADPr were injected into the nuclei of oocytes together with the Ca2+ indicator calcium green dextran; their photoreleasing elicited nuclear calcium spikes which, in the case of cADPr, had repetitive behaviour. The spikes were abolished by the nuclear injection of antagonists or antibodies to the InsP3 or cADPr-sensitive Ca2+ channels. cADPr-modulated channels were localized on the membranes of the nuclear envelope using specific antibodies conjugated with IgG-gold complexes.

NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes

We have explored the role of the recently discovered second messenger nicotinic acid adenine nucleotide phosphate (NAADP+)inCa2+ swings that accompany the fertilization process in starfish oocytes. The injection of NAADP+ deep into the cytoplasm of oocytes matured by the hormone 1‐methylad‐enine (1‐MA), mobilized Ca2+ exclusively in the cortical layer, showing that the NAADP+‐sensitive Ca2+ pool is restricted to the subplasma membrane region of the cell. At variance with this, InsP3 initiated the liberation of Ca2+ next to the point of injection in the center of the cell. The initial cortical Ca2+ liberation induced by NAADP+ was followed by a spreading of the Ca2+ wave to the remainder of the cell and by a massive cortical granule exocytosis similar to that routinely observed on injection of InsP3. A striking difference in the responses to NAADP+ and InsP3 was revealed by the removal of the nucleus from immature oocytes, i.e., from oocytes not treated with 1‐MA. Whereas the Ca2+ response and the cortical granule exocytosis induced by NAADP+ were unaffected by the removal of the nucleus, the Ca2+ response promoted by InsP3 was significantly slowed. In addition, the cortical granule exocy‐tosis was completely abolished. When enucleated oocytes were fertilized, the spermatozoon still promoted the Ca2+ wave and normal cortical exocytosis, strongly suggesting that the Ca2+ response was mediated by NAADP+ and not by InsP3. InsP3‐sensitive Ca2+ stores may mediate the propagation of the wave initiated by NAADP+ since its spreading was strongly affected by removal of the nucleus.—Lim, D., Kyozuka, K., Gragnaniello, G., Carafoli, E., Santella, L. NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes. FASEB J. 15, 2257–2267 (2001)

Calcium, protease action, and the regulation of the cell cycle.

Proteolysis is a key event in the control of the cell cycle. Most of the proteins which are degraded at specific cycle points, e.g. cyclins A, B, and E, are substrates of the ubiquitin/proteasome pathway. The Ca2+ dependent neutral protease calpain also cleaves cell cycle proteins, among them cyclin D1 and the c-mos proto-oncogene product which is a component of the CSF. The proteasome itself, however, may be under Ca2+ control through the binding of Ca2+ to its 29 kDa regulatory subunit. Calpain undergoes relocation among cell compartments during the various steps of the mitotic and meitotic cycles. It promotes the initiation and the progression of mitosis when injected into the perinuclear space of synchronized PtK1 cells, and the resumption of meiosis when directly injected into the nuclei of prophase-arrested starfish oocytes. Apart from the proteins mentioned above, most of the substrates of calpain which become cleaved during mitosis and meiosis are still unknown. Microtubule-associated proteins are likely candidates.

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.

Serotonergic Ligands Induce Spawning but not Oocyte Maturation in the Bivalve Mactra chinensis from Central Japan

We examined the spawning sensitivity to serotonin and serotonergic ligands in the Japanese bivalve Mactra chinensis. Spawning was induced by both injected and externally applied scrotonin (5-hydroxytryptamine. 5-HT). The vertebrate 5-HT2 receptor agonist alpha-methyl 5-HT and the selective 5HT1A agonist 8-OH-DPAT were also effective at inducing spawning. However TFMPP (m-trifluoromethylphenylpiperazine, a verterbrate 5-HT1 receptor agonist) and 1-methyl-chlorophenyl biguanide (a vertebrate 5-HT3 agonist) were not effective spawning inducers. The 5-HT-induced spawning was blocked by mianserin (a vertebrate 5-HT2 antagonist). The rank order of potency of the agonists was: 5-HT > alpha-methyl 5-HT > 8-OH-DPAT > TFMPP > 1-methyl-chlorophenyl biguanide; these data support a growing body of literature invoking a mixed 5-HT1/5-HT2 pharmacological profile for serotonin receptors mediating reproductive processes in bivalves. However, neither 5-HT nor 8-OH-DPAT induced germinal vesicle breakdown (GVBD) in Mactra oocytes. Sperm induced GVBD in a high percentage of oocytes. This is the first report of a bivalve in which spawning, but not GVBD, can be induced by 5-HT. This result might be expected because Mactra spawns germinal vesicle oocytes that normally undergo GVBD upon fertilization, but is in contrast to the case of the closely related Spisula spp. in which serotonin induces both processes. The ability of 5-HT to induce spawning but not GVBD makes Mactra chinensis a model organism for studying spawning and meiotic mechanisms in bivalves.

Serotonin stimulates [Ca2+]i elevation in ciliary ectodermal cells of echinoplutei through a serotonin receptor cell network in the blastocoel.

SUMMARY A full-length serotonin receptor mRNA from the 5Hthpr gene was sequenced from larvae of the sea urchin, Hemicentrotus pulcherrimus. The DNA sequence was most similar to 5HT-1A of the sea urchin Strongylocentrotus purpuratus found by The Sea Urchin Genome Project, and the protein sequence predicted the presence of seven transmembrane domains. Immunohistochemistry with anti-5HThpr antibodies indicated that the protein was expressed on blastocoelar cells that comprised the major blastocoelar network (serotonin receptor cell network). These network cells inserted their processes into the ectoderm in various regions, including the ciliary band region. Serotonin injected into the blastocoel stimulated a transient elevation of cytoplasmic Ca2+ concentration ([Ca2+]i) in the ectoderm, as detected by Oregon-Green dextran, injected earlier in development. The calcium transient propagated as a wave at about 175 μm s–1, but was not detectable in the serotonin receptor-positive cell network. In larvae treated with p-chlorophenylalanine, a potent and irreversible serotonin synthesis inhibitor, serotonin application did not stimulate [Ca2+]i, the serotonin receptor cell network did not develop properly, and the swimming behavior of the larvae was abnormal. However, formation of a different nervous system comprising synaptotagmin-possessed neurites was not affected by p-chlorophenylalanine treatment. These results imply that serotonin secreted from the apical ganglion into the blastocoel stimulates the elevation of [Ca2+]i in the larval ectodermal cells through the serotonin receptor cell network.

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

Background Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca2+ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca2+ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. Methodology/Principal Findings By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca2+ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca2+; iii) GDPβS has little effect on the amplitude of the Ca2+ peak, but significantly expedites the initial Ca2+ waves induced by InsP3 photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca2+ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca2+ signaling. Conclusions/Significance Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca2+ release.

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.

Novel two-step Ca2+ increase and its mechanisms and functions at fertilization in oocytes of the annelidan worm Pseudopotamilla occelata

Mature oocytes of the annelidan worm Pseudopotamilla occelata have a wide perivitelline space between the oocyte surface and the vitelline envelope and are arrested at the first metaphase (MI). We found a novel two‐step Ca2+ increase in normally fertilized oocytes. The first Ca2+ increase originated at a cortex situated underneath a fertilizing sperm on the vitelline envelope, but failed to propagate beyond the center of the oocyte. The first localized Ca2+ increase was then followed by a larger Ca2+ increase starting from the whole oocyte cortex and spreading inwardly to the center. The first localized Ca2+ increase at fertilization was suppressed by the phospholipase C inhibitor U73122, and a similar Ca2+ change was induced by inositol 1,4,5‐trisphosphate (IP3). On the other hand, the second global Ca2+ increase in fertilized oocytes was blocked by removal of external Ca2+ or the voltage‐gated Ca2+ channel blocker D‐600, and a similar Ca2+ change could be mimicked by addition of excess K+ only when external Ca2+ was present. These results suggest that the first localized Ca2+ increase and the second global Ca2+ increase at fertilization are regulated by Ca2+ release from IP3‐sensitive stores and Ca2+ influx via voltage‐gated Ca2+ channels, respectively. Our data also demonstrated that the localized Ca2+ increase induces the formation of large cytoplasmic protrusion, which helps the fertilizing sperm to enter the oocyte, whereas the following global Ca2+ increase is a prerequisite for the retraction of the cytoplasmic protrusion and the resumption of meiosis from MI.

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening

Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca(2+)-dependent and attributed to the late Ca(2+) rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (t(p)) which corresponds to peak [Ca(2+)](i), suggesting that NO is not related to any other ionic changes besides [Ca(2+)](i). We measured O(2) consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O(2) consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H(2)O(2) and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H(2)O(2) production and consolidates FE hardening by H(2)O(2).