Keiichiro Kyozuka

Publications (11)35.82 Total impact

• Source

  Available from: PubMed Central

  Article: Guanine nucleotides in the meiotic maturation of starfish oocytes: regulation of the actin cytoskeleton and of Ca(2+) signaling.

  Keiichiro Kyozuka, Jong T Chun, Agostina Puppo, Gianni Gragnaniello, Ezio Garante, Luigia Santella
  [show abstract] [hide abstract]
  ABSTRACT: 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 Ca(2+) 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 Ca(2+) signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPgammaS) or inactive (GDPbetaS) forms of G-proteins, we have demonstrated that: i) GTPgammaS induces Ca(2+) release that mimics the effect of 1-MA; ii) GDPbetaS completely blocks 1-MA-induced Ca(2+); iii) GDPbetaS has little effect on the amplitude of the Ca(2+) peak, but significantly expedites the initial Ca(2+) waves induced by InsP(3) photoactivation, iv) GDPbetaS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca(2+) release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPbetaS, or actinase E, all led to significant changes of 1-MA-induced Ca(2+) signaling. 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 Ca(2+) release.
  PLoS ONE 02/2009; 4(7):e6296. · 4.09 Impact Factor
• Article: Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening.

  Tatsuma Mohri, Masahiro Sokabe, Keiichiro Kyozuka
  [show abstract] [hide abstract]
  ABSTRACT: 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).
  Developmental Biology 08/2008; 322(2):251-62. · 4.07 Impact Factor
• Article: Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

  Keiichiro Kyozuka, Jong T Chun, Agostina Puppo, Gianni Gragnaniello, Ezio Garante, Luigia Santella
  [show abstract] [hide abstract]
  ABSTRACT: 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.
  Developmental Biology 07/2008; 320(2):426-35. · 4.07 Impact Factor
• Article: Two phases of calcium requirement during starfish meiotic maturation.

  Hiroaki Tosuji, Yukari Seki, Keiichiro Kyozuka
  [show abstract] [hide abstract]
  ABSTRACT: During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.
  Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 07/2007; 147(2):432-7. · 2.23 Impact Factor
• Article: Serotonin stimulates [Ca2+]i elevation in ciliary ectodermal cells of echinoplutei through a serotonin receptor cell network in the blastocoel.

  Hideki Katow, Shunsuke Yaguchi, Keiichiro Kyozuka
  [show abstract] [hide abstract]
  ABSTRACT: 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 Ca(2+) concentration ([Ca(2+)](i)) in the ectoderm, as detected by Oregon-Green dextran, injected earlier in development. The calcium transient propagated as a wave at about 175 microm 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 [Ca(2+)](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 [Ca(2+)](i) in the larval ectodermal cells through the serotonin receptor cell network.
  Journal of Experimental Biology 03/2007; 210(Pt 3):403-12. · 3.00 Impact Factor
• Source

  Available from: dmlim.net

  Article: Modulation of calcium signalling by the actin-binding protein cofilin.

  Gilda A Nusco, Jong Tai Chun, Emanuela Ercolano, Dmitry Lim, Gianni Gragnaniello, Keiichiro Kyozuka, Luigia Santella
  [show abstract] [hide abstract]
  ABSTRACT: Cofilin is a small protein that belongs to the family of actin-depolymerizing factors (ADF). The main cellular function of cofilin is to change cytoskeletal dynamics and thus to modulate cell motility and cytokinesis. We have recently demonstrated that the actin cytoskeleton is involved in the modulation of Ca(2+) signalling in starfish oocytes. To extend these observations, we have explored whether cofilin influences Ca(2+) signalling in the oocytes. Here we show that microinjection of the functionally active cofilin alters the Ca(2+) signalling mediated by the three major second messengers, InsP(3), NAADP, and cADPr. Cofilin intensifies the Ca(2+) signals induced by InsP(3) and NAADP, and delays those induced by cADPr. Furthermore, the injection of cofilin increases the Ca(2+) signals during hormone-induced oocyte maturation and fertilization. The results suggest that the dynamic regulation of F-actin by its binding proteins may play an important role in the modulation of intracellular Ca(2+) signalling.
  Biochemical and Biophysical Research Communications 10/2006; 348(1):109-14. · 2.48 Impact Factor
• Article: A novel oocyte maturation arresting factor in the central nervous system of scallops inhibits serotonin-induced oocyte maturation and spawning of bivalve mollusks.

  Toru Tanabe, Makoto Osada, Keiichiro Kyozuka, Kazuo Inaba, Akihiro Kijima
  [show abstract] [hide abstract]
  ABSTRACT: Serotonin (5-hydroxytriptamine; 5-HT) is a major neurotransmitter that triggers oocyte maturation and sequential spawning in bivalve mollusks. A proteinous and heat-labile substance that proved to be a novel inhibitor of 5-HT-induced egg release from ovarian tissue was found in the cerebral and pedal ganglia (CPG) of the scallop Patinopecten yessoensis. The same inhibitory activity was also observed in the proteinous fraction from the supernatant of hemolymph. Histological observation demonstrated that the novel inhibitor prevented 5-HT from inducing oocyte maturation in the scallop ovary and that no prostaglandin F2alpha (PGF2alpha) inhibited 5-HT-induced oocyte maturation, although PGF2alpha strongly prohibited 5-HT-induced egg release through the gonoduct from ovarian tissue. The novel inhibitor from the scallop CPG also prohibited 5-HT-induced oocyte maturation of other bivalve species as well as scallops. The novel inhibitor, mediated through a receptor mechanism on oocyte membranes, blocked extracellular Ca2+ uptake into oocytes, which was observed in 5-HT-induced oocyte maturation. It is suggested that the novel inhibitor with a molecular mass of 60 kDa, named oocyte maturation arresting factor, which appears to be a universal substance for bivalve species, may be transported from the CPG to the ovary via hemolymph and may prohibit 5-HT-induced oocyte maturation due to the interference of extracellular Ca2+ influx into oocytes, eventually resulting in the inhibition of spawning. On the other hand, it seems that PGF2alpha inhibits 5-HT-induced transport of mature eggs through the gonoduct.
  General and Comparative Endocrinology 08/2006; 147(3):352-61. · 3.27 Impact Factor
• Source

  Available from: dmlim.net

  Article: NAADP and InsP3 play distinct roles at fertilization in starfish oocytes.

  Francesco Moccia, Gilda A Nusco, Dmitry Lim, Keiichiro Kyozuka, Luigia Santella
  [show abstract] [hide abstract]
  ABSTRACT: NAADP participates in the response of starfish oocytes to sperm by triggering the fertilization potential (FP) through the activation of a Ca2+ current which depolarizes the membrane to the threshold of activation of the voltage-gated Ca2+ channels. The aim of this study was to investigate whether this Ca2+ influx is linked to the onset of the concomitant InsP3-mediated Ca2+ wave by simultaneously employing Ca2+ imaging and single-electrode intracellular recording techniques. In control oocytes, the sperm-induced membrane depolarization always preceded by a few seconds the onset of the Ca2+ wave. Strikingly, the self-desensitization of NAADP receptors either abolished the Ca2+ response or resulted in abnormal oocyte activation, i.e., the membrane depolarization followed the Ca2+ wave and the oocyte was polyspermic. The inhibition of InsP3 signaling only impaired the propagation of the Ca2+ wave and shortened the FP. The duration of FP was also reduced in low-Na+ sea water. Finally, uncaged InsP3 produced a Ca2+ increase, which depolarized the membrane upon the activation of a Ca2+-sensitive cation current. These results support the hypothesis that Ca2+ entry during the NAADP-triggered FP is required for the onset of the Ca2+ wave at fertilization. The InsP3-mediated Ca2+ wave, in turn, may interact with the NAADP-evoked depolarization by activating a Ca2+-dependent Na+ entry.
  Developmental Biology 07/2006; 294(1):24-38. · 4.07 Impact Factor
• Source

  Available from: dmlim.net

  Article: NAADP triggers the fertilization potential in starfish oocytes.

  Francesco Moccia, Dmitry Lim, Keiichiro Kyozuka, Luigia Santella
  [show abstract] [hide abstract]
  ABSTRACT: In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes. The changes in membrane potential induced by the sperm were measured in oocytes held at a low resting potential, so that the Ca2+-action potential was inactivated and only the initial slower depolarization caused by the sperm could be studied. Decreasing extracellular Na+ concentration did not prevent the onset of the fertilization potential, while removal of external Ca2+ abolished it. The pre-incubation with SK&F 96365 and verapamil and the pre-injection of BAPTA inhibited the fertilization potential, while the injection of heparin only reduced its duration. The biophysical and pharmacological properties of the sperm-elicited depolarization were similar to those displayed by the NAADP-activated Ca2+-mediated current recently described in starfish oocytes. Indeed, the desensitization of NAADP-receptors prevented the onset of the fertilization potential. Taken together, these data suggest that NAADP could trigger the fertilization potential in starfish oocytes.
  Cell Calcium 01/2005; 36(6):515-24. · 3.77 Impact Factor
• Article: Association of Calmodulin with Nuclear Structures in Starfish Oocytes and Its Role in the Resumption of Meiosis

  Luigia Santella, Keiichiro Kyozuka
  [show abstract] [hide abstract]
  ABSTRACT: The resumption of meiosis in prophase-arrested starfish oocytes is induced by the hormone 1-methyl-adenine, which has been shown previously to induce a calcium transient in the nucleus which at this stage is called the germinal vesicle. This transient precedes the breakdown of the germinal vesicle (GVBD). Experiments were performed to establish whether nuclear calmodulin (CaM) was involved in the progression of the meiotic cycle. CaM antagonists, antibodies, and an inhibitory peptide corresponding to the CaM-binding domain of myosin-light-chain kinase have been injected into the nucleus of prophase-arrested starfish oocytes. The antagonists failed to affect the final response to 1-methyladenine, i.e. GVBD, although two antagonists delayed it, whereas the peptide inhibitor and the antibodies completely inhibited it. The antibodies suppressed the nuclear Ca2+ spikes that were shown by previous work to be induced by the photoreleasing of caged adenosine 3′,5′-(cyclic)diphosphate ribose in the germinal vesicle. Immunofluorescence staining of isolated starfish oocyte nuclei with CaM antibodies showed CaM in the envelope and in the nucleolus. Immunogold labelling of oocytes revealed aggregates of CaM and of a 36-kDa protein, of the heterogeneous ribonucleoprotein particles (hnRNP), in electron-dense hnRNP in the nuclear matrix. 1-Methyladenine induced the disappearance of these hnRNP from the nucleoplasm and the translocation of CaM and the 36-kDa protein previously associated with them to the cytoplasm, prior to the breakdown of the nuclear envelope.
  European Journal of Biochemistry. 07/2004; 246(3):602 - 610.
• Article: The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton.

  Dmitri Lim, Emanuela Ercolano, Keiichiro Kyozuka, Gilda A Nusco, Francesco Moccia, Klaus Lange, Luigia Santella
  [show abstract] [hide abstract]
  ABSTRACT: The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.
  Journal of Biological Chemistry 11/2003; 278(43):42505-14. · 4.77 Impact Factor