理学修士, 東京大学

博士（医学）, 東京女子医科大学

Bioimaging

Cell Biology

バイオイメージング

細胞生理学

Life sciences, Clinical pharmacy

Life sciences, Physiology

Life sciences, Cell biology

Life sciences, Biophysics

01 Apr. 2010
The University of Electro-Communications, 准教授

01 Apr. 2007 - 31 Mar. 2010
電気通信大学 電気通信学部, 准教授

01 Oct. 2005 - 31 Mar. 2007
The University of Electro-Communications, Associate Professor

01 Jul. 2002 - 30 Sep. 2005
Tokyo Women's Medical University, Assistant Professor

01 Apr. 1991 - 30 Jun. 2002
Tokyo Women's Medical University, Instructor

31 Mar. 1991
The University of Tokyo, Graduate School, Division of Science, 動物学

31 Mar. 1989
University of Tsukuba, Second Cluster of College, 生物学類

01 Apr. 1981 - 31 Mar. 1984
私立武蔵高等学校

2000
評議員, 日本生理学会, Society

Eccentric contraction increases hydrogen peroxide levels and alters gene expression through Nox2 in skeletal muscle of male mice
Ryotaro Kano; Tatsuya Kusano; Reo Takeda; Hideki Shirakawa; David C. Poole; Yutaka Kano; Daisuke Hoshino
Journal of Applied Physiology, American Physiological Society, 137, 3, 778-788, 01 Sep. 2024, This in vivo model successfully characterized the effects of eccentric (ECC) and concentric (CONC) contractions on cytosolic and mitochondrial [H₂O₂] in mouse skeletal muscle. Compared with CONC, ECC induced higher and more sustained [H₂O₂]_cyto—an effect that was abolished by Nox2 inhibition. ECC-induced [H₂O₂]_cyto elevations were requisite for altered gene expression.
Scientific journal

In vivo cytosolic H2O2 changes and Ca2+ homeostasis in mouse skeletal muscle.
Ryotaro Kano; Ayaka Tabuchi; Yoshinori Tanaka; Hideki Shirakawa; Daisuke Hoshino; David C Poole; Yutaka Kano
American journal of physiology. Regulatory, integrative and comparative physiology, 30 Oct. 2023, Peer-reviwed, True, Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1. Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2]cyto) leads to a concentration-dependent increase in cytosolic Ca2+concentration ([Ca2+]cyto) through its effect on RyR1. 2. In hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2]cyto induce Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in C57BL/6J male mice under four conditions: Elevated exogenous H2O2, Cardiac arrest, Twitch and Tetanic contractions. Exogenous H2O2 (0.1-100mM) induced a concentration-dependent increase in [H2O2]cyto (+55%,0.1mM; +280%,100mM) and an increase in [Ca2+]cyto (+3%,1.0mM; +8%,10mM). This increase in [Ca2+]cyto was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2]cyto (+33%) and [Ca2+]cyto (+20%) 50min post-cardiac arrest. Compared to exogenous 1.0mM H2O2 condition, [H2O2]cyto after tetanic contractions rose less than one-tenth as much, while [Ca2+]cyto was 4.7-fold higher. In conclusion, substantial increases in [H2O2]cyto levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2]cyto from contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.
Scientific journal, English

Ryanodine receptors mediate high intracellular Ca²⁺ and some myocyte damage following eccentric contractions in rat fast twitch skeletal muscle
Tabuchi, A; Tanaka, Y; Takagi, R; Shirakawa, H; Shibaguchi, T; Sugiura, T; Poole, D.C; Kano, T
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 322, 1, R14-R27, 01 Jan. 2022, Peer-reviwed, True, Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from the extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e., ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 h post-ECC (5 × 40 contractions). Separate groups of rats received RyR inhibitor dantrolene (DAN; 10 mg/kg ip) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared with control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased percent total area of high [Ca2+]i sites (operationally defined as R ≥ 1.39, i.e., ≥1 SD of mean control) 5 h post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, P < 0.01). DAN substantially reduced the high [Ca2+]i area 5 h post-ECC (ECC5h + DAN: 6.4 ± 3.1%, P < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h + DAN: 29.1 ± 2.2%, P < 0.01). Temporal and spatially amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs. ECC + DAN, P > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage, whereas the [Ca2+]i fluctuation is an RyR-independent phenomenon.
Scientific journal, English

Dual-FRET imaging of IP₃ and Ca²⁺ revealed Ca²⁺-induced IP₃ production maintains long lasting Ca²⁺ oscillations in fertilized mouse eggs
Matsu-ura, T; Shirakawa, H; Suzuki, K.G.N; Miyamoto, A; Sugiura, K; Michikawa, T; Kusumi, A; Mikoshiba, K
Scientific Reports, 9, 1, 4829, 18 Mar. 2019, Peer-reviwed
Scientific journal, English

In vitro Ca²⁺ dynamics induced by Ca²⁺ injection in individual rat skeletal muscle fibers.
Wakizaka, M; Eshima, H; Tanaka, T; Shirakawa, H; Poole, D.C; Kano, Y
Physiological Reports, 5, 5, e13180, 13 Mar. 2017, Peer-reviwed, True, In contrast to cardiomyocytes, store overload-induced calcium ion (Ca2+) release (SOICR) is not considered to constitute a primary Ca2+ releasing system from the sarcoplasmic reticulum (SR) in skeletal muscle myocytes. In the latter, voltage-induced Ca2+ release (VICR) is regarded as the dominant mechanism facilitating contractions. Any role of the SOICR in the regulation of cytoplasmic Ca2+ concentration ([Ca2+]i) and its dynamics in skeletal muscle in vivo remains poorly understood. By means of in vivo single fiber Ca2+ microinjections combined with bioimaging techniques, we tested the hypothesis that the [Ca2+]i dynamics following Ca2+ injection would be amplified and fiber contraction facilitated by SOICR. The circulation-intact spinotrapezius muscle of adult male Wistar rats (n = 34) was exteriorized and loaded with Fura-2 AM to monitor [Ca2+]i dynamics. Groups of rats underwent the following treatments: (1) 0.02, 0.2, and 2.0 mmol/L Ca2+ injections, (2) 2.0 mmol/L Ca2+ with inhibition of ryanodine receptors (RyR) by dantrolene sodium (DAN), and (3) 2.0 mmol/L Ca2+ with inhibition of SR Ca2+ ATPase (SERCA) by cyclopiazonic acid (CPA). A quantity of 0.02 mmol/L Ca2+ injection yielded no detectable response, whereas peak evoked [Ca2+]i increased 9.9 ± 1.8% above baseline for 0.2 mmol/L and 23.8 ± 4.3% (P < 0.05) for 2.0 mmol/L Ca2+ injections. The peak [Ca2+]i in response to 2.0 mmol/L Ca2+ injection was largely abolished by DAN and CPA (-85.8%, -71.0%, respectively, both P < 0.05 vs. unblocked) supporting dependence of the [Ca2+]i dynamics on Ca2+ released by SOICR rather than injected Ca2+ itself. Thus, this investigation demonstrates the presence of a robust SR-evoked SOICR operant in skeletal muscle in vivo.
Scientific journal, English

Calcium Signaling in Mammalian Eggs at Fertilization
Hideki Shirakawa; Takashi Kikuchi; Masahiko Ito
CURRENT TOPICS IN MEDICINAL CHEMISTRY, BENTHAM SCIENCE PUBL LTD, 16, 24, 2664-2676, 2016, Peer-reviwed, The innovation and development of live-cell fluorescence imaging methods have revealed the dynamic aspects of intracellular Ca2+ in a wide variety of cells. The fertilized egg, the very first cell to be a new individual, has long been under extensive investigations utilizing Ca2+ imaging since its early days, and spatiotemporal Ca2+ dynamics and underlying mechanisms of Ca2+ mobilization, as well as physiological roles of Ca2+ at fertilization, have become more or less evident in various animal species. In this article, we illustrate characteristic patterns of Ca2+ dynamics in mammalian gametes and molecular basis for Ca2+ release from intracellular stores leading to the elevation in cytoplasmic Ca2+ concentration, and describe the identity and properties of sperm-borne egg-activating factor in relation to the induction of Ca2+ waves and Ca2+ oscillations, referring to its potential use in artificial egg activation as infertility treatment. In addition, a possible Ca2+ influx-driven mechanism for slow and long-lasting Ca2+ oscillations characteristic of mammalian eggs is proposed, based on the recent experimental findings and mathematical modeling. Cumulative knowledge about the roles of Ca2+ in the egg activation leading to early embryogenesis is summarized, to emphasize the diversity of functions that Ca2+ can perform in a single type of cell.
Scientific journal, English

Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs
Tooru Takahashi; Takashi Kikuchi; Yusuke Kidokoro; Hideki Shirakawa
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, 430, 1, 60-65, Jan. 2013, Peer-reviwed, On mammalian fertilization, long-lasting Ca2+ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca2+ increase in Ca2+ concentration ([Ca2+) in the cytosol is due to Ca2+ release from the endoplasmic reticulum (ER), Ca2+ influx from outside is required for Ca2+ oscillations to persist. In this study, we investigated how Ca2+ influx is interrelated to the cycle of Ca2+ release and uptake by the intracellular Ca2+ stores during Ca2+ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca2+] with fura-2, the influx rate was evaluated using Ma(2+) quenching technique, and the change in [Ca2+] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca2+ release and then diminished gradually to the basal level, and demonstrated that the ER Ca2+ stores once depleted by Ca2+ release were gradually refilled until the next Ca2+ transient to be initiated. Experiments altering extracellular [Ca2+] in the middle of Ca2+ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca2+ influx, indicating the crucial role of Ca2+ influx in determining the intervals of Ca2+ transients. As for the influx pathway supporting Ca2+ oscillations to persist, STIM1/Orai1-mediated store-operated Ca2+ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate. (C) 2012 Elsevier Inc. All rights reserved.
Scientific journal, English

Characterization of store-operated Ca²⁺ entry in mouse eggs
Takahashi, T; Shirakawa, H
Comparative Physiology and Biochemistry, 28, Suppl, 158, 2011
International conference proceedings, English

FUNCTIONAL ROLE OF CRAC CHANNELS IN THE GENERATION AND MAINTENANCE OF Ca2+ OSCILLATIONS IN MAMMALIAN EGGS
Atsushi Takahashi; Yusuke Kidokoro; Hideki Shirakawa
JOURNAL OF PHYSIOLOGICAL SCIENCES, SPRINGER TOKYO, 59, Suppl. 1, 244-244, 2009
International conference proceedings, English

CHARACTERIZATION OF Ca2+ INFLUX PATHWAY ACTIVATED DURING Ca2+ OSCILLATIONS IN MOUSE EGGS
Tooru Takahashi; Hideki Shirakawa
JOURNAL OF PHYSIOLOGICAL SCIENCES, SPRINGER TOKYO, 59, Suppl. 1, 244-244, 2009
International conference proceedings, English

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe
H Shirakawa; M Ito; M Sato; Y Umezawa; S Miyazaki
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, 345, 2, 781-788, Jun. 2006, Peer-reviwed, Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3](i)) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase C zeta (PLC zeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of PPA in fertilized eggs, and the enhancement of PLC-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLC zeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLC zeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations. (c) 2006 Elsevier Inc. All rights reserved.
Scientific journal, English

Measurement of IP₃ in mouse eggs with FRET-based probe
Shirakawa, H; Ito, M; Miyazaki, S
International Symposium on "Cell Signaling in Gamete Activation - from Basic Research to ART", 116, 2006
International conference proceedings, English

The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase C xi
Z Kouchi; T Shikano; Y Nakamura; H Shirakawa; K Fukami; S Miyazaki
JOURNAL OF BIOLOGICAL CHEMISTRY, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 280, 22, 21015-21021, Jun. 2005, Peer-reviwed, Sperm-specific phospholipase C-xi(PLC xi) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLC xi has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at similar to 100 nM Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408 - 10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2)- hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLC xi activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLC xi activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3) P and, to the lesser extent, to PI(5) P but not to PI(4,5)P-2 or acidic phospholipids. PI(3) P and PI(5) P reduced PLC xi activity in vitro, suggesting that the interaction could play a role for negative regulation of PLC xi.
Scientific journal, English

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development
Y Sone; M Ito; H Shirakawa; T Shikano; H Takeuchi; K Kinoshita; S Miyazaki
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, 330, 3, 690-694, May 2005, Peer-reviwed, Phospholipase C-zeta (PLC zeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLC zeta. Changes in the localization of expressed PLC zeta were investigated by tagging with a fluorescent protein. PLC zeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLC zeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated I-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLC zeta was recognized in every embryo up to blastocyst. Thus, PLC zeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis. (c) 2005 Elsevier Inc. All rights reserved.
Scientific journal, English

Ca2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation
A Yoda; S Oda; T Shikano; Z Kouchi; T Awaji; H Shirakawa; K Kinoshita; S Miyazaki
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC ELSEVIER SCIENCE, 268, 2, 245-257, Apr. 2004, Peer-reviwed, Sperm-specific phospholipase C zeta (PLCzeta) is known to induce intracellular Ca2+ oscillations and egg, activation when expressed in mouse eggs by injection of RNA encoding PLCzeta. We investigated the expression level and spatial distribution of PLCzeta in the egg in real time and in relation to the initiation and termination of Ca2+ oscillations by monitoring fluorescence of a yellow fluorescent protein 'Venus' fused with PLCzeta. Ca2+ oscillations similar to those at fertilization were induced at 40-50 min after RNA injection, when expressed PLCzeta reached 10-40 x 10(-15) g in the egg. PLCzeta-Venus increased up to 3 h and attained a steady level at 4-5 h. Interestingly, PLCzeta-Venus is accumulated to the pronucleus (PN) formed at 5-6 h and continuously increased there. Ca2+ oscillations stopped in most eggs before initiation of the accumulation. A variant of PLCzeta that lacks three EF hand domains was much less effective in induction of Ca2+ oscillations and little accumulated in the pronucleus, indicating a critical role of those domains. The ability of the accumulation to the pronucleus qualifies PLCzeta for a strong candidate of the Ca2+ oscillation-inducing sperm factor, which is introduced into the ooplasm upon sperm-egg fusion and concentrated to the pronucleus after inducing egg activation. (C) 2004 Elsevier Inc. All rights reserved.
Scientific journal, English

Blind spectral decomposition of single-cell fluorescence by parallel factor analysis
H Shirakawa; S Miyazaki
BIOPHYSICAL JOURNAL, BIOPHYSICAL SOCIETY, 86, 3, 1739-1752, Mar. 2004, Peer-reviwed, Simultaneous measurement of multiple signaling molecules is essential to investigate their relations and interactions in living cells. Although a wide variety of fluorescent probes are currently available, the number of probes that can be applied simultaneously is often limited by the overlaps among their fluorescence spectra. We developed the experimental system to measure and analyze many overlapping fluorescent components in single cells. It is based on the recording of two-dimensional single-cell fluorescence spectra and on the blind spectral decomposition of fluorescence data by method of parallel factor analysis. Because this method does not require any preknowledge about the shapes of individual component spectra, it can be applied to the specimens that contain fluorescent components with unknown spectra. By examining the performance using the mixture solutions of fluorescent indicators, it was confirmed that >10 largely overlapping spectral components could be easily separated. The effectiveness in the physiological experiments was proven in the applications to the temporal analysis of intracellular Ca2+ concentration and pH, as well as the intrinsic fluorescent components, in single mouse oocytes.
Scientific journal, English

PLCζ expressed in the mouse egg induces Ca²⁺ oscillations and is accumulated into the pronucleus
Yoda, A; Oda, S; Shikano, T; Kouchi, Z; Awaji, T; Shirakawa, H; Miyazaki, S
The 3rd Japanese Biochemical Society Biofrontier Symposium on New Aspect of Phospholipid Biology, 95, 2004
International conference proceedings, English

Characterization of PLC-zeta: Ca²⁺ oscillation-inducing sperm factor
Oda, S; Kouchi, Z; Yoda, A; Awaji, T; Shirakawa, H; Miyazaki, S
The 4th International Symposium on "The Molecular and Cell Biology of Egg- and Embryo-coats", 55, 2004
International conference proceedings, English

広波長域２次元蛍光スペクトル顕微測光にによる多因子分離解析法の細胞生理学的応用
白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 24, 17-18, 2004
Research institution, Japanese

広波長域２次元蛍光スペクトル顕微測光に基づく多因子同時解析システムの構築
白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 23, 19-20, 2003
Research institution, Japanese

可視光励起プローブによる細胞内カルシウムのレシオメトリック測定
白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 22, 23-25, 2002
Research institution, Japanese

Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains
T Awaji; A Hirasawa; H Shirakawa; G Tsujimoto; S Miyazaki
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC, 289, 2, 457-462, Nov. 2001, Peer-reviwed, To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha (1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function. (C) 2001 Elsevier Science.
Scientific journal, English

Analysis of Mn2+/Ca2+ influx and release during Ca2+ oscillations in mouse eggs injected with sperm extract
T Mohri; H Shirakawa; S Oda; MS Sato; K Mikoshiba; S Miyazaki
CELL CALCIUM, CHURCHILL LIVINGSTONE, 29, 5, 311-325, May 2001, Peer-reviwed, Repetitive Ca2+ release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn2+ and Ca2+ during Ca2+ oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn2+-quenching of intracellular Fura-2 after adding Mn2+ to external medium. Mn2+/Ca2+ influx was detected at the resting state. A marked Mn2+/Ca2+ influx occurred during the first Ca2+ release upon SE injection, and persistently facilitated Mn2+/Ca2+ influx was observed during steady Ca2+ oscillations. As intracellular Mn2+ concentration ([Mn2+](i)) increased progressively, periodic [Mn2+](i) rises appeared, corresponding to each Ca2+ transient but taking a slower time course. A numerical simulation based on continuous Mn2+/Ca2+ influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn2+ oscillations calculated from experimental data, strongly suggesting that repetitive Mn2+ release develops after Mn2+ entry and uptake into the ER. In other experiments, a marked Mn2+ influx occurred upon Mn2+ addition to Ca2+-free medium after depletion of the ER using an ER Ca2+ pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn2+ influx was induced by injection of SE, InsP(3), or Ca2+, when Ca2+ release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca2+ influx is activated during the initial large Ca2+ release possibly by a capacitative mechanism and kept facilitated during steady Ca2+ oscillations. The finding that repetitive Mn2+ release is caused by continuous Mn2+ entry suggests that continuous Ca2+ influx may play a critical role in refilling the ER and, thereby, maintaining Ca2+ oscillations in mammalian fertilization. (C) 2001 Harcourt Publishers Ltd.
Scientific journal, English

IP₃誘発性Ca遊離に伴う小胞体膜電位変化の光学的解析
白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 21, 18-19, 2001
Research institution, Japanese

Ca2+/Mn2+ dynamics during Ca2+ oscillations in mouse eggs injected with sperm extract
T Mohri; H Shirakawa; S Oda; MS Sato; S Miyazaki
MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, 11, 407A-407A, Dec. 2000
International conference proceedings, English

Spatiotemporal analysis of Ca2+ waves in relation to the sperm entry site and animal-vegetal axis during Ca2+ oscillations in fertilized mouse eggs
R Deguchi; H Shirakawa; S Oda; T Mohri; S Miyazaki
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC, 218, 2, 299-313, Feb. 2000, Peer-reviwed, Fertilized mouse eggs exhibit repetitive rises in intracellular Ca2+ concentration ([Ca2+](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca2+](i) rise were investigated by high-speed Ca2+ imaging during early development of monospermic eggs. Every [Ca2+](i) rise involved a Ca2+ wave. In the first Ca2+ transient, [Ca2+](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca2+ release mechanisms. The first step was a Ca2+ wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, similar to 20 mu m/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca2+](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca2+ wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca2+ oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation (similar to 3 h after fertilization). (1) The duration of Ca2+ transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca2+](i) rise became greater and wave speeds increased up to 80-100 mu m/s or more. (4) The transient cytoplasmic movement always resulted from the Ca2+ wave, although its displacement became smaller. (5) The Ca2+ wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature. (C) 2000 Academic Press.
Scientific journal, English

Spatiotemporal analysis of Ca2+ waves in relation to the sperm entry site and animal-vegetal axis during Ca2+ oscillations in fertilized mouse eggs
R Deguchi; H Shirakawa; S Oda; T Mohri; S Miyazaki
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC, 218, 2, 299-313, Feb. 2000, Fertilized mouse eggs exhibit repetitive rises in intracellular Ca2+ concentration ([Ca2+](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca2+](i) rise were investigated by high-speed Ca2+ imaging during early development of monospermic eggs. Every [Ca2+](i) rise involved a Ca2+ wave. In the first Ca2+ transient, [Ca2+](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca2+ release mechanisms. The first step was a Ca2+ wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, similar to 20 mu m/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca2+](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca2+ wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca2+ oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation (similar to 3 h after fertilization). (1) The duration of Ca2+ transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca2+](i) rise became greater and wave speeds increased up to 80-100 mu m/s or more. (4) The transient cytoplasmic movement always resulted from the Ca2+ wave, although its displacement became smaller. (5) The Ca2+ wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature. (C) 2000 Academic Press.
Scientific journal, English

Numerical simulation of Mn²⁺ quenching of fura-2 during Ca²⁺ oscillations in mouse eggs.
Shirakawa, H; Mohri, T; Miyazaki, S
SEIRIKEN International Symposium "Mechanisms of Cell Signaling in Early Development", P11, 2000
International conference proceedings, English

Dual-wavelength ratiometric fluorescence measurement of endolasmic reticulum membrane potential using voltage-sensitive dyes
Shirakawa, H; Miyazaki, S
SEIRIKEN International Symposium "Mechanisms of Cell Signaling in Early Development", P12, 2000
International conference proceedings, English

Effect of FK506 on ATP-induced intracellular calcium oscillations in cow tracheal epithelium
S Kanoh; M Kondo; J Tamaoki; H Shirakawa; K Aoshiba; S Miyazaki; H Kobayashi; N Nagata; A Nagai
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, AMER PHYSIOLOGICAL SOC, 276, 6, L891-L899, Jun. 1999, To elucidate the effect of FK506 on Ca2+ oscillations in airway epithelium, we investigated cultured cow tracheal epithelial cells with a Ca2+ image-analysis system. ATP (1 mu M) induced long-lasting Ca2+ oscillations, having nearly constant peak values (300-400 nM) and intervals (20-40 s) in subconfluent cells but not in confluent cells. These responses were gradually attenuated and abolished by the addition of FK506. Rapamycin, which binds the FK506-binding protein (FKBP), likewise inhibited Ca2+ oscillations, whereas cyclosporin A, a calcineurin inhibitor, did not. Treatment of cells with FK506 decreased Ca2+ content in thapsigargin-sensitive stores, suggesting that the partial depletion of the stores causes the inhibition of Ca2+ oscillations. Immunocytochemistry revealed the existence of cytoplasmic FKBP-like immunoreactivities. The expression of a 12-kDa FKBP was greater in subconfluent cells than in confluent cells as determined by Western blotting, suggesting that the 12-kDa FKBP may be one of the factors that regulates Ca2+ oscillations. Therefore, FK506 possesses an inhibitory action on the Ca2+ response via intracellular FKBP but not via calcineurin, which may result in modification of airway epithelial functions.
Scientific journal, English

Effect of FK506 on ATP-induced intracellular calcium oscillations in cow tracheal epithelium
S Kanoh; M Kondo; J Tamaoki; H Shirakawa; K Aoshiba; S Miyazaki; H Kobayashi; N Nagata; A Nagai
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, AMER PHYSIOLOGICAL SOC, 276, 6, L891-L899, Jun. 1999, Peer-reviwed, To elucidate the effect of FK506 on Ca2+ oscillations in airway epithelium, we investigated cultured cow tracheal epithelial cells with a Ca2+ image-analysis system. ATP (1 mu M) induced long-lasting Ca2+ oscillations, having nearly constant peak values (300-400 nM) and intervals (20-40 s) in subconfluent cells but not in confluent cells. These responses were gradually attenuated and abolished by the addition of FK506. Rapamycin, which binds the FK506-binding protein (FKBP), likewise inhibited Ca2+ oscillations, whereas cyclosporin A, a calcineurin inhibitor, did not. Treatment of cells with FK506 decreased Ca2+ content in thapsigargin-sensitive stores, suggesting that the partial depletion of the stores causes the inhibition of Ca2+ oscillations. Immunocytochemistry revealed the existence of cytoplasmic FKBP-like immunoreactivities. The expression of a 12-kDa FKBP was greater in subconfluent cells than in confluent cells as determined by Western blotting, suggesting that the 12-kDa FKBP may be one of the factors that regulates Ca2+ oscillations. Therefore, FK506 possesses an inhibitory action on the Ca2+ response via intracellular FKBP but not via calcineurin, which may result in modification of airway epithelial functions.
Scientific journal, English

Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida
H Shirakawa; S Miyazaki
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC, 208, 1, 70-78, Apr. 1999, The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+](i)). Spatiotemporal aspects of the [Ca2+](i) increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+](i) occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+](i) was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle similar to 20 s after the onset of the [Ca2+](i) rise. High-resolution imaging revealed that the [Ca2+](i) rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+](i) rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+](i) rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR. (C) 1999 Academic Press.
Scientific journal, English

Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida
H Shirakawa; S Miyazaki
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC, 208, 1, 70-78, Apr. 1999, Peer-reviwed, The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+](i)). Spatiotemporal aspects of the [Ca2+](i) increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+](i) occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+](i) was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle similar to 20 s after the onset of the [Ca2+](i) rise. High-resolution imaging revealed that the [Ca2+](i) rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+](i) rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+](i) rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR. (C) 1999 Academic Press.
Scientific journal, English

Sperm-induced signaling in mouse eggs.
Miyazaki, S; Oda, S; Shirakawa, H; Mohri, T; Sato, M.S; Deguchi, R
The Susumu Hagiwara Memorial Symposium, 24, 1999
International conference proceedings, English

Erythromycin inhibits ATP-Induced intracellular calcium responses in bovine tracheal epithelial cells
M Kondo; S Kanoh; J Tamaoki; H Shirakawa; S Miyazaki; A Nagai
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, AMER LUNG ASSOC, 19, 5, 799-804, Nov. 1998, Erythromycin (EM) therapy is known to decrease airway secretion in chronic inflammatory airway diseases such as diffuse panbronchiolitis. Airway secretion is regulated by intracellular Ca2+ concentration ([Ca2+](i)). To elucidate the intracellular site of action of EM in airway epithelium, we examined the effect of EM on Ca2+ dynamics in cultured bovine tracheal epithelial cells using fura-2. EM per se did not cause any change in [Ca2+](i). Adenosine triphosphate (ATP; 10(-4) M) induced a biphasic [Ca2+](i) increase, consisting of a transient response followed by a sustained response. Pretreatment of cells with EM had little effect on the ATP-induced transient Ca2+ response but substantially reduced the sustained response in a dose-dependent manner. Clarithromycin, another 14-membered ring macrolide, likewise showed the inhibitory effect, but ampicillin and cephasolin did not. Uridine triphosphate (UTP; 10(-4) M) induced a biphasic [Ca2+](i) increase similar to ATP, and the UTP-induced sustained Ca2+ response was also inhibited by EM. In Ca2+-deficient medium (1 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid [EGTA]) or in the presence of La3+, the sustained Ca2+ response disappeared, suggesting that EM may inhibit Ca2+ influx induced by P-2u purinoceptor stimulation. In single-cell Ca2+ image analysis, low concentration of ATP (10(-6) M) induced Ca2+ oscillations, which were also inhibited by EM. The disappearance of [Ca2+](i) oscillations after addition of EM was similar to that after addition of EGTA. These results suggest that EM may decrease Ca2+-dependent airway secretion by inhibiting agonist-stimulated Ca2+ influx.
Scientific journal, English

Erythromycin inhibits ATP-Induced intracellular calcium responses in bovine tracheal epithelial cells
M Kondo; S Kanoh; J Tamaoki; H Shirakawa; S Miyazaki; A Nagai
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, AMER LUNG ASSOC, 19, 5, 799-804, Nov. 1998, Peer-reviwed, Erythromycin (EM) therapy is known to decrease airway secretion in chronic inflammatory airway diseases such as diffuse panbronchiolitis. Airway secretion is regulated by intracellular Ca2+ concentration ([Ca2+](i)). To elucidate the intracellular site of action of EM in airway epithelium, we examined the effect of EM on Ca2+ dynamics in cultured bovine tracheal epithelial cells using fura-2. EM per se did not cause any change in [Ca2+](i). Adenosine triphosphate (ATP; 10(-4) M) induced a biphasic [Ca2+](i) increase, consisting of a transient response followed by a sustained response. Pretreatment of cells with EM had little effect on the ATP-induced transient Ca2+ response but substantially reduced the sustained response in a dose-dependent manner. Clarithromycin, another 14-membered ring macrolide, likewise showed the inhibitory effect, but ampicillin and cephasolin did not. Uridine triphosphate (UTP; 10(-4) M) induced a biphasic [Ca2+](i) increase similar to ATP, and the UTP-induced sustained Ca2+ response was also inhibited by EM. In Ca2+-deficient medium (1 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid [EGTA]) or in the presence of La3+, the sustained Ca2+ response disappeared, suggesting that EM may inhibit Ca2+ influx induced by P-2u purinoceptor stimulation. In single-cell Ca2+ image analysis, low concentration of ATP (10(-6) M) induced Ca2+ oscillations, which were also inhibited by EM. The disappearance of [Ca2+](i) oscillations after addition of EM was similar to that after addition of EGTA. These results suggest that EM may decrease Ca2+-dependent airway secretion by inhibiting agonist-stimulated Ca2+ influx.
Scientific journal, English

Sperm-induced local [Ca2+](i) rise separated from the Ca2+ wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin
T Mohri; S Miyazaki; H Shirakawa; S Ikegami
DEVELOPMENT, COMPANY OF BIOLOGISTS LTD, 125, 2, 293-300, Jan. 1998, An increase in intracellular Ca2+ concentration ([Ca2+](i)) at a focal plane was recorded simultaneously with sperm-egg binding and membrane current upon insemination of sea urchin Hemicentrotus pulcherrimus eggs, No change in current and [Ca2+](i) occurred in the presence of jaspisin, a novel substance that inhibits metallo-endoproteinase and sperm-egg membrane fusion (S. Ikegami, H. Kobayashi, Y. Myotoishi, S. Ohta and K. H. Kato (1994) J. Biol, Chem. 269, 23262-23267), With low doses of jaspisin, a spermatozoon first produced a step inward current (Ion) as an indication of gamete membrane fusion and then induced a local [Ca2+](i) rise at the site of sperm attachment 6-10 seconds after Ion, The sperm, however, soon detached from the egg, Increasing inward current was abruptly cut off (I-off) within 9-15 seconds and the local [Ca2+](i) rise began to decline 1-3 seconds after I-off, In most cases, no further responses or an elevation of fertilization envelope (FE) occurred, In some cases, [Ca2+](i) at the sperm attachment site increased again even after the sperm detached and triggered a Ca2+ wave which caused an activation current and FE formation, This recording of a gamete membrane-fusion-induced local [Ca2+](i) rise, separated from the Ca2+ wave, is a key phenomenon for elucidating the initial sperm stimulation of the egg at fertilization.
Scientific journal, English

Sperm-induced local [Ca2+](i) rise separated from the Ca2+ wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin
T Mohri; S Miyazaki; H Shirakawa; S Ikegami
DEVELOPMENT, COMPANY OF BIOLOGISTS LTD, 125, 2, 293-300, Jan. 1998, Peer-reviwed, An increase in intracellular Ca2+ concentration ([Ca2+](i)) at a focal plane was recorded simultaneously with sperm-egg binding and membrane current upon insemination of sea urchin Hemicentrotus pulcherrimus eggs, No change in current and [Ca2+](i) occurred in the presence of jaspisin, a novel substance that inhibits metallo-endoproteinase and sperm-egg membrane fusion (S. Ikegami, H. Kobayashi, Y. Myotoishi, S. Ohta and K. H. Kato (1994) J. Biol, Chem. 269, 23262-23267), With low doses of jaspisin, a spermatozoon first produced a step inward current (Ion) as an indication of gamete membrane fusion and then induced a local [Ca2+](i) rise at the site of sperm attachment 6-10 seconds after Ion, The sperm, however, soon detached from the egg, Increasing inward current was abruptly cut off (I-off) within 9-15 seconds and the local [Ca2+](i) rise began to decline 1-3 seconds after I-off, In most cases, no further responses or an elevation of fertilization envelope (FE) occurred, In some cases, [Ca2+](i) at the sperm attachment site increased again even after the sperm detached and triggered a Ca2+ wave which caused an activation current and FE formation, This recording of a gamete membrane-fusion-induced local [Ca2+](i) rise, separated from the Ca2+ wave, is a key phenomenon for elucidating the initial sperm stimulation of the egg at fertilization.
Scientific journal, English

High-resolution analysis of Ca²⁺ rise in hamster spermatozoa induced by solubilized zona pellucida
Shirakawa, H; Miyazaki, S
The International Symposium on the Molecular and Cell Biology of Fertilization, 65, 1998
International conference proceedings, English

マウス卵細胞室内精子注入により誘発される細胞内カルシウム増加の解析
中野義弘; 白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 18, 20-21, 1998
Research institution, Japanese

Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon
Y Nakano; H Shirakawa; N Mitsuhashi; Y Kuwabara; S Miyazaki
MOLECULAR HUMAN REPRODUCTION, OXFORD UNIV PRESS, 3, 12, 1087-1093, Dec. 1997, Oscillatory rises in intracellular Ca2+ concentration ([Ca2+](i)) are the pivotal signal in the fertilization of mammalian eggs. The spatiotemporal dynamics of [Ca2+](i) rises in mouse eggs subjected to intracytoplasmic sperm injection (ICSI) were analysed by Ca2+ imaging and compared with those subjected to in-vitro fertilization (IVF). The first Ca2+ transient occurred 15-30 min after ICSI in most eggs, and was followed by Ca2+ oscillations which lasted for at least 6 h at intervals of similar to 10 min. The pattern of Ca2+ oscillations, an initial relatively larger Ca2+ transient followed by smaller Ca2+ transients, was similar to that at fertilization. Confocal Ca2+ imaging during early Ca2+ transients showed that, in fertilized eggs, [Ca2+](i) increased in a wave which started from the sperm attachment site and propagated across the egg cytoplasm. In eggs subjected to ICSI, [Ca2+](i) increased gradually and then a Ca2+ spike was generated when [Ca2+](i) reached a certain level. The [Ca2+](i) rise occurred in the whole egg, associated with neither a wave nor significant heterogeneity between the cortical and central regions. It is suggested that cytosolic factor(s) may leak from the injected spermatozoon, diffuse slowly in the egg cytoplasm, and then cause a synchronous Ca2+ release from intracellular Ca2+ stores.
Scientific journal, English

Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon
Y Nakano; H Shirakawa; N Mitsuhashi; Y Kuwabara; S Miyazaki
MOLECULAR HUMAN REPRODUCTION, OXFORD UNIV PRESS, 3, 12, 1087-1093, Dec. 1997, Peer-reviwed, Oscillatory rises in intracellular Ca2+ concentration ([Ca2+](i)) are the pivotal signal in the fertilization of mammalian eggs. The spatiotemporal dynamics of [Ca2+](i) rises in mouse eggs subjected to intracytoplasmic sperm injection (ICSI) were analysed by Ca2+ imaging and compared with those subjected to in-vitro fertilization (IVF). The first Ca2+ transient occurred 15-30 min after ICSI in most eggs, and was followed by Ca2+ oscillations which lasted for at least 6 h at intervals of similar to 10 min. The pattern of Ca2+ oscillations, an initial relatively larger Ca2+ transient followed by smaller Ca2+ transients, was similar to that at fertilization. Confocal Ca2+ imaging during early Ca2+ transients showed that, in fertilized eggs, [Ca2+](i) increased in a wave which started from the sperm attachment site and propagated across the egg cytoplasm. In eggs subjected to ICSI, [Ca2+](i) increased gradually and then a Ca2+ spike was generated when [Ca2+](i) reached a certain level. The [Ca2+](i) rise occurred in the whole egg, associated with neither a wave nor significant heterogeneity between the cortical and central regions. It is suggested that cytosolic factor(s) may leak from the injected spermatozoon, diffuse slowly in the egg cytoplasm, and then cause a synchronous Ca2+ release from intracellular Ca2+ stores.
Scientific journal, English

Role of inositol trisphosphate and tetrakisphosphate in Ca²⁺ release and Ca²⁺ influx in hamster eggs
Shirakawa, H; Miyazaki, S
The 39th Yamada Conference, B64, 1997
International conference proceedings, English

Spatiotemporal analysis of calcium dynamics in hamster spermatozoa during acrosome reaction observed with confocal microscopy
Miyazaki, S; Shirakawa, H
C112, 1997
International conference proceedings, English

共焦点レーザー走査顕微鏡を用いたハムスター精子細胞内カルシウム増加の解析
白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 17, 18-19, 1997
Research institution, Japanese

Spatiotemporal analysis of calcium dynamics in the nucleus of hamster oocytes
H Shirakawa; S Miyazaki
JOURNAL OF PHYSIOLOGY-LONDON, CAMBRIDGE UNIV PRESS, 494, 1, 29-40, Jul. 1996, 1. Subcellular Ca2+ dynamics inside and around the nucleus of immature hamster oocytes were analysed with confocal Ca2+ imaging.
2. The ratio value between emission intensity of two injected fluorescent Ca2+ indicators, Calcium Green and Fura Red, was almost uniform over the entire oocyte, suggesting that nucleoplasmic Ca2+ concentration ([Ca2+](n)) is comparable to cytoplasmic Ca2+ concentration ([Ca2+](c)) at the resting state.
3. When Ca2+ was iontophoretically injected into the nucleoplasm or the perinuclear cytoplasm, it diffused across the nuclear envelope (NE), and perinuclear [Ca2+](c) and [Ca2+](n) reached the same level within 2 s, although the NE worked as a weak but detectable barrier for Ca2+ diffusion.
4. Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the NE through the inner membrane was not detected, even when a large amount of IP3 was delivered in close proximity to the inner nuclear membrane.
5. When an oocyte was uniformly stimulated by photolysis of caged IP3, a Ca2+ rise was initiated in the perinuclear cytoplasm. The [Ca2+](n) rise was always delayed with respect to, but rapidly equilibrated with, the [Ca2+](c) rise.
6. Clusters of the endoplasmic reticulum were located in the perinuclear cytoplasm and served as the trigger zone of IP3-induced Ca2+ release.
7. The results indicate that the [Ca2+](n) rise occurs as the consequence of the influx of Ca2+ which was released in the perinuclear cytoplasm, not Ca2+ release from NE to the nucleoplasm.
Scientific journal, English

Spatiotemporal analysis of calcium dynamics in the nucleus of hamster oocytes
H Shirakawa; S Miyazaki
JOURNAL OF PHYSIOLOGY-LONDON, CAMBRIDGE UNIV PRESS, 494, 1, 29-40, Jul. 1996, Peer-reviwed, 1. Subcellular Ca2+ dynamics inside and around the nucleus of immature hamster oocytes were analysed with confocal Ca2+ imaging.
2. The ratio value between emission intensity of two injected fluorescent Ca2+ indicators, Calcium Green and Fura Red, was almost uniform over the entire oocyte, suggesting that nucleoplasmic Ca2+ concentration ([Ca2+](n)) is comparable to cytoplasmic Ca2+ concentration ([Ca2+](c)) at the resting state.
3. When Ca2+ was iontophoretically injected into the nucleoplasm or the perinuclear cytoplasm, it diffused across the nuclear envelope (NE), and perinuclear [Ca2+](c) and [Ca2+](n) reached the same level within 2 s, although the NE worked as a weak but detectable barrier for Ca2+ diffusion.
4. Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the NE through the inner membrane was not detected, even when a large amount of IP3 was delivered in close proximity to the inner nuclear membrane.
5. When an oocyte was uniformly stimulated by photolysis of caged IP3, a Ca2+ rise was initiated in the perinuclear cytoplasm. The [Ca2+](n) rise was always delayed with respect to, but rapidly equilibrated with, the [Ca2+](c) rise.
6. Clusters of the endoplasmic reticulum were located in the perinuclear cytoplasm and served as the trigger zone of IP3-induced Ca2+ release.
7. The results indicate that the [Ca2+](n) rise occurs as the consequence of the influx of Ca2+ which was released in the perinuclear cytoplasm, not Ca2+ release from NE to the nucleoplasm.
Scientific journal, English

ハムスター卵核内カルシウム動態の解析
白川英樹; 宮崎俊一
東京女子医科大学総合研究所紀要, 16, 18-19, 1996
Research institution, Japanese

DEVELOPMENTAL-CHANGES IN THE DISTRIBUTION OF THE ENDOPLASMIC-RETICULUM AND INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS AND THE SPATIAL PATTERN OF CA2+ RELEASE DURING MATURATION OF HAMSTER OOCYTES
K SHIRAISHI; A OKADA; H SHIRAKAWA; S NAKANISHI; K MIKOSHIBA; S MIYAZAKI
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 170, 2, 594-606, Aug. 1995, During maturation of hamster oocytes, the distribution of the endoplasmic reticulum (ER) and inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) was found to change dramatically, as observed using confocal microscopy with DiI and electron microscopy for the ER and immunohistochemistry for InsP(3)Rs. In immature oocytes at the germinal vesicle (GV) stage, ER and InsP(3)Rs were located predominantly in several large masses near the surface and also in the perinuclear region near the surface. In contrast, fine ER networks and few-density InsP(3)Rs were present in the inner cytoplasm. The ER appeared to be formed as vesicles from annulate lamellae (AL) in the subcortical area. Rises in Ca2+ concentration occurred in the cytoplasm and the GV when immature oocytes were inseminated, but clear Ca2+ waves did not occur. Ca2+ rises began preferentially from the perinuclear region in response to low doses of serotonin or to uniform stimulation of InsP(3)Rs with photocleavage of caged InsP(3). Serum also induced inhomogeneous Ca2+ release, shown by nonpropagating Ca2+ rises at multiple surface sites. Between the GV stage and pro-metaphase I the number and size of the surface ER masses increased, and the AL disappeared. This quantitative ER maturation was followed by a second step, spatial maturation. After prometaphase I, surface ER masses gradually dispersed to a number of much smaller ER clusters near the surface and, together with the perinuclear mass, were incorporated into thicker ER networks, resulting in a reticular pattern of the ER with small patches of InsP(3)Rs throughout the mature egg. The ER shifted to the peripheral surface with apposition to cortical granules. These developmental changes in ER Ca2+ stores may account, at least partly, for the acquisition of the ability of an egg to undergo normal fertilization. (C) 1995 Academic Press, Inc.
Scientific journal, English

DEVELOPMENTAL-CHANGES IN THE DISTRIBUTION OF THE ENDOPLASMIC-RETICULUM AND INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS AND THE SPATIAL PATTERN OF CA2+ RELEASE DURING MATURATION OF HAMSTER OOCYTES
K SHIRAISHI; A OKADA; H SHIRAKAWA; S NAKANISHI; K MIKOSHIBA; S MIYAZAKI
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 170, 2, 594-606, Aug. 1995, Peer-reviwed, During maturation of hamster oocytes, the distribution of the endoplasmic reticulum (ER) and inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) was found to change dramatically, as observed using confocal microscopy with DiI and electron microscopy for the ER and immunohistochemistry for InsP(3)Rs. In immature oocytes at the germinal vesicle (GV) stage, ER and InsP(3)Rs were located predominantly in several large masses near the surface and also in the perinuclear region near the surface. In contrast, fine ER networks and few-density InsP(3)Rs were present in the inner cytoplasm. The ER appeared to be formed as vesicles from annulate lamellae (AL) in the subcortical area. Rises in Ca2+ concentration occurred in the cytoplasm and the GV when immature oocytes were inseminated, but clear Ca2+ waves did not occur. Ca2+ rises began preferentially from the perinuclear region in response to low doses of serotonin or to uniform stimulation of InsP(3)Rs with photocleavage of caged InsP(3). Serum also induced inhomogeneous Ca2+ release, shown by nonpropagating Ca2+ rises at multiple surface sites. Between the GV stage and pro-metaphase I the number and size of the surface ER masses increased, and the AL disappeared. This quantitative ER maturation was followed by a second step, spatial maturation. After prometaphase I, surface ER masses gradually dispersed to a number of much smaller ER clusters near the surface and, together with the perinuclear mass, were incorporated into thicker ER networks, resulting in a reticular pattern of the ER with small patches of InsP(3)Rs throughout the mature egg. The ER shifted to the peripheral surface with apposition to cortical granules. These developmental changes in ER Ca2+ stores may account, at least partly, for the acquisition of the ability of an egg to undergo normal fertilization. (C) 1995 Academic Press, Inc.
Scientific journal, English

EVIDENCE FOR INOSITOL TETRAKISPHOSPHATE-ACTIVATED CA2+ INFLUX PATHWAY REFILLING INOSITOL TRISPHOSPHATE-SENSITIVE CA2+ STORES IN HAMSTER EGGS
H SHIRAKAWA; S MIYAZAKI
CELL CALCIUM, CHURCHILL LIVINGSTONE, 17, 1, 1-13, Jan. 1995, To identify the Ca2+ influx pathway responsible for maintaining Ca2+ oscillations in hamster eggs, changes in intracellular Ca2+ concentration ([Ca2+](i)) were recorded using the Fura-2 fluorescent imaging technique during iontophoretic injection of inositol phosphates under voltage clamp. Both inositol 1,4,5-trisphosphate (InsP(3)) and 1,3,4,5-tetrakisphosphate (InsP(4)) caused repetitive Ca2+ transients when injected continuously into eggs, although the latter was much less effective. These Ca2+ transients were inhibited by the monoclonal antibody 18A10 to the InsP(3) receptor/Ca2+ channel. In Ca2+-free medium, InsP(4)-induced Ca2+ transients were absent or much less frequent than in normal medium. A small but persistent increase in [Ca2+](i) during InsP(4) injection was revealed when Ca2+ uptake into InsP(3)-sensitive Ca2+ stores was suppressed by thapsigargin. This Ca2+ rise is due to Ca2+ entry, but not Ca2+ release, because it was: (i) increased by raising the extracellular Ca2+ concentration and abolished in Ca2+-free medium; (ii) larger at more negative membrane potentials which provide greater electrical driving force for Ca2+ entry; and (iii) not affected by 18A10. A moderate dose of InsP(3) did not cause substantial Ca2+ entry, as tested in thapsigargin- and 18A10-treated eggs. InsP(4) facilitated the restoration of Ca2+ stores after Ca2+ releases induced by pulsatile InsP(3) injections. Thus, we obtained evidence for a Ca2+ influx pathway activated by InsP(4) which provides Ca2+ to refill InsP(3)-sensitive Ca2+ stores in intact cells.
Scientific journal, English

EVIDENCE FOR INOSITOL TETRAKISPHOSPHATE-ACTIVATED CA2+ INFLUX PATHWAY REFILLING INOSITOL TRISPHOSPHATE-SENSITIVE CA2+ STORES IN HAMSTER EGGS
H SHIRAKAWA; S MIYAZAKI
CELL CALCIUM, CHURCHILL LIVINGSTONE, 17, 1, 1-13, Jan. 1995, Peer-reviwed, To identify the Ca2+ influx pathway responsible for maintaining Ca2+ oscillations in hamster eggs, changes in intracellular Ca2+ concentration ([Ca2+](i)) were recorded using the Fura-2 fluorescent imaging technique during iontophoretic injection of inositol phosphates under voltage clamp. Both inositol 1,4,5-trisphosphate (InsP(3)) and 1,3,4,5-tetrakisphosphate (InsP(4)) caused repetitive Ca2+ transients when injected continuously into eggs, although the latter was much less effective. These Ca2+ transients were inhibited by the monoclonal antibody 18A10 to the InsP(3) receptor/Ca2+ channel. In Ca2+-free medium, InsP(4)-induced Ca2+ transients were absent or much less frequent than in normal medium. A small but persistent increase in [Ca2+](i) during InsP(4) injection was revealed when Ca2+ uptake into InsP(3)-sensitive Ca2+ stores was suppressed by thapsigargin. This Ca2+ rise is due to Ca2+ entry, but not Ca2+ release, because it was: (i) increased by raising the extracellular Ca2+ concentration and abolished in Ca2+-free medium; (ii) larger at more negative membrane potentials which provide greater electrical driving force for Ca2+ entry; and (iii) not affected by 18A10. A moderate dose of InsP(3) did not cause substantial Ca2+ entry, as tested in thapsigargin- and 18A10-treated eggs. InsP(4) facilitated the restoration of Ca2+ stores after Ca2+ releases induced by pulsatile InsP(3) injections. Thus, we obtained evidence for a Ca2+ influx pathway activated by InsP(4) which provides Ca2+ to refill InsP(3)-sensitive Ca2+ stores in intact cells.
Scientific journal, English

ハムスター未成熟卵でのIP₃依存性カルシウム遊離機構の局在とその卵成熟に伴う変化
白川英樹; 白石浩一; 宮崎俊一
東京女子医科大学総合研究所紀要, 15, 25-26, 1995
Research institution, Japanese

Sperm-egg fusion in the sea urchin is blocked in Mg2+-free seawater
Hideo Mohri; Yukihisa Hamamchi; Miyako S. Hamasruchi; Kiyoshi Sano; Hideki Shirakawa; Ken Nakada; Shunichi Miyazaki
Zygote, 2, 2, 149-157, 1994, Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al. Dev. Growth Differ. 22, 531-41,1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+concentration of the egg was observed after insemination in the absence of Mg2, although both could be observed after the addition of Mg2. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane. © 1994, Cambridge University Press. All rights reserved.
Scientific journal, English

精子ー卵相互作用のシグナル伝達メカニズム
宮崎俊一; 白川英樹
生体の科学, 金原一郎記念医学医療振興財団, 45, 1, 79-90, 1994
Japanese

共焦点レーザー顕微鏡 その原理と応用
白川英樹; 宮崎俊一
東京女子医科大学雑誌, 東京女子医科大学学会, 64, 12, 1043-1048, 1994, Peer-reviwed
Japanese

細胞内Ca²⁺を見る 「電子顕微鏡基礎技術と応用1994～物質のナノ局在解析～」
学際企画, 190-199, 1994

Sperm-egg fusion in the sea urchin is blocked in Mg2+-free seawater
Hideo Mohri; Yukihisa Hamamchi; Miyako S. Hamasruchi; Kiyoshi Sano; Hideki Shirakawa; Ken Nakada; Shunichi Miyazaki
Zygote, 2, 2, 149-157, 1994, Peer-reviwed, Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al. Dev. Growth Differ. 22, 531-41,1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+concentration of the egg was observed after insemination in the absence of Mg2, although both could be observed after the addition of Mg2. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane. © 1994, Cambridge University Press. All rights reserved.
Scientific journal, English

ハムスター卵受精時のカルシウム動態の解析
宮崎俊一; 白石浩一; 白川英樹
東京女子医科大学総合研究所紀要, 14, 20-21, 1994
Research institution, Japanese

ESSENTIAL ROLE OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR/CA2+ RELEASE CHANNEL IN CA2+ WAVES AND CA2+ OSCILLATIONS AT FERTILIZATION OF MAMMALIAN EGGS
S MIYAZAKI; H SHIRAKAWA; K NAKADA; Y HONDA
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC ELSEVIER SCIENCE, 158, 1, 62-78, Jul. 1993
English

DEVELOPMENT OF INOSITOL TRISPHOSPHATE-INDUCED CALCIUM RELEASE MECHANISM DURING MATURATION OF HAMSTER OOCYTES
T FUJIWARA; K NAKADA; H SHIRAKAWA; S MIYAZAKI
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 156, 1, 69-79, Mar. 1993
Scientific journal, English

DEVELOPMENT OF INOSITOL TRISPHOSPHATE-INDUCED CALCIUM RELEASE MECHANISM DURING MATURATION OF HAMSTER OOCYTES
T FUJIWARA; K NAKADA; H SHIRAKAWA; S MIYAZAKI
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 156, 1, 69-79, Mar. 1993, Peer-reviwed
Scientific journal, English

CA2+-INDUCED CA2+ RELEASE MEDIATED BY THE IP3 RECEPTOR IS RESPONSIBLE FOR CA2+ WAVES AND CA2+ OSCILLATIONS AT FERTILIZATION OF MAMMALIAN EGGS
S MIYAZAKI; H SHIRAKAWA
BIOMEDICAL RESEARCH-TOKYO, BIOMEDICAL RESEARCH PRESS LTD, 14, Suppl2, 35-38, 1993, Ca2+ waves and Ca2+ oscillations occur at fertilization of hamster eggs, and a regenerative propagating Ca2+ release is induced by injection of either IP3 or Ca2+ in unfertilized eggs. All these Ca2+ responses were inhibited by a monoclonal antibody, 18A10, against the inositol 1,4,5-trisphosphate receptor (IP3R), whereas ryanodine receptor (RyR) mediated Ca2+ release was not detected. Findings in hamster eggs support the view that Ca2+-induced Ca2+ release mediated by IP3R but not RyR is caused by the sensitizing effect of Ca2+ on IP3R and is responsible for the spatiotemporal pattern of Ca2+ signaling. In addition, Ca2+ influx from outside the cell is necessary to maintain Ca2+ oscillations by refilling the stores. As a candidate of the Ca2+ influx pathway, a small but sustained Ca2+ rise was detected following injection of inositol 1,3,4,5-tetrakisphosphate (IP4) in thapsigargin-treated hamster eggs.
Scientific journal, English

CA2+-INDUCED CA2+ RELEASE MEDIATED BY THE IP3 RECEPTOR IS RESPONSIBLE FOR CA2+ WAVES AND CA2+ OSCILLATIONS AT FERTILIZATION OF MAMMALIAN EGGS
S MIYAZAKI; H SHIRAKAWA
BIOMEDICAL RESEARCH-TOKYO, BIOMEDICAL RESEARCH PRESS LTD, 14, 35-38, 1993, Peer-reviwed, Ca2+ waves and Ca2+ oscillations occur at fertilization of hamster eggs, and a regenerative propagating Ca2+ release is induced by injection of either IP3 or Ca2+ in unfertilized eggs. All these Ca2+ responses were inhibited by a monoclonal antibody, 18A10, against the inositol 1,4,5-trisphosphate receptor (IP3R), whereas ryanodine receptor (RyR) mediated Ca2+ release was not detected. Findings in hamster eggs support the view that Ca2+-induced Ca2+ release mediated by IP3R but not RyR is caused by the sensitizing effect of Ca2+ on IP3R and is responsible for the spatiotemporal pattern of Ca2+ signaling. In addition, Ca2+ influx from outside the cell is necessary to maintain Ca2+ oscillations by refilling the stores. As a candidate of the Ca2+ influx pathway, a small but sustained Ca2+ rise was detected following injection of inositol 1,3,4,5-tetrakisphosphate (IP4) in thapsigargin-treated hamster eggs.
Scientific journal, English

Regulation of calcium influx pathway by inositol 1,3,4,5-tetrakisphosphate in hamster eggs
Shirakawa, H; Miyazaki, S
The 32nd International Congress of Physiological Sciences, Thu102, 1993
International conference proceedings, English

Confocal Ca²⁺ imaging of hamster oocytes following fertilization or IP₃ injection
Shirakawa, H; Miyazaki, S
Gordon Research Conferences, C298, 1993
International conference proceedings, English

ANTIBODY TO THE INOSITOL TRISPHOSPHATE RECEPTOR BLOCKS THIMEROSAL-ENHANCED CA2+-INDUCED CA2+ RELEASE AND CA2+ OSCILLATIONS IN HAMSTER EGGS
S MIYAZAKI; H SHIRAKAWA; K NAKADA; Y HONDA; M YUZAKI; S NAKADE; K MIKOSHIBA
FEBS LETTERS, ELSEVIER SCIENCE BV, 309, 2, 180-184, Sep. 1992, The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP, receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca2+-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.
Scientific journal, English

EVIDENCE THAT METALLOENDOPROTEASES ARE INVOLVED IN GAMETE FUSION OF CIONA-INTESTINALIS, ASCIDIA
R DESANTIS; H SHIRAKAWA; K NAKADA; S MIYAZAKI; M HOSHI; R MARINO; MR PINTO
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 153, 1, 165-171, Sep. 1992
Scientific journal, English

ANTIBODY TO THE INOSITOL TRISPHOSPHATE RECEPTOR BLOCKS THIMEROSAL-ENHANCED CA2+-INDUCED CA2+ RELEASE AND CA2+ OSCILLATIONS IN HAMSTER EGGS
S MIYAZAKI; H SHIRAKAWA; K NAKADA; Y HONDA; M YUZAKI; S NAKADE; K MIKOSHIBA
FEBS LETTERS, ELSEVIER SCIENCE BV, 309, 2, 180-184, Sep. 1992, Peer-reviwed, The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP, receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca2+-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.
Scientific journal, English

EVIDENCE THAT METALLOENDOPROTEASES ARE INVOLVED IN GAMETE FUSION OF CIONA-INTESTINALIS, ASCIDIA
R DESANTIS; H SHIRAKAWA; K NAKADA; S MIYAZAKI; M HOSHI; R MARINO; MR PINTO
DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 153, 1, 165-171, Sep. 1992, Peer-reviwed
Scientific journal, English

BLOCK OF CA2+ WAVE AND CA2+ OSCILLATION BY ANTIBODY TO THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN FERTILIZED HAMSTER EGGS
S MIYAZAKI; M YUZAKI; K NAKADA; H SHIRAKAWA; S NAKANISHI; S NAKADE; K MIKOSHIBA
SCIENCE, AMER ASSOC ADVANCEMENT SCIENCE, 257, 5067, 251-255, Jul. 1992, The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (IICR) and Ca2+-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both IICR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2 release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca2+-sensitized IICR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.
Scientific journal, English

Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs
Shun-Ichi Miyazaki; Michisuke Yuzaki; Ken Nakada; Hideki Shirakawa; Setsuko Nakanishi; Shinji Nakade; Katsuhiko Mikoshiba
Science, 257, 5067, 251-255, 1992, Peer-reviwed, The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (NCR) and Ca2+-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both NCR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2 release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca 2+-sensitized NCR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.
Scientific journal, English

In vivo環境下におけるマウス骨格筋ミトコンドリア内Ca²⁺動態の可視化—In vivo visualization of mitochondrial Ca²⁺ dynamics in mouse skeletal muscle during contractions
田中 嘉法; 田渕 絢香; 白川 英樹; 狩野 豊
東京 : 北隆館, Jan. 2024, アグリバイオ = Agricultural biotechnology, 8, 1, 60-64, Japanese, 2432-5511, AA12770259

In vivo環境下におけるマウス骨格筋ミトコンドリア内Ca²⁺動態の可視化—In vivo visualization of mitochondrial Ca²⁺ dynamics in mouse skeletal muscle during contractions
田中 嘉法; 田渕 絢香; 白川 英樹; 狩野 豊
東京 : ニューサイエンス社, Apr. 2023, 細胞, 55, 5, 335-338, Japanese, 1346-7557, AA11382662

哺乳類卵活性化精子蛋白質候補ホスホリパーゼCゼータ細胞内動態および機能解析
ITO MASAHIKO; SHIKANO TOMOHIDE; SHIRAKAWA HIDEKI; AWAJI TAKEO; MIYAZAKI SHUN'ICHI
東京女子医科大学総合研究所, Oct. 2007, 東京女子医科大学総合研究所紀要, 27, 21-22, Japanese, 0911-4491, 2010188584, 200902200411154883

FRET型プローブを用いた哺乳類卵細胞内IP3濃度変化の測定
SHIRAKAWA HIDEKI; ITO MASAHIKO; MIYAZAKI SHUN'ICHI
東京女子医科大学総合研究所, Jul. 2006, 東京女子医科大学総合研究所紀要, 26, 20-21, Japanese, 0911-4491, 2010188510, 200902219578957743

マウス胚初期発生におけるホスホリパーゼCゼーターの核移行
曽根 淑恵; 熊切 順; 武内 裕之; 木下 勝之; 伊藤 昌彦; 白川 英樹; 鹿野 朝秀; 宮崎 俊一
(一社)日本生殖医学会, Oct. 2005, 日本不妊学会雑誌, 50, 4, 511-511, Japanese, 0029-0629, 2006063756

卵活性化精子因子候補PLC‐ζのマウスはい初期発生における核移行能の解析
伊藤昌彦; 曽根淑恵; 白川英樹; 鹿野朝秀; 宮崎俊一
May 2005, 日本発生生物学会大会発表要旨集, 38th, 198, Japanese, 200902280626774711

多変量解析的手法による蛍光スペクトル成分分離法とその細胞生理学的実験への応用
白川英樹
2004, 日本生理学会雑誌, 66, 12, 381-390, Japanese, Introduction other

精子ー卵相互作用のシグナル伝達メカニズム
宮崎俊一; 白川英樹
金原一郎記念医学医療振興財団, 1994, 生体の科学, 45, 1, 79-90, Japanese, Introduction other, 0370-9531, 40002063003, AN10360633

共焦点レーザー顕微鏡 その原理と応用
白川英樹; 宮崎俊一
1994, 東京女子医科大学雑誌, 64, 1043-1048, Japanese, Peer-reviwed, Introduction other

ESSENTIAL ROLE OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR/CA2+ RELEASE CHANNEL IN CA2+ WAVES AND CA2+ OSCILLATIONS AT FERTILIZATION OF MAMMALIAN EGGS
S MIYAZAKI; H SHIRAKAWA; K NAKADA; Y HONDA
ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 1993, DEVELOPMENTAL BIOLOGY, 158, 1, 62-78, English, Book review, 0012-1606, 1095-564X, WOS:A1993LM14800005

細胞内カルシウム実験プロトコール
白川英樹; 宮崎俊一
Japanese, Joint work, 卵細胞のカルシウム：ハムスター, 羊土社, 1996

電子顕微鏡基礎技術と応用～物質のナノ局在解析～
白川英樹; 宮崎俊一
Japanese, Joint work, 細胞内Ca²⁺をみる, 学際企画, 1994

Biology of Germ Lines in Animals and Man
Miyazaki, S; Nakada, K; Shirakawa, H
English, Joint work, Signal transduction of gamete interaction and intracellular calcium release at fertilization of mammalina eggs, Japan Scientific Societies Press, 1993

Calcium-induced reorganization of cortical actin cytoskeleton in mammalian oocytes at fertilization
Hideki Shirakawa; Yumiya Kato; Ryo Yonekura
Poster presentation, English, 第101回日本生理学会大会
30 Mar. 2024
28 Mar. 2024- 30 Mar. 2024

In vivo mitochondrial Ca²⁺ dynamics during tetanic muscle contraction in male and female mice
Yoshinori Tanaka; Ayaka Tabuchi; Hideki Shirakawa; Yutaka Kano
English, 第100回日本生理学会大会
16 Mar. 2023

Regulation of cortical actin dynamics and cytoplasmic flow by intracellular Ca²⁺ in mouse oocytes
Shirakawa, H; Yonekura, R; Kondo, K
English, 第100回日本生理学会大会
15 Mar. 2023

In vivo環境下における筋収縮負荷時のマウスミトコンドリア内Ca²⁺動態の可視化
田中 嘉法; 田渕 絢香; 白川 英樹; 狩野 豊
第76回日本体力医学会
21 Sep. 2022

The organization and dynamics of cortical actin cytoskeleton regulated by cytosolic Ca²⁺ in mammalian eggs.
Shirakawa, H; Kondo, K; Muratani, H
Poster presentation, English, 第99回日本生理学会合同大会, Domestic conference
16 Mar. 2022

The organization and dynamics of cortical actin cytoskeleton regulated by cytosolic Ca²⁺ in mammalian eggs.resumption in mammalian eggs
Shirakawa, H; Kondo, K; Muratani, H
Poster presentation, English, 第99回日本生理学会合同大会, Domestic conference
16 Mar. 2022

伸張性収縮後の筋細胞内カルシウムイオン時空間変化と根損傷の関係
田渕絢香; 田中嘉法; 高木領; 白川英樹; 狩野豊
第76回日本体力医学会
17 Sep. 2021

Repetitive Ca²⁺ increases coordinate the reorganization of cortical actin cytoskeleton and meiotic resumption in mammalian eggs
Shirakawa, H; Kondo, K
Poster presentation, English, 第126回日本解剖学会第98回日本生理学会合同大会, Domestic conference
30 Mar. 2021

Luminal Ca²⁺ dynamics in the endoplasmic reticulum during Ca²⁺ oscillations in mouse eggs analyzed using a fluorescent probe with improved subcellular localization
Kikuchi, T; Yokoyama, T; Shirakawa, H
Poster presentation, English, 第126回日本解剖学会第98回日本生理学会合同大会, Domestic conference
28 Mar. 2021

運動誘発性の損傷骨格筋に特徴的な細胞内カルシウムイオン変動パターン
田渕絢香; 田中嘉法; 高木領; 白川英樹; 狩野豊
第75回日本体力医学会
24 Sep. 2020

Differential regulation of cortical actin cytoskeleton by intracellular calcium in mouse eggs
Shirakawa, H; Arakawa, S; Yoshida, T
Poster presentation, English, 第97回日本生理学会, Domestic conference
18 Mar. 2020

Improvement of genetically encoded probe to measure Ca²⁺ dynamics in subcellular compartments
Murooka, N; Kikuchi, T; Shirakawa, H
Poster presentation, English, The 9th Federation of the Asian and Oceanian Physiological Societies Congress
30 Mar. 2019

Calcium-dependent regulation of cortical actin filaments in mouse eggs
Arakawa, S; Yoshida, T; Shirakawa, H
Poster presentation, English, The 9th Federation of the Asian and Oceanian Physiological Societies Congress
29 Mar. 2019

損傷再生過程における細胞内カルシウムイオンダイナミクスとmTOR系の変化
田渕絢香; 白川英樹; 杉浦崇夫; 狩野豊
第73回日本体力学会
Sep. 2018

Manipulating intracellular concentration of inositol trisphosphate by photoactivatable enzymes
Enokida, Y; Yamada, N; Yanagisawa, R; Shirakawa, H
Poster presentation, English, 第95回日本生理学会, Domestic conference
28 Mar. 2018

Dynamic reconstruction of Golgi apparatus during mammalian oocyte maturation
Iguchi, T; Shirakawa, H
Poster presentation, English, 第95回日本生理学会, Domestic conference
28 Mar. 2018

Role of luminal Ca²⁺ binding proteins in the pattern formation of Ca²⁺ oscillations in mammalian eggs
Murata, T; Kikuchi, T; Shirakawa, H
Poster presentation, English, 第94回日本生理学会, Domestic conference
30 Mar. 2017

Functional requirements of interdomain interactions for the enzymatic activity of PLCzeta
Yamada, N; Tsuda, T; Shirakawa, H
Poster presentation, English, The 22nd International Congress of Zoology, International conference
18 Nov. 2016

Calcium oscillation-dependent dynamics of cortical actin filaments in mouse eggs
Yoshida, T; Shirakawa, H
Poster presentation, English, The 22nd International Congress of Zoology, International conference
18 Nov. 2016

Mechanism for the generation of fast and slow Ca²⁺ oscillations in mouse eggs
Hideki Shirakawa; Takashi Kikuchi; Noriyuki Yamada; Takuya Tsuda
Invited oral presentation, English, Oocyte Maturation and Fertilization Meeting IV, International conference
17 Jun. 2015

Measurement and analysis of changes in the ER Ca²⁺ concentration during Ca²⁺ oscillations in mouse eggs
Takashi Kikuchi; Takasuke Murata; Hideki Shirakawa
Poster presentation, English, Oocyte Maturation and Fertilization Meeting IV, International conference
17 Jun. 2015

Mechanism for the generation of fast and slow Ca²⁺ oscillations in mouse eggs.
Shirakawa, H; Kikuchi, T; Yamada, Y; Tsuda, T
Invited oral presentation, English, Oocyte Maturation and Fertilization Meeting IV, Invited, International conference
17 Jun. 2015

Measurement and analysis of changes in the ER Ca²⁺ concentrations during Ca²⁺ oscillations in mouse eggs.
Kikuchi, T; Murata, T; Shirakawa, H
Poster presentation, English, Oocyte Maturation and Fertilization Meeting IV, International conference
17 Jun. 2015

Analysis of interdomain interactions in PLCzeta by the combined expression of split mutants
Tsuda, T; Yamada, R; Kawamoto, R; Shirakawa, H
Poster presentation, English, 第92回日本生理学会
21 Mar. 2015

Analysis of changes in the Ca²⁺ concentration in the endoplasmic reticulum during Ca²⁺ oscillations in mammalian eggs
Kikuchi, T; Murata, T; Shirakawa, H
Poster presentation, English, 第92回日本生理学会
21 Mar. 2015

Analysis of biphasic Ca²⁺ uptake by the endoplasmic reticulum during Ca²⁺ oscillations in mouse eggs
Kikuchi, T; Shirakawa, H
Poster presentation, English, 第91回日本生理学会
18 Mar. 2014

Numerical simulation of fast and slow Ca²⁺ oscillations in fertilized mouse eggs.
Shirakawa H; Tan M
Poster presentation, English, 第91回日本生理学会
18 Mar. 2014

Measurement of Ca²⁺ in the endoplasmic reticulum during Ca²⁺ oscillations in mammalian eggs.
Kikuchi, T; Takahashi, T; Shirakawa, H
Oral presentation, Japanese, J. Physiol. Sci.
Mar. 2013

On the role of STIM/Orai pathway in Ca²⁺ entry during Ca²⁺ oscillations in fertilized mouse eggs.
Takahashi, T; Kidokoro, Y; Shirakawa, H
Oral presentation, Japanese, J. Physiol. Sci.
Mar. 2012

Visualization of intracellular IP₄ dynamics using a novel fluorescent probe
Tanaka, S; Takahashi, T; Shirakawa, H
Oral presentation, Japanese, J. Physiol. Sci.
Mar. 2012

On the functional role of STIM/Orai proteins in the regulation of intracellular Ca²⁺ in mouse eggs.
Kidokoro, Y; Shirakawa, H
Oral presentation, English, Journal of Physiological Sciences
Mar. 2011

Time-lapse observation of cytoplasmic actin filament in mouse eggs during fertilization.
Kumakura, Y; Manabe, S; Shirakawa, H
Oral presentation, English, Journal of Physiological Sciences
Mar. 2011

Pharmacological characterization of store-operated Ca²⁺ entry in mouse eggs.
Takahashi, T; Shirakawa, H
Oral presentation, English, Journal of Physiological Sciences
Mar. 2011

ユートロフィンを用いたマウス卵受精時の細胞内F-アクチンの経時的観察および解析
熊倉裕紀; 白川英樹
Oral presentation, Japanese, 日本動物学会,第８２回大会
2011

細胞内イノシトール四リン酸動態可視化のための蛍光プローブの開発
田中理子; 高橋徹; 白川英樹
Oral presentation, Japanese, 日本動物学会,第８２回大会
2011

バイオサイエンスにおける光技術の応用
白川英樹
Invited oral presentation, Japanese, 第１７回レーザー夏の学校, レーザー夏の学校2010実行委員会
Aug. 2010

Evaluation and application of novel method for expression of extrinsic proteins in mouse oocytes using RNA with oligo(A) repeats.
Shirakawa, H; Kidokoro, Y; Hatanaka, T; Takahashi, T
Oral presentation, English, Journal of Physiological Sciences
2010

マウス受精卵におけるCa²⁺オシレーション発生時のCa²⁺流入の解析
高橋徹; 畑中貴之; 原悠輔; 白川英樹
Oral presentation, Japanese, 日本動物学会,第８０回大会
Sep. 2009

マウス卵におけるSTIM/Oraiタンパク質による細胞内Ca²⁺濃度調節
木所佑介; 日高裕華; 白川英樹
Oral presentation, Japanese, 日本動物学会,第８０回大会
Sep. 2009

哺乳類受精時の細胞内Ca²⁺振動の発生・維持に対するミトコンドリアの関与
今井修; 宮川薫; 熊倉裕紀; 白川英樹
Oral presentation, Japanese, 日本動物学会,第８０回大会
Sep. 2009

哺乳類受精時のCa²⁺反応におけるCa²⁺流入の機能的な関与の検証
高橋徹; 白川英樹
Oral presentation, Japanese, 日本動物学会,日本動物学会第７９回大会
2008

Pharmacological characterization of Ca²⁺ entry pathway during Ca²⁺ oscillations in fertilized mouse eggs.
Takahashi, T; Shirakawa, H
Oral presentation, English, Journal of Physiological Sciences
2008

非翻訳領域を付加したRNAを用いたマウス卵での外来タンパク質発現方法の検討
高橋篤; 白川英樹
Oral presentation, Japanese, 日本動物学会,日本動物学会第７８回大会
2007

Analysis of IP₃ dynamics during the intracellular Ca²⁺ oscillations in mammalina eggs.
Shirakawa, H; Sato, M; Umezawa, Y; Miyazaki, S
Oral presentation, English, Journal of Physiological Sciences,第８５回日本生理学会大会
2006

Physiological applications of blind spectral unmixing method based on EEM fluorescence imaging and multivariate analysis
Shirakwa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2005

The role of EF-hand domains in the enzymatic activity and calcium oscillation-inducing activity of phospholipase C-zeta
Kouchi, Z; Shikano, T; Shirakawa, H; Ito, M; Fukami, K; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2005

Nuclear translocation of phospholipase C-zeta during embryonic development of the mouse
Sone, Y; Ito, M; Shirakawa, H; Shikano, T; Kinoshita, K; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2005

Application of blind spectral decomposition method to the measurement of membrane potential with voltage-sensitive dyes
Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2004

Phospholipase C-zeta has the ability of Ca²⁺ oscillation induction and nuclear translocation in mouse eggs
Yoda, A; Oda, S; Shikano, T; Kouchi, Z; Awaji, T; Shirakawa, H; Kinoshita, K; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2004

フォスフォリパーゼＣゼータの特性とCa²⁺オシレーション誘発
宮崎俊一; 河内全; 尾田正二; 依田綾子; 淡路健雄; 白川英樹; 鹿野朝秀
Invited oral presentation, Japanese, 第８１回大会, 日本生理学会
2004

多変量解析的手法による蛍光スペクトル分離法の細胞生物学的実験への応用
白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本生物物理学会,第４２回年会
2004

Multicomponent measurement based on wide-range two-dimensional fluorescence microspectroscopy
Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2003

広波長域２次元蛍光スペクトル顕微測光に基づく卵細胞内因子の多成分同時解析
白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本生物物理学会,第４１回年会
2003

２次元蛍光スペクトル顕微測光による細胞内多因子同時測定
白川英樹; 宮崎俊一
Oral presentation, Japanese, 生理学研究所研究会,生理学研究所研究会「細胞内シグナル伝達機構の多角的・包括的理解」
2003

Ca²⁺/Mn²⁺ dynamics during Ca²⁺ oscillations in mouse eggs
Mohri, T; Shirakawa, H; Oda, S; Sato, M.S; Mikoshiba, K; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2001

Dual emission ratiometric measurement of intracellular calcium with visible-light excitation
Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2001

Novel GFP-based ratiometric indicators for monitoring intracelluar pH
Awaji, T; Hirasawa, A; Shirakawa, H; Tsujimoto, G; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2001

GFPを利用した定量的pHプローブの開発と応用
淡路健雄; 平澤明; 白川英樹; 辻本豪三; 宮崎俊一
Oral presentation, Japanese, 日本分子生物学会,第２４回大会
2001

Optical measurement of endoplasmic reticulum membrane potential during Ca²⁺ release
Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
2000

IP₃誘発性Ca遊離に伴う小胞体膜電位変化
白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本生物物理学会,第３８回年会
2000

精子抽出物顕微注入によるCa²⁺振動時のマウス卵のCa²⁺/Mn²⁺ダイナミクス
毛利達磨; 白川英樹; 尾田正二; 佐藤真則; 御子柴克彦; 宮崎俊一
Oral presentation, Japanese, 日本細胞生物学会,第５３回大会
2000

マウス卵のCa²⁺ oscillationにおけるCa²⁺/Mn²⁺ influx/release
毛利達磨; 白川英樹; 尾田正二; 宮崎俊一
Oral presentation, Japanese, 生理学研究所研究会,生理学研究所研究会「Ca²⁺シグナルと膜輸送体の発現および機構調節」
2000

精子因子によるCa²⁺オシレーションと卵活性化
宮崎俊一; 尾田正二; 出口竜作; 白川英樹; 鹿野朝秀; 佐藤真則; 吉友美樹; 毛利達磨
Oral presentation, Japanese, 生理学研究所研究会,生理学研究所研究会「細胞内シグナルの時・空間的制御」
1999

Spatiotemporal analysis of intracellular calcium increases in hamster sperm induced by solubilized zona pellucida
Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
1997

哺乳類精子先体反応におけるカルシウム動態の解析
白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本発生生物学会,第３０回大会
1997

The use of an agonist of inositol 1,4,5-trisphosphate receptor, adenophostin, for activation of mouse eggs injected with round spermatids
Sato, Y; Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
1997

円形精子細胞注入マウス卵の活性化法：非分解性IP₃アナログの利用
佐藤雄一; 中野義弘; 豊成由佳; 淡路正則; 竹内裕之; 三橋直樹; 桑原慶紀; 白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本産婦人科学会,第４９回学術講演会
1997

マウスICSI卵におけるカルシウム画像解析
中野義弘; 佐藤雄一; 豊成由佳; 淡路正則; 竹内裕之; 三橋直樹; 桑原慶紀; 白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本受精着床学会,第１４回学術講演会
1996

Changes in the distribution of the endoplasmic reticulum and inositol 1,4,5-trisphosphate receptors during meiotic maturation of hamster oocytes
Shiraishi, K; Shirakawa, H; Honda, Y; Okada, A; Nakanishi, S; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
1995

Spatiotemporal analysis of the increases of nuclear and cytoplasmic Ca²⁺ induced by IP₃ in hamster oocytes.
Shirakawa, H; Shiraishi, K; Miyazaki, S
Oral presentation, Japanese, Japanese Journal of Physiology
1995

ハムスター未成熟卵核でのCa²⁺動態の解析
白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本生物物理学会,第３３回年会
1995

マウス卵細胞室内精子注入法における卵細胞内カルシウムイオン画像解析
中野義弘; 佐藤雄一; 豊成由佳; 淡路正則; 竹内裕之; 三橋直樹; 桑原慶紀; 白川英樹; 宮崎俊一
Oral presentation, Japanese, 日本産婦人科学会,第４８回学術講演会
1995

Analysis of Ca²⁺ wave in hamster eggs using confocal microscopy
Shiraishi, K; Shirakawa, H; Yonda, Y; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
1994

卵細胞内Ca²⁺の画像解析と共焦点レーザー顕微鏡
白川英樹; 白石浩一; 宮崎俊一
Invited oral presentation, Japanese, 第３５回ワークショップ「卵子研究における新技術」, 哺乳類卵子学会
1994

Activation of calcium influx pathway by inositol 1,3,4,5-tetrakisphosphate in hamster eggs
Shirakawa, H; Honda, Y; Nakada, K; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
1993

Increase in sensitivity of the inositol trisphosphate receptor during maturation of hamster oocytes
Nakada, K; Fujiwarak T; Shirakawa, H; Miyazaki, S
Oral presentation, English, Japanese Journal of Physiology
1993

Monoclonal antibody to inositol 1,4,5-trisphosphate receptor blocks Ca²⁺ wave and Ca²⁺ oscillation in fertilized hamster eggs
Nakada, K; Shirakawa, H; Miyazaki, S; Yuzaki, M; Nakanishi, S; Nakade, S; Mikoshiba, K
Oral presentation, English, Japanese Journal of Physiology
1992

ハムスター卵受精時のCa waveおよびCa oscillationsはIP₃依存性Ca遊離に基づく
白川英樹; 中田健; 宮崎俊一; 柚崎通介; 中出真嗣; 御子柴克彦
Oral presentation, Japanese, 日本発生生物学会,第２５回大会
1992

受精現象におけるIP₃レセプターの役割
粂昭苑; 武藤彩; 有賀純; 岡野栄之; 宮脇敦史; 古市貞一; 白川英樹; 中田健; 宮崎俊一; 柚崎通介; Nuccitelli, R; 御子柴克彦
Oral presentation, Japanese, 日本分子生物学会,年会
1992

ハムスター卵受精時の細胞内カルシウムイオン遊離機構
中田健; 白川英樹; 宮崎俊一
Oral presentation, Japanese, 家畜繁殖学会,第８２回大会
1992

日本生理学会

日本発生生物学会

日本生物物理学会

Biophysical Society（アメリカ）

日本動物学会

生細胞で二次メッセンジャー濃度を自由自在に制御する手法の開発と応用
Challenging Research (Exploratory), Principal investigator, 22K19401
Apr. 2022 - Mar. 2025

Development of IP3 clamp method
Shirakawa Hideki
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, The University of Electro-Communications, Grant-in-Aid for Challenging Exploratory Research, Protein-based molecular tools were developed, aiming to establish the IP3 clamp method, a novel technique to control the intracellular IP3 concentrations in living cells by light. It was shown that the combination of human phospholipase C and plant photoreceptor proteins could give the photoactivatability to an IP3-producing enzyme. We also developed fluorescent IP3 probes that have favorable excitation/emission wavelengths to be used with photoactivatable enzymes in the IP3 clamp system., 15K15027
01 Apr. 2015 - 31 Mar. 2017

Functional coupling between Ca^<2+> influx anf Ca^<2+> release during Ca^<2+> oscillatons in fertilized mammalian eggs
SHIRAKAWA Hideki
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), The University of Electro-Communications, Grant-in-Aid for Scientific Research (C), To elucidate the regulatory mechanism for Ca^<2+> influx pathway in fertilized mammalian eggs, we conducted a series of molecular and physiological experiments to interfere the interactions between Ca^<2+> influx channels on the plasma membrane, TRPC and Orai, and proteins on the ER membrane, STIM. The results suggested that, although none of these proteins participate functionally in the maintenance of the Ca^<2+> oscillations by activating Ca^<2+> influx, STIM1 may have an novel role other than as an activator of store-operated Ca^<2+> entry., 21590229
2009 - 2011

Advanced Imaging Method of Light Source Distributions in Biological Tissues
YAMADA Yukio; OKAWA Shinpei; SHIRAKAWA Hideki; HOSHI Yoko; TANIKAWA Yukari; GAO Feng
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), The University of Electro-Communications, Grant-in-Aid for Scientific Research (B), 新薬開発などにおけるコストと犠牲動物の低減を目指し,分子イメージング技術の一つである蛍光トモグラフィー法の高度化を目的として,画像再構成法の開発とシミュレーション,および,生体模擬試料やマウスを用いた実験を行った.画像再構成法の新しい手法を開発し,実験によりその手法を実証することができた.結果として,マウス体内に埋め込んだ蛍光物質の濃度分布に関する断層画像を得た., 19360098
2007 - 2009

生細胞分光イメージングと多変量解析法による多重蛍光の定量的分離解析システムの開発
白川 英樹
日本学術振興会, 科学研究費助成事業 特定領域研究, 電気通信大学, 特定領域研究, 多数の蛍光分子種の生細胞・組織内での時空間的動態を定量的に測定できるイメージング手法として、Excitation-Emission Matrix (EEM)イメージングとParallel Factor Analysis (PARAFAC)によるスペクトル分離手法に基づく多重蛍光同時測定システムの開発・構築を試みた。 平成18年度には、従来型の蛍光顕微鏡をべースにして、励起波長と蛍光波長を数種類のバンドパスフィルターで切り換えて高感度ビデオカメラで記録する光学系を構築した。また、多チャンネル分光センサを搭載した共焦点顕微鏡をベースにしたシステムも併せて検討した。これらの測定系で得られたEEM画像について励起光強度やフィルタ透過率についての補正を行った後、昨年度までに開発したPARAFACに基づくスペクトル分離アルゴリズムを画像データ用に改変したものを用いて、各蛍光成分画像の分離を行った。マウス卵細胞に各種蛍光色素や蛍光タンパク質を導入したものを対象にして、上述のシステムで単一細胞レベルでのEEM画像を経時的に記録・解析した結果、いずれの系においても導入した複数の蛍光成分と内因性蛍光成分のそれぞれが正確に分離できることが確認できた。具体的には例えば、IP_3産生酵素であるPLC、細胞内カルシウムイオン、およびミトコンドリアの細胞内動態を、それぞれGFP、カルシウムプローブ、フラビン由来の自家蛍光により、同時に測定することが出来た。また、従来のLinear unmixingによる成分分離法より分離結果が正確であることも確かめられた。各成分スペクトルに関する先見情報が不要な方法なので、特に未知の成分を含む試料に関して有用なシステムであると考えられるが、現時点では分離可能な蛍光成分数が3から4個と少なく、今後更なる改良が必要である。, 18038014
2006 - 2006

Characterization and functional analysis of phospholipase C-zeta, a candidate of mammalian egg-activating sperm factor
MIYAZAKI Shunichi; ITO Masahiko; AWAJI Takeo; SHIRAKAWA Hideki; ODA Shoji
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Tokyo Women's Medical University, Grant-in-Aid for Scientific Research (B), Repetitive transient increase in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) called Ca^<2+> oscillations occur at fertilization of mammalian eggs, and trigger egg activation. Each [Ca^<2+>]_i rise is due to Ca^<2+> release from the endoplasmic reticulum mainly through inositol 1,4,5-trisphosphate (IP_3) receptors. Evidence indicates that Ca^<2+> oscillations are caused by cytosolic sperm factor driven into the ooplasm upon sperm-egg fusion. The Ca^<2+> oscillation-inducing factor is the egg-activating protein (EAP). A current strong candidate of EAP is a novel isozyme of IP_3-producing enzyme, phospholipase C-zeta (PLCζ). This study aims to characterize PLCζ and confirm whether PLCζ operates as EAP at fertilization. The following results were obtained. 1) Injection of recombinant PLCζ that was synthesized by baculovirus/Sf9 cell expression system induced Ca^<2+> oscillations in mouse eggs at low concentrations. 2) PLCζ has such a high Ca^<2+>-sensitivity of PLC activity that the enzyme can be active in resting cells at 〜100 nM [Ca^<2+>]_i, suitable for a putative EAP to be introduced into the egg at fertilization. 3) The N-terminal EF-hand domain 1 (EF1) and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca^<2+> sensitivity. 4) C2 domain has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P. 5) Ca^<2+> oscillations were induced by injection of RNA encoding PLCζ fused with a fluorescent protein "Venus". The expressed PLCζ corresponded to the estimated amount contained in a single spermatozoon. 6) Expressed PLCζtranslocated into the formed pronucleus. 7) The nuclear localization signal was located at the X-Y linker region of PLCζ molecule. A highly coordinated three-dimensional structure involving a folding in the middle is thought to be required for not only Ca^<2+> oscillation-inducing activity but also nuclear translocation ability. These results support PLCζ as a strong candidate of EAP. To confirm whether PLCζ operates as EAP at fertilization, PLCζ-knockout mice were produced, and PLCζ-transgenic mice were prepared for reserve PLCζ in the knockout mice sperm., 16390055
2004 - 2006

カルシウム遊離に伴うin situ小胞体の電気的挙動に関する光学的手法による解析
白川 英樹
日本学術振興会, 科学研究費助成事業 奨励研究(A), 東京女子医科大学, 奨励研究(A), IP_3受容体Caチャネルを介した小胞体(ER)からのCa遊離の過程を理解するためには、ER内外のCa濃度差だけでなく、電位差(=ER膜電位)をも考慮して電気化学的現象としてとらえることが重要である。本研究では、膜電位感受性蛍光色素を用いてハムスター卵細胞におけるIP_3依存性Ca遊離の際のER膜電位変化を光学的に測定する、というアプローチを行っている。ハムスター卵は、リアノジン受容体系は存在せずIP_3受容体系のみである、細胞内のERの分布が均一である、などモデル系としての利点がある。 昨年度までの研究において、(1)Stylyl系膜電位依存性蛍光色素di-18:2-ANEPPSによるER膜電位の選択的な光学的測定系を確立し、さらに(2)IP_3によるCa遊離に伴うER内陰性の向きのER膜電位変化の存在の確認、(3)Kチャネル阻害剤によるIP_3誘発性Ca遊離の部分的抑制など、Ca遊離の制御因子としてのER膜電位の機能を示唆する実験結果を得た。 本年度は、さらに以下のような成果を得、その一部をすでに発表した。 (1)膜電位との同時測定に使用可能な蛍光Ca指示薬をスクリーニングし、その中でFura Redが可視光域での1波長励起2波長蛍光のratiometricな測定に用いることができることを見いだした。 (2)多波長同時記録型の蛍光検出器を導入し、多波長励起・多波長蛍光の単一細胞顕微蛍光測定システムを構築した。これにより、自家蛍光成分を排除してより定量的な蛍光測定を行うこと、また複数種の蛍光プローブを用いて複数の細胞内因子を同時に測定することが可能になる、と期待される。 (3)Ca動態についての包括的な数値モデルの構築に向け、その予備的段階のモデルを作成しマウス卵のCaオシレーションをシミュレートした。, 12770025
2000 - 2001

哺乳類精子活性化過程で機能する二次メッセンジャーとイオンチャネルの同定
白川 英樹
日本学術振興会, 科学研究費助成事業 奨励研究(A), 東京女子医科大学, 奨励研究(A), 前年度に引き続き、主としてハムスター精子に対し先体反応誘発物質として卵透明帯を可溶化したものを投与した際の細胞内Q反応について、Ca上昇と先体胞開口分泌の時間的関係および電位依存性L型Caチャネルの関与について解析を進めた。またサイクリックヌクレオチド(cAMP、cGMP)の効果についても検討した。 [Ca上昇と先体胞開口分泌の時間的関係]可溶化卵透明帯の投与に対し、精子細胞内Ca濃度は頭部内赤道部付近から上昇が始まり約0.6秒以内に先体胞を除く頭部全体に伝播した後、2分以上にわたり高いレベルに保たれた。その間に、先体胞の蛍光強度が急激に減少する現象が観察された。これは開口分泌により先体胞内部の蛍光色素が細胞外に流出したためと考えられる。Ca上昇の開始から先体胞開口分泌開始までの平均時間は22秒であった。 [電位依存性L型Caチャネルの関与の検討]L型Caチャネル阻害剤の存在下では、透明帯によるCa反応の増加相のパターンや速度、およびピークの大きさは影響されなかったが、Ca上昇の持続時間が有意に短くなった。また、先体胞開口分泌も阻害されたことから、L型Caチャネルによって細胞内Caが長時間高濃度に維持されることが開口分泌に必要であることが示唆された。 [サイクリックヌクレオチドの効果]予備的な実験において、細胞膜透過性のサイクリックヌクレオチドのアナログ、8-br-cGrS4Pや8-br-cAMPの投与により精子内Ca上昇が観察された例があったが、ケイジドcGMP(膜透過性)をロードした精子のどの部分に紫外線照射しても、Ca上昇は誘発されなかった。サイクリックヌクレオチドと精子内Caの関係については、さらに検討が必要である。, 09770033
1997 - 1998

精子活性化の細胞内メカニズムの解明への直接的なアプローチ
宮崎 俊一; 白川 英樹
日本学術振興会, 科学研究費助成事業 萌芽的研究, 東京女子医科大学, 萌芽的研究, 前年度に引き続き、主としてハムスター精子に対し先体反応誘発物質として卵透明帯を可溶化したものを投与した際の細胞内Ca反応について、Ca上昇と先体胞開口分泌の時間的関係および電位依存性L型Caチャネルの関与について解析を進めた。またサイクリックヌクレオチド(cAMP、cGMP)の効果についても検討した。 [Ca上昇と先体胞開口分泌の時間的関係]可溶化卵透明帝の投与に対し、精子細胞内Ca濃度は頭部内赤道部付近から上昇が始まり約0.6秒以内に先体胞を除く頭部全体に伝播した後、2分以上にわたり高いレベルに保たれた。その間に、先体胞の蛍光強度が急激に減少する現象が観察された。これは開口分泌により先体胞内部の蛍光色素が細胞外に流出したためと考えられる。Ca上昇の開始から先体胞開口分泌開始までの平均時間は22秒であった。 [電位依存性L型Caチャネルの関与の検討]L型Caチャネル阻害剤の存在下では、透明帯によるCa反応の増加相のパターンや速度、およびピークの大きさは影響されなかったが、Ca上昇の持続時間が有意に短くなった。また、先体胞開口分泌も阻害されたことから、L型QCaチャネルによって細胞内Caが長時間高濃度に維持されることが開口分泌に必要であることが示唆された。 [サイクリックヌクレオチドの効果]予備的な実験において、細胞膜透過性のサイクリックヌクレオチドのアナログ、8-br-cGMPや8-br-cAMPの投与により精子内Ca上昇が観察された例があったが、ケイジドcGMP(膜透過性)をロードした精子のどの部分に紫外線照射しても、Ca上昇は誘発されなかった。サイクリックヌクレオチドと精子内Caの関係については、さらに検討が必要である。, 09878171
1997 - 1998

Identification of sperm-derived egg-activating factor and analysis of molecular mechanisms of intracellular Ca^<2+> increase at fertilization of mammalian eggs
MIYAZAKI Shunichi; AWAJI Takeo; SHIRAISHI Koichi; SHIRAKAWA Hideki; ODA Shoji
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Tokyo Women's Medical College, Grant-in-Aid for Scientific Research (B), This study was performed as a step to elucidate the molecular mechanism by which the sperm induces an increase in intracellular Ca^<2+>, the pivotal signal for egg activation at fertilization. The following results were obtainde by microinjection techniques, Ca^<2+> image analysis, and confocal laser scanning microscopy. 1)As a phenomenological observation, we established the experimental condition in which sperm-egg fusion is formed only transiently without sperm entry into the sea urchin egg in the presence of a gamete fusion inhibitor, jaspisin. A localized Ca^<2+> rise restricted at the sperm binding site was recorded, separated fron the Ca^<2+> wave which spreads throughout the egg at normal fertilization. Spatiotemporal aspects of the sprem-induced localized Ca^<2+> rise were characterized in detail. 2) Injection of a mouse spermatozoon into an egg (intracytoplsmic sperm injection, ICSI) caused repetitive transient Ca^<2+> rises (Ca^<2+> oscillalions) as seen at fertilization, starting 15 to 30 minutes after sperm injection and persisting for several hours. Each Ca^<2+> rise occurred almost synchronously in the whole egg. The results suggested that a sperm cytosolic factor leaks out from the injected spermatozoon into the egg cytoplasm and can induce Ca^<2+> oscillations, even if sperm-egg binding is bypassed by ICSI. 3) We found that injection of a precursor sperm, round spermatids without flagellum (round spermatid injection, ROSI), can not induce any Ca^<2+> response in the egg but that combined injection of a potent agonist of the inositol 1,4,5-trisphosphate (IP_3) receptor resulted in egg activation (fertilization) associated with male and female pronucleus formation. About 25% of two-cell embryos transplanted into foster mothers developed to normal offspring. The newborn infants grew up to normal adults, which reproduced normal second generations. 4)We partially purified a cytosolic protein from hamster and ascidian spermatozoa that possesses the activity of inducing Ca^<2+> oscillations when injected into hamster, mouse, and ascidian eggs. Microinjection of the sperm factor into a localized region of the egg showed that the peripheral cytoplasm is more sensitive to the sperm factor to generate Ca^<2+> increase (possibly due to Ca^<2+> release from the endoplasmic reticulum through IP_3 receptors), compared with the central region of the egg. These results suggested that, at fertilization, a sperm cytosolic factor could be introduced into the cortical region of the egg cytoplasm through cytoplasmic continuity formed between the sperm and egg, and induce Ca^<2+> release in the egg. This study provided useful information for advancing further studies in future., 08457016
1996 - 1997

Physiological Study of the Mechanism Involved in Ca^<2+> Waves and Ca^<2+> Oscillations in Fertilized Mammalian Eggs
MIYAZAKI Shunichi; ODA Shoji; SHIRAISHI Koichi; SHIRAKAWA Hideki
Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B), Tokyo Women's Medical College, Grant-in-Aid for General Scientific Research (B), An increase in intracellular calcium ion concentration (Ca) in eggs at fertilization triggers egg activation. Fertilized mammalian eggs show propagating Ca waves and repetitive Ca transients (Ca oscillations). The present study aimed to analyze the mechanism involved in the generation of these spatial and temporal Ca signals. 1. How does sperm-egg interaction generate signals leading to Ca responses in eggs? Molecules on the hamster egg plasma membrane that are suggested to mediate sperm-egg adhesion or binding were stimulated by thier ligands or antibodies, but no Ca response was not. The inhibitors of tyrosin kinases which are supposed to be associated with the membrane receptors did not block sperm-induced Ca responses. Thus evidence for the signal genetation in surface molecules was not obtained, leading us to think that cytoplas mic sperm factor (S) introduced into the egg may play more important role in the signal transduction of sperm-egg interaction. 2. IP_4-activatee Ca influx pathway. We physiologicaly identified inositol 1,3,4,5-tetrakis phosphate (IP_4)-activated Ca influx pathway in hamster eggs. The Ca influx provides Ca to refill the endoplasmic reticulum (ER) from which Ca has been released by the action of inositol 1,4,5-trisphosphate (IP_3), giving rise to repeated Ca release. 3. We observed using a confocal laser scanning microscope (CLSM) that the Ca wave in the fertilized hamster egg propagates deep in the cytoplasm, and that Ca in the nucleus also increases comparably with that in the surrouding cytoplasm. 4. The ER was stained by injected lipophilic fluorescent dye DiI and observed with CLSM.The ER was found to form a fine network throughout the egg, consistent with the distribution of IP_3 receptors. In immature oocytes the ER as well as IP_3 receptors was distributed inhomogeneously as dence patches in the surface area and aound the nucleus. Ca release induced by photocleavage of preinjected caged IP_3 occurred predominantly in the nuclear zone. These findings indicate that the redistribution of the ER and IP_3 receptors during oocyte maturation is a prerequisite factor for acquisition of the ability to undergo normal fertilization with the Ca waves., 05454141
1993 - 1994

哺乳動物卵に於ける細胞内Caイオン遊離機構の生理学的解析
宮崎 俊一; 中田 健; 白川 英樹
日本学術振興会, 科学研究費助成事業 一般研究(C), 東京女子医科大学, 一般研究(C), 細胞内カルシウムイオン(Ca)は,二次メッセンジャ-として種々の重要な細胞機能を制御する。受精卵に於ては,表層顆粒の開口分泌を誘発して複数の精子の侵入を防ぐとともに,休止していた卵を代謝的に活性化して細胞分裂をもたらす引き金として重要な機機能を果たす。Ca増加機序として細胞内Ca貯蔵部位(Caストア)からの遊離があり,イノシト-ル三リン酸(IP_3)によって誘発されるIP_3ーinduced Carelease(IICR)と,Ca自身によって誘発されるCaーinduced Ca release(CICR)が知られている。本研究では,Caストア膜上に存在するIP_3受容体に対する単クロ-ン抗体(mAb)(大阪大学蛋白研究所,御子柴教授の研究室による)がIICRを機能的にブロックするという重要な知見を得,ハムスタ-卵に於けるCa遊離機構を生理学的に解析した。予めmAbとCa指示薬fura2を卵細胞内に注入し,1〜2時間後にIP_3をマイクロピペットから電流パルスで細胞内に注入し,遊離したCaをfura2蛍光による画像解析により記録した。mAb,の一つでIP_3受容体のCaチャンネル部位附近を認識する18A10は,濃度依存性にIICRを非競合的に抑制することを明らかにした。ハムスタ-卵は受精時に精子附着部位からの伝播性のCa増加と,くり返すCa増加を示す。抗体18A10は,このCa waveとCa oscillationをともに濃度依存的にブロックすることを明らかにした。他方,Ca注入によっても伝播性のCa遊離がおこり,CICRの存在を示唆するが,これもIP_3受容体抗体でブロックされることを見出した。このように,哺乳動物卵受精には,IP_3によるCa遊離機構が一義的なメカニズムとして実際に機能しており,IP_3受容体がCa waveとCa oscillationという,空間的,時間的シグナル伝達に主要な役割を果たすことを初めて明らかにした。研究結果を論文としてまとめあげ,‘Science'誌に投稿した。, 03670044
1991 - 1991