白川 英樹
| 基盤理工学専攻 | 准教授 |
| Ⅲ類（理工系） | 准教授 |
研究者情報
経歴
研究活動情報
論文
- 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, 出版日 2023年10月30日, 査読付, 国際誌, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ R14-R27, 出版日 2022年01月01日, 査読付, 国際誌, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 4829, 出版日 2019年03月18日, 査読付
研究論文（学術雑誌）, 英語 - 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巻, 5号, 掲載ページ e13180, 出版日 2017年03月13日, 査読付, 国際誌, 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.
研究論文（学術雑誌）, 英語 - Calcium Signaling in Mammalian Eggs at Fertilization
Hideki Shirakawa; Takashi Kikuchi; Masahiko Ito
CURRENT TOPICS IN MEDICINAL CHEMISTRY, BENTHAM SCIENCE PUBL LTD, 16巻, 24号, 掲載ページ 2664-2676, 出版日 2016年, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 60-65, 出版日 2013年01月, 査読付, 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.
研究論文（学術雑誌）, 英語 - Characterization of store-operated Ca2+ entry in mouse eggs
Takahashi, T; Shirakawa, H
Comparative Physiology and Biochemistry, 28巻, Suppl号, 掲載ページ 158, 出版日 2011年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, Suppl. 1号, 掲載ページ 244-244, 出版日 2009年
研究論文（国際会議プロシーディングス）, 英語 - CHARACTERIZATION OF Ca2+ INFLUX PATHWAY ACTIVATED DURING Ca2+ OSCILLATIONS IN MOUSE EGGS
Tooru Takahashi; Hideki Shirakawa
JOURNAL OF PHYSIOLOGICAL SCIENCES, SPRINGER TOKYO, 59巻, Suppl. 1号, 掲載ページ 244-244, 出版日 2009年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 2号, 掲載ページ 781-788, 出版日 2006年06月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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", 掲載ページ 116, 出版日 2006年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 22号, 掲載ページ 21015-21021, 出版日 2005年06月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 3号, 掲載ページ 690-694, 出版日 2005年05月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 2号, 掲載ページ 245-257, 出版日 2004年04月, 査読付, 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.
研究論文（学術雑誌）, 英語 - Blind spectral decomposition of single-cell fluorescence by parallel factor analysis
H Shirakawa; S Miyazaki
BIOPHYSICAL JOURNAL, BIOPHYSICAL SOCIETY, 86巻, 3号, 掲載ページ 1739-1752, 出版日 2004年03月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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, 掲載ページ 95, 出版日 2004年
研究論文（国際会議プロシーディングス）, 英語 - 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", 掲載ページ 55, 出版日 2004年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 掲載ページ 17-18, 出版日 2004年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 掲載ページ 19-20, 出版日 2003年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 掲載ページ 23-25, 出版日 2002年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 2号, 掲載ページ 457-462, 出版日 2001年11月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 5号, 掲載ページ 311-325, 出版日 2001年05月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 掲載ページ 18-19, 出版日 2001年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 掲載ページ 407A-407A, 出版日 2000年12月
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 2号, 掲載ページ 299-313, 出版日 2000年02月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 2号, 掲載ページ 299-313, 出版日 2000年02月, 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.
研究論文（学術雑誌）, 英語 - 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", 掲載ページ P11, 出版日 2000年
研究論文（国際会議プロシーディングス）, 英語 - 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", 掲載ページ P12, 出版日 2000年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 6号, 掲載ページ L891-L899, 出版日 1999年06月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 6号, 掲載ページ L891-L899, 出版日 1999年06月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 70-78, 出版日 1999年04月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 70-78, 出版日 1999年04月, 査読付, 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.
研究論文（学術雑誌）, 英語 - Sperm-induced signaling in mouse eggs.
Miyazaki, S; Oda, S; Shirakawa, H; Mohri, T; Sato, M.S; Deguchi, R
The Susumu Hagiwara Memorial Symposium, 掲載ページ 24, 出版日 1999年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 5号, 掲載ページ 799-804, 出版日 1998年11月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 5号, 掲載ページ 799-804, 出版日 1998年11月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 2号, 掲載ページ 293-300, 出版日 1998年01月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 2号, 掲載ページ 293-300, 出版日 1998年01月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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, 掲載ページ 65, 出版日 1998年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 掲載ページ 20-21, 出版日 1998年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 12号, 掲載ページ 1087-1093, 出版日 1997年12月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 12号, 掲載ページ 1087-1093, 出版日 1997年12月, 査読付, 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.
研究論文（学術雑誌）, 英語 - Role of inositol trisphosphate and tetrakisphosphate in Ca2+ release and Ca2+ influx in hamster eggs
Shirakawa, H; Miyazaki, S
The 39th Yamada Conference, 掲載ページ B64, 出版日 1997年
研究論文（国際会議プロシーディングス）, 英語 - Spatiotemporal analysis of calcium dynamics in hamster spermatozoa during acrosome reaction observed with confocal microscopy
Miyazaki, S; Shirakawa, H
掲載ページ C112, 出版日 1997年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 掲載ページ 18-19, 出版日 1997年
研究論文（大学，研究機関等紀要）, 日本語 - Spatiotemporal analysis of calcium dynamics in the nucleus of hamster oocytes
H Shirakawa; S Miyazaki
JOURNAL OF PHYSIOLOGY-LONDON, CAMBRIDGE UNIV PRESS, 494巻, 1号, 掲載ページ 29-40, 出版日 1996年07月, 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.
研究論文（学術雑誌）, 英語 - Spatiotemporal analysis of calcium dynamics in the nucleus of hamster oocytes
H Shirakawa; S Miyazaki
JOURNAL OF PHYSIOLOGY-LONDON, CAMBRIDGE UNIV PRESS, 494巻, 1号, 掲載ページ 29-40, 出版日 1996年07月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 掲載ページ 18-19, 出版日 1996年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 2号, 掲載ページ 594-606, 出版日 1995年08月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 2号, 掲載ページ 594-606, 出版日 1995年08月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 1-13, 出版日 1995年01月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 1-13, 出版日 1995年01月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 掲載ページ 25-26, 出版日 1995年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 2号, 掲載ページ 149-157, 出版日 1994年, 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.
研究論文（学術雑誌）, 英語 - 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, 掲載ページ 190-199, 出版日 1994年 - 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巻, 2号, 掲載ページ 149-157, 出版日 1994年, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 掲載ページ 20-21, 出版日 1994年
研究論文（大学，研究機関等紀要）, 日本語 - 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巻, 1号, 掲載ページ 62-78, 出版日 1993年07月
英語 - 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巻, 1号, 掲載ページ 69-79, 出版日 1993年03月
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 69-79, 出版日 1993年03月, 査読付
研究論文（学術雑誌）, 英語 - 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巻, Suppl2号, 掲載ページ 35-38, 出版日 1993年, 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.
研究論文（学術雑誌）, 英語 - 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巻, 掲載ページ 35-38, 出版日 1993年, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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, 掲載ページ Thu102, 出版日 1993年
研究論文（国際会議プロシーディングス）, 英語 - Confocal Ca2+ imaging of hamster oocytes following fertilization or IP3 injection
Shirakawa, H; Miyazaki, S
Gordon Research Conferences, 掲載ページ C298, 出版日 1993年
研究論文（国際会議プロシーディングス）, 英語 - 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巻, 2号, 掲載ページ 180-184, 出版日 1992年09月, 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.
研究論文（学術雑誌）, 英語 - Evidence That Metalloendoproteases Are Involved in Gamate Fusion of ┣DBCiona(/)-┫DB ┣DBintestinalis(/)-┫DB, Ascidia
R DESANTIS; H SHIRAKAWA; K NAKADA; S MIYAZAKI; M HOSHI; R MARINO; MR PINTO
Developmental Biology, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 153巻, 1号, 掲載ページ 165-171, 出版日 1992年09月
研究論文（学術雑誌）, 英語 - 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巻, 2号, 掲載ページ 180-184, 出版日 1992年09月, 査読付, 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.
研究論文（学術雑誌）, 英語 - 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巻, 1号, 掲載ページ 165-171, 出版日 1992年09月, 査読付
研究論文（学術雑誌）, 英語 - 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巻, 5067号, 掲載ページ 251-255, 出版日 1992年07月, 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.
研究論文（学術雑誌）, 英語 - 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巻, 5067号, 掲載ページ 251-255, 出版日 1992年, 査読付, 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.
研究論文（学術雑誌）, 英語
MISC
- 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, 出版日 2024年01月, アグリバイオ = Agricultural biotechnology, 8巻, 1号, 掲載ページ 60-64, 日本語, 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, 出版日 2023年04月, 細胞, 55巻, 5号, 掲載ページ 335-338, 日本語, 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
伊藤昌彦; 鹿野朝秀; 白川英樹; 淡路健雄; 宮崎俊一
東京女子医科大学総合研究所, 出版日 2007年10月, 東京女子医科大学総合研究所紀要, 27巻, 掲載ページ 21-22, 日本語, 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
白川英樹; 伊藤昌彦; 宮崎俊一
東京女子医科大学総合研究所, 出版日 2006年07月, 東京女子医科大学総合研究所紀要, 26巻, 掲載ページ 20-21, 日本語, 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, 出版日 2005年10月, 日本不妊学会雑誌, 50巻, 4号, 掲載ページ 511-511, 日本語, 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
出版日 2005年05月, 日本発生生物学会大会発表要旨集, 38th巻, 掲載ページ 198, 日本語, 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
出版日 2004年, 日本生理学会雑誌, 66巻, 12号, 掲載ページ 381-390, 日本語, 記事・総説・解説・論説等（その他） - 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, 出版日 1994年, 生体の科学, 45巻, 1号, 掲載ページ 79-90, 日本語, 記事・総説・解説・論説等（その他）, 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
出版日 1994年, 東京女子医科大学雑誌, 64巻, 掲載ページ 1043-1048, 日本語, 査読付, 記事・総説・解説・論説等（その他） - 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, 出版日 1993年07月, DEVELOPMENTAL BIOLOGY, 158巻, 1号, 掲載ページ 62-78, 英語, 書評論文,書評,文献紹介等, 0012-1606, 1095-564X, WOS:A1993LM14800005
書籍等出版物
- 細胞内カルシウム実験プロトコール
白川英樹; 宮崎俊一
日本語, 共著, 卵細胞のカルシウム：ハムスター, 羊土社, 出版日 1996年 - 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
日本語, 共著, 細胞内Ca2+をみる, 学際企画, 出版日 1994年 - Biology of Germ Lines in Animals and Man
Miyazaki, S; Nakada, K; Shirakawa, H
英語, 共著, Signal transduction of gamete interaction and intracellular calcium release at fertilization of mammalina eggs, Japan Scientific Societies Press, 出版日 1993年
講演・口頭発表等
- 哺乳類卵における表層部アクチン細胞骨格の細胞内Ca2+による制御
白川英樹; 加藤良弥; 米倉遼
ポスター発表, 英語, 第101回日本生理学会大会
発表日 2024年03月30日
開催期間 2024年03月28日- 2024年03月30日 - In vivo mitochondrial Ca2+ dynamics during tetanic muscle contraction in male and female mice
Yoshinori Tanaka; Ayaka Tabuchi; Hideki Shirakawa; Yutaka Kano
英語, 第100回日本生理学会大会
発表日 2023年03月16日 - Regulation of cortical actin dynamics and cytoplasmic flow by intracellular Ca2+ in mouse oocytes
Shirakawa, H; Yonekura, R; Kondo, K
英語, 第100回日本生理学会大会
発表日 2023年03月15日 - 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
発表日 2022年09月21日 - The organization and dynamics of cortical actin cytoskeleton regulated by cytosolic Ca2+ in mammalian eggs.
Shirakawa, H; Kondo, K; Muratani, H
ポスター発表, 英語, 第99回日本生理学会合同大会, 国内会議
発表日 2022年03月16日 - 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
ポスター発表, 英語, 第99回日本生理学会合同大会, 国内会議
発表日 2022年03月16日 - 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
発表日 2021年09月17日 - Repetitive Ca2+ increases coordinate the reorganization of cortical actin cytoskeleton and meiotic resumption in mammalian eggs
Shirakawa, H; Kondo, K
ポスター発表, 英語, 第126回日本解剖学会第98回日本生理学会合同大会, 国内会議
発表日 2021年03月30日 - 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
ポスター発表, 英語, 第126回日本解剖学会第98回日本生理学会合同大会, 国内会議
発表日 2021年03月28日 - 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
発表日 2020年09月24日 - Differential regulation of cortical actin cytoskeleton by intracellular calcium in mouse eggs
Shirakawa, H; Arakawa, S; Yoshida, T
ポスター発表, 英語, 第97回日本生理学会, 国内会議
発表日 2020年03月18日 - Improvement of genetically encoded probe to measure Ca2+ dynamics in subcellular compartments
Murooka, N; Kikuchi, T; Shirakawa, H
ポスター発表, 英語, The 9th Federation of the Asian and Oceanian Physiological Societies Congress
発表日 2019年03月30日 - Calcium-dependent regulation of cortical actin filaments in mouse eggs
Arakawa, S; Yoshida, T; Shirakawa, H
ポスター発表, 英語, The 9th Federation of the Asian and Oceanian Physiological Societies Congress
発表日 2019年03月29日 - 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
発表日 2018年09月 - Manipulating intracellular concentration of inositol trisphosphate by photoactivatable enzymes
Enokida, Y; Yamada, N; Yanagisawa, R; Shirakawa, H
ポスター発表, 英語, 第95回日本生理学会, 国内会議
発表日 2018年03月28日 - Dynamic reconstruction of Golgi apparatus during mammalian oocyte maturation
Iguchi, T; Shirakawa, H
ポスター発表, 英語, 第95回日本生理学会, 国内会議
発表日 2018年03月28日 - Role of luminal Ca2+ binding proteins in the pattern formation of Ca2+ oscillations in mammalian eggs
Murata, T; Kikuchi, T; Shirakawa, H
ポスター発表, 英語, 第94回日本生理学会, 国内会議
発表日 2017年03月30日 - Functional requirements of interdomain interactions for the enzymatic activity of PLCzeta
Yamada, N; Tsuda, T; Shirakawa, H
ポスター発表, 英語, The 22nd International Congress of Zoology, 国際会議
発表日 2016年11月18日 - Calcium oscillation-dependent dynamics of cortical actin filaments in mouse eggs
Yoshida, T; Shirakawa, H
ポスター発表, 英語, The 22nd International Congress of Zoology, 国際会議
発表日 2016年11月18日 - Mechanism for the generation of fast and slow Ca2+ oscillations in mouse eggs
Hideki Shirakawa; Takashi Kikuchi; Noriyuki Yamada; Takuya Tsuda
口頭発表（招待・特別）, 英語, Oocyte Maturation and Fertilization Meeting IV, 国際会議
発表日 2015年06月17日 - Measurement and analysis of changes in the ER Ca2+ concentration during Ca2+ oscillations in mouse eggs
Takashi Kikuchi; Takasuke Murata; Hideki Shirakawa
ポスター発表, 英語, Oocyte Maturation and Fertilization Meeting IV, 国際会議
発表日 2015年06月17日 - Mechanism for the generation of fast and slow Ca2+ oscillations in mouse eggs.
Shirakawa, H; Kikuchi, T; Yamada, Y; Tsuda, T
口頭発表（招待・特別）, 英語, Oocyte Maturation and Fertilization Meeting IV, 招待, 国際会議
発表日 2015年06月17日 - Measurement and analysis of changes in the ER Ca2+ concentrations during Ca2+ oscillations in mouse eggs.
Kikuchi, T; Murata, T; Shirakawa, H
ポスター発表, 英語, Oocyte Maturation and Fertilization Meeting IV, 国際会議
発表日 2015年06月17日 - Analysis of interdomain interactions in PLCzeta by the combined expression of split mutants
Tsuda, T; Yamada, R; Kawamoto, R; Shirakawa, H
ポスター発表, 英語, 第92回日本生理学会
発表日 2015年03月21日 - Analysis of changes in the Ca2+ concentration in the endoplasmic reticulum during Ca2+ oscillations in mammalian eggs
Kikuchi, T; Murata, T; Shirakawa, H
ポスター発表, 英語, 第92回日本生理学会
発表日 2015年03月21日 - Analysis of biphasic Ca2+ uptake by the endoplasmic reticulum during Ca2+ oscillations in mouse eggs
Kikuchi, T; Shirakawa, H
ポスター発表, 英語, 第91回日本生理学会
発表日 2014年03月18日 - Numerical simulation of fast and slow Ca2+ oscillations in fertilized mouse eggs.
Shirakawa H; Tan M
ポスター発表, 英語, 第91回日本生理学会
発表日 2014年03月18日 - Measurement of Ca2+ in the endoplasmic reticulum during Ca2+ oscillations in mammalian eggs.
Kikuchi, T; Takahashi, T; Shirakawa, H
口頭発表（一般）, 日本語, 第90回日本生理学会
発表日 2013年03月 - On the role of STIM/Orai pathway in Ca2+ entry during Ca2+ oscillations in fertilized mouse eggs.
Takahashi, T; Kidokoro, Y; Shirakawa, H
口頭発表（一般）, 日本語, J. Physiol. Sci.,第８９回日本生理学会
発表日 2012年03月 - Visualization of intracellular IP4 dynamics using a novel fluorescent probe
Tanaka, S; Takahashi, T; Shirakawa, H
口頭発表（一般）, 日本語, 第８９回日本生理学会
発表日 2012年03月 - On the functional role of STIM/Orai proteins in the regulation of intracellular Ca2+ in mouse eggs.
Kidokoro, Y; Shirakawa, H
口頭発表（一般）, 英語, 第８８回日本生理学会大会
発表日 2011年03月 - Time-lapse observation of cytoplasmic actin filament in mouse eggs during fertilization.
Kumakura, Y; Manabe, S; Shirakawa, H
口頭発表（一般）, 英語, 第８８回日本生理学会大会
発表日 2011年03月 - Pharmacological characterization of store-operated Ca2+ entry in mouse eggs.
Takahashi, T; Shirakawa, H
口頭発表（一般）, 英語, 第８８回日本生理学会大会
発表日 2011年03月 - 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
口頭発表（一般）, 日本語, 日本動物学会,第８２回大会
発表日 2011年 - 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
口頭発表（一般）, 日本語, 日本動物学会,第８２回大会
発表日 2011年 - バイオサイエンスにおける光技術の応用
白川英樹
口頭発表（招待・特別）, 日本語, 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
発表日 2010年08月 - 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
口頭発表（一般）, 英語, 第８７回日本生理学会大会
発表日 2010年 - 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
口頭発表（一般）, 日本語, 日本動物学会,第８０回大会
発表日 2009年09月 - 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
口頭発表（一般）, 日本語, 日本動物学会,第８０回大会
発表日 2009年09月 - 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
口頭発表（一般）, 日本語, 日本動物学会,第８０回大会
発表日 2009年09月 - 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
口頭発表（一般）, 日本語, 日本動物学会,日本動物学会第７９回大会
発表日 2008年 - Pharmacological characterization of Ca2+ entry pathway during Ca2+ oscillations in fertilized mouse eggs.
Takahashi, T; Shirakawa, H
口頭発表（一般）, 英語, 第８５回日本生理学会大会
発表日 2008年 - 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
口頭発表（一般）, 日本語, 日本動物学会,日本動物学会第７８回大会
発表日 2007年 - Analysis of IP3 dynamics during the intracellular Ca2+ oscillations in mammalina eggs.
Shirakawa, H; Sato, M; Umezawa, Y; Miyazaki, S
口頭発表（一般）, 英語, Journal of Physiological Sciences,第８５回日本生理学会大会
発表日 2006年 - Physiological applications of blind spectral unmixing method based on EEM fluorescence imaging and multivariate analysis
Shirakwa, H; Miyazaki, S
口頭発表（一般）, 英語, 第８２回日本生理学会大会
発表日 2005年 - 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
口頭発表（一般）, 英語, 第８２回日本生理学会大会
発表日 2005年 - Nuclear translocation of phospholipase C-zeta during embryonic development of the mouse
Sone, Y; Ito, M; Shirakawa, H; Shikano, T; Kinoshita, K; Miyazaki, S
口頭発表（一般）, 英語, 第８２回日本生理学会大会
発表日 2005年 - Application of blind spectral decomposition method to the measurement of membrane potential with voltage-sensitive dyes
Shirakawa, H; Miyazaki, S
口頭発表（一般）, 英語, 第８１回日本生理学会年会
発表日 2004年 - 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
口頭発表（一般）, 英語, 第８１回日本生理学会大会
発表日 2004年 - 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
口頭発表（招待・特別）, 日本語, 第８１回大会, 日本生理学会
発表日 2004年 - 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
口頭発表（一般）, 日本語, 日本生物物理学会,第４２回年会
発表日 2004年 - Multicomponent measurement based on wide-range two-dimensional fluorescence microspectroscopy
Shirakawa, H; Miyazaki, S
口頭発表（一般）, 英語, 第８０回日本生理学会大会
発表日 2003年 - 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
口頭発表（一般）, 日本語, 日本生物物理学会,第４１回年会
発表日 2003年 - 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
口頭発表（一般）, 日本語, 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」
発表日 2003年 - Ca2+/Mn2+ dynamics during Ca2+ oscillations in mouse eggs
Mohri, T; Shirakawa, H; Oda, S; Sato, M.S; Mikoshiba, K; Miyazaki, S
口頭発表（一般）, 英語, 第７７回日本生理学会年会
発表日 2001年 - Dual emission ratiometric measurement of intracellular calcium with visible-light excitation
Shirakawa, H; Miyazaki, S
口頭発表（一般）, 英語, 第７８回日本生理学会年会
発表日 2001年 - Novel GFP-based ratiometric indicators for monitoring intracelluar pH
Awaji, T; Hirasawa, A; Shirakawa, H; Tsujimoto, G; Miyazaki, S
口頭発表（一般）, 英語, 第７８回日本生理学会大会
発表日 2001年 - 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
口頭発表（一般）, 日本語, 日本分子生物学会,第２４回大会
発表日 2001年 - Optical measurement of endoplasmic reticulum membrane potential during Ca2+ release
Shirakawa, H; Miyazaki, S
口頭発表（一般）, 英語, 第７７回日本生理学会大会
発表日 2000年 - 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
口頭発表（一般）, 日本語, 日本生物物理学会,第３８回年会
発表日 2000年 - 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
口頭発表（一般）, 日本語, 日本細胞生物学会,第５３回大会
発表日 2000年 - 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
口頭発表（一般）, 日本語, 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」
発表日 2000年 - 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
口頭発表（一般）, 日本語, 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」
発表日 1999年 - Spatiotemporal analysis of intracellular calcium increases in hamster sperm induced by solubilized zona pellucida
Shirakawa, H; Miyazaki, S
口頭発表（一般）, 英語, 第７４回日本生理学会年会
発表日 1997年 - 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
口頭発表（一般）, 日本語, 日本発生生物学会,第３０回大会
発表日 1997年 - 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
口頭発表（一般）, 英語, 第７４回日本生理学会大会
発表日 1997年 - 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
口頭発表（一般）, 日本語, 日本産婦人科学会,第４９回学術講演会
発表日 1997年 - 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
口頭発表（一般）, 日本語, 日本受精着床学会,第１４回学術講演会
発表日 1996年 - 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
口頭発表（一般）, 英語, 第７２回日本生理学会大会
発表日 1995年 - Spatiotemporal analysis of the increases of nuclear and cytoplasmic Ca2+ induced by IP3 in hamster oocytes.
Shirakawa, H; Shiraishi, K; Miyazaki, S
口頭発表（一般）, 日本語, 第７２回日本生理学会大会
発表日 1995年 - 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
口頭発表（一般）, 日本語, 日本生物物理学会,第３３回年会
発表日 1995年 - 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
口頭発表（一般）, 日本語, 日本産婦人科学会,第４８回学術講演会
発表日 1995年 - Analysis of Ca2+ wave in hamster eggs using confocal microscopy
Shiraishi, K; Shirakawa, H; Yonda, Y; Miyazaki, S
口頭発表（一般）, 英語, 第７１回日本生理学会大会
発表日 1994年 - 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
口頭発表（招待・特別）, 日本語, 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
発表日 1994年 - Activation of calcium influx pathway by inositol 1,3,4,5-tetrakisphosphate in hamster eggs
Shirakawa, H; Honda, Y; Nakada, K; Miyazaki, S
口頭発表（一般）, 英語, 第７０回日本生理学会大会
発表日 1993年 - Increase in sensitivity of the inositol trisphosphate receptor during maturation of hamster oocytes
Nakada, K; Fujiwarak T; Shirakawa, H; Miyazaki, S
口頭発表（一般）, 英語, 第７０回日本生理学会大会
発表日 1993年 - 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
口頭発表（一般）, 英語, 第６９回日本生理学会大会
発表日 1992年 - 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
口頭発表（一般）, 日本語, 日本発生生物学会,第２５回大会
発表日 1992年 - 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
口頭発表（一般）, 日本語, 日本分子生物学会,年会
発表日 1992年 - 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
口頭発表（一般）, 日本語, 家畜繁殖学会,第８２回大会
発表日 1992年
共同研究・競争的資金等の研究課題
- 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
挑戦的研究（萌芽）, 研究代表者, 22K19401
研究期間 2022年04月 - 2025年03月 - ＩＰ３クランプ法の開発
白川 英樹
日本学術振興会, 科学研究費助成事業 挑戦的萌芽研究, 電気通信大学, 挑戦的萌芽研究, 生細胞内のIP3を光照射によって任意の濃度に固定できる実験手法（IP3クランプ法）の確立を目指し、必要な分子ツール（光活性化型酵素、蛍光性プローブ）の作成を行った。ホスホリパーゼＣと植物由来の光感受性タンパク質を組み合わせることで、可視光照射によってIP3産生活性が変化する酵素が得られることを示した。また、従来の蛍光性IP3プローブの励起・蛍光波長を改変し、IP3クランプ法において光活性化型酵素との併用が可能なプローブを得た。, 15K15027
研究期間 2015年04月01日 - 2017年03月31日 - 受精卵CaオシレーションにおけるCa流入とCa遊離の機能的共役機構
白川 英樹
日本学術振興会, 科学研究費助成事業 基盤研究(C), 電気通信大学, 基盤研究(C), 哺乳類受精時の卵活性化に関わる細胞内Ca^<2+>濃度の制御機構、特に細胞外からのCa^<2+>流入の実体とその調節機構の解明を目指し、マウス受精卵におけるCa^<2+>オシレーションにおけるTRPC、OraiおよびSTIMタンパク質の機能的関与を想定して実験を行った。種々のペプチドを用いた阻害実験では、これらのタンパク質のいずれも受精時のCa^<2+>流入には関与していないことを示す結果となった。一方でSTIM1はCa^<2+>流入の活性化以外の機能を示すことが新たに示唆された。, 21590229
研究期間 2009年 - 2011年 - 生体内光源物質特性分布を画像化する技術の高度化に関する研究
山田 幸生; 大川 晋平; 白川 英樹; 星 詳子; 谷川 ゆかり; 高 峰
日本学術振興会, 科学研究費助成事業 基盤研究(B), 電気通信大学, 基盤研究(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
研究期間 2007年 - 2009年 - 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
研究期間 2006年 - 2006年 - 哺乳類卵活性化精子蛋白質候補ホスホリパーゼCゼータの検証と機能解析
宮崎 俊一; 伊藤 昌彦; 淡路 健雄; 白川 英樹; 尾田 正二; 河内 全
日本学術振興会, 科学研究費助成事業 基盤研究(B), 東京女子医科大学, 基盤研究(B), 哺乳類の受精時に卵細胞内カルシウムイオンが反復増加し(Ca^<2+>オシレーション),卵活性化(第二減数分裂の再開,第二極体の放出,雌雄前核の形成)の引き金となる.Ca^<2+>の増加はイノシトール3リン酸(IP_3)受容体を介する小胞体からのCa^<2+>遊離による.Ca^<2+>遊離は精子細胞質にあるCa^<2+>オシレーション誘起物質,即ち卵活性化蛋白質(Egg Activating Protein, EAP)が精子-卵融合時に卵内に移入することによる.IP3産生酵素であるホスホリパーゼCの新タイプであるゼータ(PLCζ)がEAPの有力候補とされている.本研究はPLCζの特性を解析し,PLCζが哺乳類のEAPであるか否かを検証することを目的として行われ,以下の結果を得た. 1)バキュロウイルス/Sf9細胞系で合成したPLCζ蛋白質をマウス卵に注入すると,低い濃度でCa^<2+>オシレーションが誘発された.2)PLCζのin vitro酵素活性アッセイで,非常に高い感受性を有し,静止時の1)バキュロウイルス/Sf9細胞系で合成したPLCζ蛋白質をマウス卵に注入すると,低い濃度でCa^<2+>オシレーションが誘発された.2)PLCζのin vitro酵素活性アッセイで,非常に高い感受性を有し,静止時のCa^<2+>濃度でも70%最大活性を持っていた.3)N端から1,2番目のEFハンドドメイン(EF1,EF2)は酵素活性に強く関与し,EF3は酵素活性のCa^<2+>依存性に関与する.4)C端側にあるC2ドメインはイノシトールリン脂質のうちのPI(3)P, PI(5)Pに親和性がある.5)PLCζに蛍光蛋白Venusを結合した蛋白質をコードするRNAをマウス卵に注入し,発現した低濃度のPLCζでCa^<2+>オシレーションが起る.6)発現したPLCζが卵の前核に移行する.7)核移行配列が触媒ドメインX, Yの連結部位にあり,またEF1とC2が会合するような立体構造が必須である.以上の結果は,PLCζがEAPの有力候補であることを支持するものである.さらにPLCζ欠損精子で受精時の卵活性化阻害を確認するため,PLCζの遺伝子ノックアウトマウスを作成した.また機能的な相補性を確認するためPLCζのトランスジェニックマウスを準備した., 16390055
研究期間 2004年 - 2006年 - 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
研究期間 2000年 - 2001年 - 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
研究期間 1997年 - 1998年 - 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
研究期間 1997年 - 1998年 - 哺乳動物受精時の精子由来卵活性化因子の同定と卵細胞内Ca^<2+>増加の分子機構の解明
宮崎 俊一; 淡路 健雄; 白石 浩一; 白川 英樹; 尾田 正二
日本学術振興会, 科学研究費助成事業 基盤研究(B), 東京女子医科大学, 基盤研究(B), 本研究は,受精の引き金になる細胞内カルシウムイオン(Ca)増加反応に至る精子による卵活性化の分子機構を解明するステップとして実施され,顕微細胞操作の技法,Ca画像解析装置および共焦点レーザー走査顕微鏡を用いて以下の結果を得た. 1)まず現像論として,ウニ精子-卵融合が一過性にしかおこならい条件を融合阻害剤ジャスピシン存在下で設定し,精子が最初に卵に引き起こす電流変化およびCa増加反応を同時記録し,精子結合部位直下の卵細胞内で局所内におこるCa増加を分離して記録することに成功し,その時・空間的特性を解析した.2)マウス成熟卵内への精子注入を行い,受精時に類似した反復性のCa増加(Ca振動)が15〜30分後に起こり初め数時間持続すること,Ca増加は卵全体で同期的に起こることを示した.精子-卵結合をバイパスしても,注入精子の細胞質因子が卵細胞質に漏出してCa増加反応,卵活性化をおこしうることを示した.3)マウス未成熟精子(円形精子細胞)の卵内注入ではCa増加反応は誘発できず卵活性化もおこらないが,IP_3受容体の強力なアゴニストであるアデノホスチンを同時注入して人為的にCa振動を誘発し,受精させることに成功した.さらに2細胞期胚を宿主母体に移植し,正常な産仔を得た.4)卵内注入によってCa振動を誘発する活性をもつ蛋白質をハムスター,ホヤ精子から抽出した.この抽出物をハムスター,マウス,ホヤ卵の表層および中心部にそれぞれ微量注入し,卵表層部細胞質が中心部に比べて精子因子に対する感受性が強いことを明らかにした. これらの実験により,受精時に卵面に結合した精子細胞質から精子由来卵活性化因子が卵表層細胞質に導入されてCa増加反応(恐らくIP_3受容体を介する小胞体から細胞質へのCa遊離)を誘発する可能性が示された.本研究は今後の研究の発展に繋がる有用な実験データを提供したことで,成果をあげたと考える., 08457016
研究期間 1996年 - 1997年 - 哺乳動物受精卵に於けるCa波とCa振動のメカニズムに関する生理学的研究
宮崎 俊一; 尾田 正二; 白石 浩一; 白川 英樹
日本学術振興会, 科学研究費助成事業 一般研究(B), 東京女子医科大学, 一般研究(B), 受精時の卵細胞内Ca^<2+>濃度(Ca)の上昇は卵賦活の引き金となる。我々は既に、哺乳動物卵のCa増加は精子付着部位から全体に伝播するCa波と、繰り返しおこるCa振動を示し、イノシトール三燐酸受容体(IP_3R)を介する小胞体(ER)からのCa遊離によることを解明した。本研究はこれら空間的、時間的Caシグナルの成因を解析した。 1.精子一卵の接着・結合に関わる細胞膜上の蛋白質に関して、リガンドや抗体による刺激はCa増加反応を誘発せず、細胞内信号伝達としてチロシンキナーゼのインヒビターは受精時のCa反応を抑制しなかった。即ち表面分子を介するシグナル発生の証拠が得られず、精子細胞質から卵内に誘導される物質が重要ではないかという結論に達した。なお精子一卵結合にMg^<2+>が重要な因子であることを明らかにした。 2.繰り返すCa振動(Ca遊離)の成因に関して、ハムスター卵でイノシトール四燐酸(Ip_<4+>)で活性化される細胞外からのCa流入経路の存在を証明し、流入したCaがERを再充填して次のCa遊離を可能にする機構を支持する結果を得た。 3.共焦点レーザー走査顕微鏡を用い、Ca波が細胞質深部に伝播すること、核内も細胞質と同様のCa増加を起こすことを確認した。 4.脂溶性蛍光色素DiIをハムスター卵内に注入し、レーザー顕微鏡で観察した結果、ERは細胞全体に亘る綱目構造を形成しており、免疫組織化学によって同定されたIP_3Rの分布と一致した。 5.米成熟卵では、ER及びIP_3R表層の細胞質と核周辺にパッチ状に密に偏在しており、低濃度のセロトニンによる刺激、予め注入したcagedIP_3の光分解によるCa遊離は、周辺の細胞質、核周辺で感受性が高いことを観察した。ERの卵全体への分数がCa波の形成に必要な成熟過程であることを明らかにした。 得られた結果は学会、学会誌に発表した。また、「哺乳動物卵受精時のCa波とCa振動とIP_3R」「IP_3Rを介する空間的・時間的Caシグナリング」に関する総説を発表した。, 05454141
研究期間 1993年 - 1994年 - 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
研究期間 1991年 - 1991年