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J. Biol. Chem., Vol. 277, Issue 4, 2763-2772, January 25, 2002

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Molecular Characterization of the Starfish Inositol 1,4,5-Trisphosphate Receptor and Its Role during Oocyte Maturation and Fertilization^*

Hirohide Iwasaki^§¶, Kazuyoshi Chiba, Tsuyoshi Uchiyama^§, Fumio Yoshikawa^**, Fumiko Suzuki, Masako Ikeda, Teiichi Furuichi^**, and Katsuhiko Mikoshiba^§

From the  Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, the ^§ Division of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8039, Japan, the  Department of Biology, Ochanomizu University, 2-1-1 Ootsuka, Bunkyo-ku, Tokyo 112-8610, Japan, ^** Laboratory for Molecular Neurogenesis, Brain Science, Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and the  Department of Biosciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

The release of calcium ions (Ca²⁺) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca²⁺ via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP₃R) and the ryanodine receptor. Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found. In this study, we utilized a novel IP₃R antagonist, the "IP₃ sponge," to investigate the role of IP₃ during fertilization of the starfish oocyte. The IP₃ sponge strongly and specifically competed with endogenous IP₃R for binding to IP₃. By injecting IP₃ sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked. To investigate the role of IP₃R in the starfish oocyte more precisely, we cloned IP₃R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP₃R has 58-68% identity to mammalian IP₃R types 1, 2, and 3. We then raised antibodies that recognize starfish IP₃R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP₃R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP₃R changes during oocyte maturation.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB071372.

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