Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization.
The release of calcium ions (Ca(2+)) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca(2+) via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP(3)R) 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 IP(3)R antagonist, the "IP(3) sponge," to investigate the role of IP(3) during fertilization of the starfish oocyte. The IP(3) sponge strongly and specifically competed with endogenous IP(3)R for binding to IP(3). By injecting IP(3) 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 IP(3)R in the starfish oocyte more precisely, we cloned IP(3)R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP(3)R has 58-68% identity to mammalian IP(3)R types 1, 2, and 3. We then raised antibodies that recognize starfish IP(3)R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP(3)R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP(3)R changes during oocyte maturation.
Iwasaki
Hirohide
H
Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. hiwasaki@ims.u-tokyo.ac.jp
Chiba
Kazuyoshi
K
Uchiyama
Tsuyoshi
T
Yoshikawa
Fumio
F
Suzuki
Fumiko
F
Ikeda
Masako
M
Furuichi
Teiichi
T
Mikoshiba
Katsuhiko
K
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005306:Fertilization; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008969:Molecular Sequence Data; D009865:Oocytes / Q000254:growth & development*; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013215:Starfish; D013997:Time Factors
D015220:Calcium Channels; D018076:DNA, Complementary; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D005982:Glutathione Transferase; D002118:Calcium