Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization

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.