In situ analysis of microtubule assembly of the centrosome

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The purpose of this study is to clarify the role of a transient increase in intracellular calcium ion concentration (Cai) and increase in intracellular pH (pHi) on sperm aster formation in the eggs of the sea urchin, Clypeaster japonicus. In order to inhibit the increase in Cai, a solution of calcium chelating substance was microinjected into sea urchin eggs and, to control pHi, a pH buffer was microinjected or sea water containing ammonium acetate was applied to sea urchin eggs. The sperm entered the unfertilized eggs which had been injected with a solution of calcium chelating substance but sperm aster formation was not observed. In the eggs injected with a solution of calcium chelating substance, pHi increase was not detected. When pHi of those eggs increased up to 7.0 or more, the sperm aster formed. In the eggs treated with Na-free sea water shortly after insemination, pHi of those eggs did not increase or sperm aster formation did not occur. However, when pHi of the eggs treated with Na-free sea water increased up to 7.0 or more, the sperm aster formed. These results indicate that the increase in pHi with or without the increase in Cai is necessary for sperm aster formation in fertilized and sperm-entered eggs. Furthermore, sperm heads were microinjected into unfertilized eggs, in which sperm aster formation never observed, and then when pHi of those eggs increased, the sperm aster formed. In this case Cai change was not detected. Therefore, it is clearly demonstrated that pHi increase activates the centrosome introduced in the egg by means of fertilization and microinjection, which results in assembly of microtubules around the centrosome, and finally in organization of the sperm aster.