In situ analysis of microtubule assembly of the centrosome

本研究的目的是阐明细胞内钙离子浓度（Cai）和细胞内pH （pHi）的短暂升高对日本海胆（Clypeaster japonicus）卵中精子形成的作用。为了抑制Cai的增加，我们在海胆卵中微量注射钙螯合物质溶液，在海胆卵中微量注射pH缓冲液或在海胆卵中施用含乙酸铵的海水来控制pHi。精子进入注射了钙螯合剂溶液的未受精卵，但未观察到精子形成。在注射钙螯合剂溶液的鸡蛋中，未检测到pHi升高。当这些卵子的pHi值增加到7.0或更高时，精子就会加速形成。在授精后不久用不含na的海水处理的卵子中，这些卵子的pHi没有增加，精子也没有形成。然而，当卵子的pHi值在不含na的海水中增加到7.0或更高时，精子加速形成。这些结果表明，在受精卵和进入精子的卵子中，无论是pHi的增加还是Cai的增加，都是精子形成的必要条件。此外，将精子头微注射到未受精卵中，在未受精卵中从未观察到精子形成，然后当这些卵子的pHi增加时，精子形成。在这种情况下，没有检测到Cai变化。因此，可以清楚地证明，通过受精和显微注射，pHi的增加激活了引入卵子的中心体，导致中心体周围的微管聚集，最终组织了精子aster。

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.