The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method: (intracellular...

The change in intracellular pH (pHi ) upon fertilization and the effects of changing the pHi by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pHi was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pHi ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pHi ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1-4), development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pHi did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pHi was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pHi . The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.