Nuclear and cytoplasmic pH increase at fertilization in Urechis caupo

Intracellular pH (pHi) was measured in Urechis caupo (Echiura) eggs during fertilization using the pH-sensitive dye BCECF [bis(carboxyethyl)carboxyfluorescein] and fluorescence microscopy. When eggs were inseminated at pH 8, pHi began to rise 22-36 sec (n = 7) after sperm contact and reached a plateau by 3 min (2.8 +/- 1 SD; n = 14). The net increased was 0.25-0.3 pH units and the alkalinization persisted through 1 hr after insemination (after second polar body formation). Separate measurements of germinal vesicles and cytoplasm revealed that pH rose dramatically within the nuclei well before germinal vesicle breakdown (GVBD), as well as in cytoplasm. When eggs were fertilized at pH 6.9 (previously shown to inhibit proton release and egg activation despite sperm entry; see Paul, 1970, 1975; Holland et al., 1984), there was no net pH increase in either cytoplasm or germinal vesicles. When fertilization was at pH 7.45, the average pHi increase in whole eggs was 58% of that at pH 8, and 61% of the eggs activated partially or completely. These results show that the pHi rise is correlated with GVBD and egg activation.