Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes

Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pH_i), which is required for spindle assembly. Herein, we describe a protocol for pH_i measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH₃COONH₄ to clamp pH_i, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pH_i values. Of note, if the actual pH_i is higher or lower than the known pH of injected standard solutions, the BCECF fluorescence intensity ratio will decrease or increase, respectively. On the other hand, the pH of the injected solution displaying no change in fluorescence intensity should be considered the actual pH_i. These methods for pH_i calibration and clamping are simple and reproducible.