Role of calcium in regulating intracellular pH following the stepwise release of the metabolic blocks at first-meiotic prophase and second-meiotic metaphase in amphibian oocytes

³¹P-NMR has been used to monitor changes in intracellular pH following the sequential release of the block at first-meiotic prophase by hormones and the block at second-meiotic metaphase by fertilization in Rana eggs and oocytes. The broad phosphoprotein signal was eliminated by a combination of spin-echo and deconvolution techniques. pH_i was determined from the pH-dependent separation of intracellular P_i and phosphocreatine resonances. Agents that release the prophase block (progesterone, insulin, D-600, La³⁺) increased pH_i from 7.38 to 7.7-7.8 within 1-3 h. Noninducers such as 17 β-estradiol were without effect. By second-metaphase arrest (ovulated, unfertilized) the pH_i had fallen to 7.1-7.2. pH_i underwent a transient increase to about 7.7 within the first 30 min at fertilization, with a slow 0.1-0.2 pH unit oscillation during early cleavage. The progesterone-induced elevation of intracellular pH is not blocked by amiloride and occurs in Na⁺-free medium. A transient rise in pH_i occurs when the prophase-arrested oocyte is transferred to Ca²⁺-free medium or when ionophore A23187 is added to the Ca²⁺-containing medium. Agents that inhibit the resumption of the first meiotic division either block the rise in pH_i (procaine, PMSF) or shorten the time-course of the rise in pH_i (ionophore A23187). Conditions that elevate intracellular Ca²⁺ levels and/or increase Ca²⁺ exchange produce an increase in pH_i, whereas those conditions that decrease intracellular Ca²⁺ levels and/or exchange produce a fall in pH_i within 1 h. The time-course of the increase in pH_i both following release of the prophase block and at fertilization coincide with a fall in intracellular cAMP and release of surface and/or intracellular Ca²⁺. These results suggest that: (1) pH_i is a function of cytosolic free Ca²⁺ levels and/or Ca²⁺ exchange across the oocyte plasma membrane, and (2) meiotic agonists (progesterone, insulin, D-600) and mitogens (sperm, ionophore A23187) modulate intracellular and/or membrane Ca²⁺ with the resulting changes in pH_i and cAMP and resumption of the meiotic divisions.