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

Abstract

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, La 3+) 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 Ca 2+-free medium or when ionophore A23187 is added to the Ca 2+-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 Ca 2+ levels and/or increase Ca 2+ exchange produce an increase in pH i, whereas those conditions that decrease intracellular Ca 2+ 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 Ca 2+. These results suggest that: (1) pH i is a function of cytosolic free Ca 2+ levels and/or Ca 2+ exchange across the oocyte plasma membrane, and (2) meiotic agonists (progesterone, insulin, D-600) and mitogens (sperm, ionophore A23187) modulate intracellular and/or membrane Ca 2+ with the resulting changes in pH i and cAMP and resumption of the meiotic divisions.