31P NMR reveals increased intracellular pH after fertilization in Xenopus eggs

Abstract

31P NMR spectra of mature eggs of the frog (Xenopus laevis) were taken prior to and after both fertilization and activation by a Ca2+/H+ ionophore (A23187). The eggs were constantly perfused with fresh well-buffered solution during the experiments, and the intracellular pH (pHi) was determined from the pH-dependent chemical shift of the internal Pi peak. The detection of this Pi peak in the presence of overlapping yolk phosphoprotein signals was accomplished by a T2 experiment which discriminated against the broader yolk phosphoprotein peak. The average pHi of the unfertilized, fertilized, and activated eggs was 7.42, 7.66, and 7.64, respectively. Thus, a cytoplasmic alkalinization of 0.24 pH unit occurs within 90 min 90 min after fertilization. These values are practically identical to pHi measurements made in this laboratory on Xenopus eggs by using pH-sensitive glass microelectrodes. These 31P NMR studies also indicate that extracellular pH changes as large as 3 pH units had no effect on pHi. We also found that phosphocreatine levels are very sensitive to metabolic perturbations such as oxygen depletion or metabolic inhibitor application. These treatments resulted in a rapid decrease in the phosphocreatine concentration; the ATP concentration declined only slowly after the phosphocreatine peak had disappeared.