The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases

Abstract

During fertilization in mouse eggs, the sperm triggers a series of intracellular Ca2+ oscillations that lead to egg activation, as indicated by pronuclear formation. We show that Ca2+ oscillations in fertilized mouse eggs can be inhibited by addition of either the Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) or the heavy-metal-ion chelator N,N,N´,N´-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) plus dithiothreitol (DTT). Both treatments inhibited Ca2+ oscillations, but they had different effects upon egg activation. Blocking Ca2+ oscillations with BAPTA-AM after the occurrence of just two Ca2+ spikes resulted in most eggs forming pronuclei. However, we found that BAPTA-AM-treated fertilizing eggs showed a decreased rate of protein synthesis, which by itself can promote egg activation. In contrast, blocking Ca2+ oscillations with TPEN plus DTT was accompanied by the inhibition of egg activation with no significant effect on protein synthesis. In eggs that were fertilized and then treated with TPEN plus DTT, there was a correlation between the number of Ca2+ spikes and the proportion of eggs that formed pronuclei, as well as between the number of Ca2+ spikes and the time taken for pronuclear formation and the first mitosis to occur. The addition of TPEN plus DTT did not block the generation of Ca2+ spikes or pronuclear formation when eggs were artificially stimulated by electroporation pulses. These data suggest that TPEN plus DTT inhibits pronuclear formation in fertilizing eggs via the inhibition of Ca2+ oscillations and that the number of Ca2+ spikes may regulate egg activation.