Fertilization of Xenopus laevis eggs triggers a wave of increased [Ca2+]i. The exact signal transduction pathway culminating in this Ca2+ wave remains unknown. To determine whether increases in tyrosine kinase activity are part of this pathway, we microinjected tyrosine kinase inhibitors into unfertilized eggs. Upon fertilization, signs of activation were monitored, such as fertilization envelope liftoff and the Ca2+ wave (for eggs microinjected with lavendustin A). Various concentrations of lavendustin A and tyrphostin B46 were microinjected, as well as inactive forms of these compounds (lavendustin B and tyrphostin A1) to provide negative controls. Peptide A, a 20-amino-acid peptide derived from the SH2 region of pp60(v-src) tyrosine kinase, was also microinjected. Peptide A inhibits tyrosine kinase activity but not PKA or PKG activity. Dose-response curves for lavendustin A, tyrphostin B46, and peptide A show clear inhibition of vitelline envelope liftoff by these three compounds. Confocal imaging of eggs coinjected with lavendustin A and Oregon Green-dextran showed that the Ca2+ wave was inhibited under normal insemination conditions but that the block of the Ca2+ wave could be overcome with very high sperm densities. A phenomenon of small local Ca2+ increases termed "hot spots" seen in lavendustin A containing eggs is also described. Since this inhibition of egg activation by tyrosine kinase inhibitors can be overcome by Ca2+ microinjection, the inhibitors must act on a step in the signal transduction cascade that is upstream of the Ca2+ wave.

Voltage-clamped mature, jelly-intact Xenopus eggs were used to carefully examine the ionic currents crossing the plasma membrane before, during, and after fertilization. The bulk of the fertilization current was transient, of large amplitude, and reversed at the predicted Cl- reversal potential. However, the large amplitude fertilization current was preceded by a small, step-like increase in holding current. This small increase in holding current is referred to in this paper as Ion to acknowledge its qualitative similarity to the Ion current previously described in the sea urchin. It was observed in both fertilized and artificially activated eggs, and was found to be unaffected by 10 mm tetra-ethyl ammonium (TEA), a concentration found to block K+ currents in Rana pipiens. Current-voltage relationships are presented for the large fertilization potential, and show that the fertilization currents have a marked outward rectification and are voltage sensitive. These properties are in contrast to the total lack of rectification and slight voltage sensitivity seen before or after the fertilization currents. The time required for sperm to fertilize the egg was found to be voltage dependent with a relatively more depolarized voltage requiring a longer time for fertilization to occur. The percentage of eggs blocked with varying potential levels was determined and this information was fitted to a modified Boltzmann equation having a midpoint of -9 mV.