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Tyrosine kinase inhibitors block sperm-induced egg activation in Xenopus laevis
1Section of Molecular and Cellular Biology, University of California, Davis, California, 95616, USA. dbglahn@ucdavis.edu
Abstract:
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.
Insights
Tyrosine kinase inhibitors block calcium waves and egg activation in Xenopus laevis. This suggests tyrosine kinase activity is crucial upstream in the fertilization signaling pathway.
Area of Science:
- Cellular and Molecular Biology
- Developmental Biology
- Reproductive Biology
Background:
- Fertilization of Xenopus laevis eggs initiates a calcium ion (Ca2+) wave, a critical event for egg activation.
- The precise signal transduction pathway responsible for this Ca2+ wave remains incompletely understood.
Purpose of the Study:
- To investigate the role of tyrosine kinase activity in the signal transduction pathway leading to the Ca2+ wave upon fertilization.
- To determine if inhibiting tyrosine kinases affects early egg activation events in Xenopus laevis.
Main Methods:
- Microinjection of unfertilized Xenopus laevis eggs with various tyrosine kinase inhibitors (lavendustin A, tyrphostin B46) and controls (lavendustin B, tyrphostin A1).
- Monitoring of egg activation signs, including fertilization envelope liftoff and Ca2+ wave propagation using confocal imaging.
- Microinjection of a peptide inhibitor (Peptide A) targeting the SH2 region of pp60(v-src) tyrosine kinase.
Main Results:
- Tyrosine kinase inhibitors (lavendustin A, tyrphostin B46) and Peptide A significantly inhibited vitelline envelope liftoff.
- Confocal imaging revealed inhibition of the Ca2+ wave by lavendustin A, which could be overcome by high sperm density.
- Microinjection of Ca2+ rescued egg activation, indicating inhibitors act upstream of the Ca2+ wave.
Conclusions:
- Tyrosine kinase activity is essential for the signal transduction cascade initiating the Ca2+ wave during Xenopus laevis egg fertilization.
- These findings place tyrosine kinases at a critical upstream step in the pathway regulating early embryonic development.