Tyrosine Kinase Inhibitors Block Sperm-Induced Egg Activation inXenopus laevis

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.

Roles of Src family kinase signaling during fertilization and the first cell cycle in the marine protostome worm Cerebratulus

“…The absence of oscillations in U73122 is not due to a lack of sperm-egg interactions, since non-oscillating specimens commonly undergo cortical flash-like increases or "hot spots" (Glahn et al 1999). Similarly, >50% of Cerebratulus oocytes treated with U73122 after fertilization discontinue their oscillations or display oscillation dampening.…”

“…1B). Among non-oscillating specimens, 9/12 exhibited cortical flash-like elevations and/ or non-propagating cortical "hot spots" (Glahn et al 1999) (Fig. 2 C,I).…”

Calcium signalling in early embryos

“…Depleting cholesterol from lipid-signalling rafts with methyl-b-cyclodextrin caused a reduction in tyrosine kinase activity and blocked the fertilization calcium transient (Sato et al 2002). The tyrosine kinase inhibitors lavendustin A and tyrphostin B46 prevented the fertilization calcium wave, as did a 20 amino acid truncation of the Src SH2 domain (Glahn et al 1999).…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part I. Protein Kinases

“…lymphocyte activation, neuronal signal transduction). In some sea invertebrates (sea urchin, starfish, ascidian, and others) (Abassi et al 2000;Belton et al 2001;Dasgupta and Garbers 1983;Giusti et al 1999a;Giusti et al 1999b;Giusti et al 2000a;Giusti et al 2003;Giusti et al 2000b;Kamel et al 1986;O'Neill et al 2004;Runft et al 2004;Runft and Jaffe 2000;Runft et al 2002;Sakuma et al 1997;Shen et al 1999;Shilling et al 1994;Stricker et al 2010a;Townley et al 2006;Townley et al 2009), fish (zebrafish) (in this case, Fyn tyrosine kinase) and frog (African clawed frog) (Glahn et al 1999;Iwasaki et al 2008;Iwasaki et al 2006;Kushima et al 2011;Mahbub Hasan et al 2011;Mahbub Hasan et al 2007;Mahbub Hasan et al 2005;Mammadova et al 2009;Sakakibara et al 2005;Sato et al 1996;Sato et al 2006a;Sato et al 1999;Sato et al 2004;Sato et al 2002;Sato et al 2001;Sato et al 2003;Sato et al 2000;Sato et al 2006b;Steele 1985;Steele et al 1989b;…”