Calcium Release at Fertilization of Xenopus Eggs Requires Type I IP3 Receptors, but Not SH2 Domain-Mediated Activation of PLCγ or Gq-Mediated Activation of PLCβ

Abstract

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). We then examine how IP(3) production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…Phenotypic analysis of such a mutant is needed to definitively conclude whether or not PLC serves as the trigger for Ca 2ϩ release in mouse. The specific PLC isoform used in Xenopus has not been resolved; PLC␥ is present and active (phosphorylated) in fertilized eggs, but its activation does not appear to occur through an egg SFK (Runft et al, 1999;Sato et al, 2000). There is some evidence for a sperm-derived PLC in Xenopus because sperm extracts from Xenopus can induce an increase in Ca 2ϩ upon injection into mouse eggs (Dong et al, 2000).…”

Ca2+ signaling differentiation during oocyte maturation

“…The Ca 2þ wave is followed by a sustained Ca 2þ plateau that lasts for several minutes (Busa and Nuccitelli, 1985;El Jouni et al, 2005). The Ca 2þ wave is due to Ca 2þ release from intracellular stores through the IP 3 receptor (IP 3 R), since it is inhibited by blocking IP 3 -dependent Ca 2þ release or PIP2 hydrolysis (Larabell and Nuccitelli, 1992;Nuccitelli et al, 1993;Runft et al, 1999). Moreover, a wave of IP 3 has been proposed to precede the Ca 2þ release wave in eggs at fertilization (Wagner et al, 2004).…”

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

“…PLCc isozymes are thought to operate in ascidian and echinoderm fertilization, as it was shown that injection of excess PLCc SH2 domain, acting in a dominant negative manner, prevented sperm-induced [Ca 2+ ] i release in eggs of these species (Carroll et al, 1997Shearer et al, 1999;Runft and Jaffe, 2000). On the contrary, using a similar approach, it was shown that [Ca 2+ ] i release in Xenopus and mouse fertilization occurred unperturbed in the presence of excess SH2 domains, suggesting that PLCc activation is not important in these species, or that it occurs by a different mechanism Runft et al, 1999). Toward this end, it is worth noting that injection of recombinant PLCc1 protein into mouse eggs resulted in the generation of fertilization-like [Ca 2+ ] i oscillations (Mehlmann et al, 2001).…”