Identification of PLCγ-Dependent and -Independent Events during Fertilization of Sea Urchin Eggs

Abstract

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.

Protein tyrosine kinase‐dependent release of intracellular calcium in the sea urchin egg

“…Our conclusion on the PLC␤-dependent Ca 2+ release in sea urchin eggs during fertilization contrasts with recent reports of inhibiting the sea urchin fertilization response by preloading eggs with a bovine PLC␥ SH2 domain fusion protein, which presumably blocked PLC␥-dependent phospholipid hydrolysis (Carroll et al 1999;Shearer et al 1999). A possible explanation for the difference in results with PTK inhibitors and fusion protein is that the level of inhibitors might still be insufficient to completely block tyrosine kinase activity (Livingston et al 1998).…”

Flipping the switch: How a sperm activates the egg at fertilization

“…In line with this result, microinjection of an antagonist of cADPR, 8-amino-cADPR, decreased the duration of the fertilization Ca 2ϩ transient, suggesting that cADPR plays a major part in sustaining the Ca 2ϩ signal. This is in contrast to IP 3 production, which precedes, and coincides with, the fertilization Ca 2ϩ wave, but which is short lived (Ciapa and Whitaker, 1986;Carroll et al, 1999;Thaler et al, 2004).…”

Calcium signalling in early embryos

“…The Src kinase stimulates PLCg to produce InsP 3 (Giusti et al 1999a(Giusti et al , 2000bShearer et al 1999;Jaffe et al 2001;Runft Linda et al 2004). SH2 domains inhibitory to PLCg and Src-family kinases will block fertilization in sea urchins, starfish, zebrafish and ascidians (Carroll et al 1997(Carroll et al , 1999Runft et al 1999Runft et al , 2002Shearer et al 1999;Rongish & Kinsey 2000;Kinsey William et al 2003), but not in mammals or frogs (Mehlmann et al 1998;Mehlmann & Jaffe 2005). Nonetheless, in frogs, Src-related tyrosine kinase co-immunoprecipitates with PLCg after fertilization and complex formation is blocked by the protein tyrosine kinase inhibitor PP1 (Sato et al 2000).…”