Ca2+ signalling during fertilization of echinoderm eggs.

The Ca2+ rise at fertilization of echinoderm eggs is initiated by a process requiring the sequential activation of a Src family kinase, phospholipase C gamma, and the inositol trisphosphate receptor/channel in the endoplasmic reticulum. The consequences of the Ca2+ rise include exocytosis of cortical granules, which establishes a block to polyspermy, and inactivation of MAP kinase, which functions in linking the Ca2+ rise to the reinitiation of the cell cycle.

Evidence that fertilization activates starfish eggs by sequential activation of a Src-like kinase and phospholipase cgamma.

Recent evidence has indicated a requirement for a Src family kinase in initiating Ca(2+) release at fertilization in starfish eggs (Giusti, A. F., Carroll, D. J., Abassi, Y. A., Terasaki, M., Foltz, K. R., and Jaffe, L. A. (1999) J. Biol. Chem. 274, 29318-29322). We now show that injection of Src protein into starfish eggs initiates Ca(2+) release and DNA synthesis, as occur at fertilization. These responses depend on the phosphorylation state of the Src protein; only the kinase active form is effective. Like Ca(2+) release at fertilization, the Ca(2+) release in response to Src protein injection is inhibited by prior injection of the SH2 domains of phospholipase Cgamma. These findings support the conclusion that in starfish, sperm-egg interaction causes egg activation by sequential activation of a Src-like kinase and phospholipase Cgamma. Injection of the SH2 domain of Src, which inhibits Ca(2+) release at fertilization, does not inhibit Ca(2+) release caused by Src protein injection. This indicates that the requirement for a Src SH2 domain interaction is upstream of Src activation in the pathway leading to Ca(2+) release at fertilization.

Evidence that Src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization in sea urchin eggs.

The initiation of Ca(2+) release from internal stores in the egg is a hallmark of egg activation. In sea urchins, PLCgamma activity is necessary for the production of IP(3), which leads to the initial rise in Ca(2+). To examine the possible function of a tyrosine kinase in activating PLCgamma at fertilization, sea urchin eggs were treated with the specific Src kinase inhibitor PP1 or microinjected with recombinant Src-family SH2-domain proteins, which act as dominant interfering inhibitors of Src-family kinase function. Both modes of inhibiting Src-family kinases resulted in a specific and dose-dependent delay in the onset of Ca(2+) release from the endoplasmic reticulum at fertilization. The rise in cytoplasmic pH at fertilization also was inhibited by microinjection of Src-family SH2-domain proteins. Further, an antibody directed against Src-type kinases recognized a protein of ca. M(r) 57K that was enriched in the membrane fraction of eggs. The kinase activity of this protein was stimulated rapidly and transiently at fertilization, as measured by autophosphorylation and by phosphorylation of an exogenous substrate. Together, these data indicate that a Src-type tyrosine kinase is necessary for the initiation of Ca(2+) release from the egg ER at fertilization and identify a Src-type p57 protein as a candidate in the signaling pathway leading to this Ca(2+) release.

Expression of a Scr/Hox5 gene in the larval central nervous system of the gastropod Haliotis, a non-segmented spiralian lophotrochozoan.

Hox genes encode a set of evolutionarily conserved transcription factors that regulate anteroposterior patterning mechanisms in insects and vertebrates and are expressed along this axis in a range of bilaterians. Here we present the developmental expression of a Scr/Hox5 gene in the gastropod mollusc Haliotis. In Haliotis, embryogenesis yields a non-feeding trochophore larva that subsequently develops into the veliger larva, which possesses many of the characteristics of the adult body plan. Quantitative RT-PCR analysis reveals that this gene, which is called Hru-Hox5, is first expressed in the trochophore larva. Hru-Hox5 transcript prevalence increases continually through larval development until metamorphic competence develops in the veliger and then again over the first four days of metamorphosis. In situ hybridization reveals that larval expression of Hru-Hox5 is restricted primarily to the primordial and newly formed branchial ganglia, located between the anterior cerebral-pleuropedal ganglionic complex and the posterior visceral ganglia. The expression of Hru-Hox5 in the central region of the abalone CNS is similar to that observed for its orthologue (Lox20) in the leech, suggesting that Hox5 genes were used, along with other Hox genes, to pattern the CNS of the ancestral spiralian lophotrochozoan.

Requirement of a Src family kinase for initiating calcium release at fertilization in starfish eggs.

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase Cgamma-mediated production of inositol trisphosphate (IP(3)), indicating that a tyrosine kinase is a likely upstream regulator. Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase Cgamma and a Src family kinase, we examined whether a Src family kinase was required for Ca(2+) release at fertilization. To inhibit the function of kinases in this family, we injected starfish eggs with the SH2 domains of Src and Fyn kinases. This inhibited Ca(2+) release in response to fertilization but not in response to injection of IP(3). We further established the specificity of the inhibition by showing that the SH2 domains of several other tyrosine kinases (Abl, Syk, and ZAP-70), and the SH3 domain of Src, were not inhibitory. Also, a point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca(2+) release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP(3)-mediated Ca(2+) release at fertilization in starfish eggs.

Evidence that a starfish egg Src family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization.

The initiation of calcium release at fertilization in the eggs of most animals relies on the production of IP3, implicating the activation of phospholipase C. Recent work has demonstrated that injection of PLC-gamma SH2 domain fusion proteins into starfish eggs specifically inhibits the initiation of calcium release in response to sperm, indicating that PLC-gamma is necessary for Ca2+ release at fertilization [Carroll et al. (1997) J. Cell Biol. 138, 1303-1311]. Here we investigate how PLC-gamma may be activated, by using the PLC-gamma SH2 domain fusion protein as an affinity matrix to identify interacting proteins. A tyrosine kinase activity and an egg protein of ca. Mr 58 K that is recognized by an antibody directed against Src family tyrosine kinases associate with PLC-gamma SH2 domains in a fertilization-dependent manner. These associations are detected by 15 s postfertilization, consistent with a function in releasing Ca2+. Calcium ionophore treatment of eggs did not cause association of the kinase activity or of the Src family protein with the PLC-gamma SH2 domains. These data identify an egg Src family tyrosine kinase as a potential upstream regulator of PLC-gamma in the activation of starfish eggs.

Surface localization of the sea urchin egg receptor for sperm.

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.