ABSTRACT
The protocols outlined here hopefully will provide researchers with healthy, beautiful echinoderm oocytes, eggs, and embryos for experimental use. The large size of echinoderm oocytes and eggs, the ease with which they can be manipulated, and (in many species) their optical clarity, make them an ideal model system for studying not only the events specific to oocyte maturation and fertilization, but also for investigating more general questions regarding cell cycle regulation in an in vivo system. The quick rate at which development proceeds after fertilization to produce transparent embryos and larva makes the echinoderm an advantageous organism for studying deuterostome embryogenesis. Continued use of the echinoderms as model systems will undoubtedly uncover exciting answers to questions regarding fertilization, cell cycle regulation, morphogenesis, and how developmental events are controlled.
Subject(s)
Aquaculture/methods , Cell Culture Techniques/methods , Echinodermata/growth & development , Embryo, Nonmammalian/embryology , Ovum/physiology , Animals , Echinodermata/embryology , Embryo, Nonmammalian/cytology , Female , Larva/cytology , Larva/growth & development , Male , Models, Animal , Ovum/cytology , Reproduction/physiologyABSTRACT
At fertilization, eggs undergo a cytoplasmic free Ca2+ rise, which is necessary for stimulating embryogenesis. In starfish eggs, studies using inhibitors designed against vertebrate proteins have shown that this Ca2+ rise requires an egg Src family kinase (SFK) that directly or indirectly activates phospholipase C-gamma (PLC-gamma) to produce IP3, which triggers Ca2+ release from the egg's endoplasmic reticulum (ER) [reviewed in Semin. Cell Dev. Biol. 12 (2001) 45]. To examine in more detail the endogenous factors in starfish eggs that are required for Ca2+ release at fertilization, an oocyte cDNA encoding PLC-gamma was isolated from the starfish Asterina miniata. This cDNA, designated AmPLC-gamma, encodes a protein with 49% identity to mammalian PLC-gamma1. A 58-kDa Src family kinase interacted with recombinant AmPLC-gamma Src homology 2 (SH2) domains in a specific, fertilization-responsive manner. Immunoprecipitations of sea urchin egg PLC-gamma using an affinity-purified antibody directed against AmPLC-gamma revealed fertilization-dependent phosphorylation of PLC-gamma. Injecting starfish eggs with the tandem SH2 domains of AmPLC-gamma (which inhibits PLC-gamma activation) specifically inhibited Ca2+ release at fertilization. These results indicate that an endogenous starfish egg PLC-gamma interacts with an egg SFK and mediates Ca2+ release at fertilization via a PLC-gamma SH2 domain-mediated mechanism.
Subject(s)
Calcium/metabolism , Fertilization/physiology , Ovum/metabolism , Starfish/metabolism , Type C Phospholipases/metabolism , Amino Acid Sequence , Animals , Molecular Sequence Data , Phospholipase C gamma , Type C Phospholipases/isolation & purification , src Homology DomainsABSTRACT
A centrally important factor in initiating egg activation at fertilization is a rise in free Ca(2+) in the egg cytosol. In echinoderm, ascidian, and vertebrate eggs, the Ca(2+) rise occurs as a result of inositol trisphosphate-mediated release of Ca(2+) from the endoplasmic reticulum. The release of Ca(2+) at fertilization in echinoderm and ascidian eggs requires SH2 domain-mediated activation of a Src family kinase (SFK) and phospholipase C (PLC)gamma. Though some evidence indicates that a SFK and PLC may also function at fertilization in vertebrate eggs, SH2 domain-mediated activation of PLC gamma appears not to be required. Much work has focused on identifying factors from sperm that initiate egg activation at fertilization, either as a result of sperm-egg contact or sperm-egg fusion. Current evidence from studies of ascidian and mammalian fertilization favors a fusion-mediated mechanism; this is supported by experiments indicating that injection of sperm extracts into eggs causes Ca(2+) release by the same pathway as fertilization.
