Different calcium-dependent pathways control fertilisation-triggered glycoside release and the cortical contraction...

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probably dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction. Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10 kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30-40 s before the cortical contraction is triggered by fertilisation or ionomycin-induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium wave triggered by the sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation of ascidian eggs appears to involve two independent pathways involving calcium.