| Abstract: | Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc. |
