Sea Urchin Fertilization

Gametes are collected as described . To remove some of their jelly coat,wash eggs several times in ASW by allowing them to settle and gently pouring off excess liquid. Transfer enough eggs to barely cover the bottom to 10 ml ASW in a 50 ml beaker. Sperm will only remain viable for a short time in water. Collect them in a dry petri dish. Before use, dilute 50 ul of sperm in 5 ml ASW (diluted sperm). Use 0.1 ml of diluted sperm to fertilize eggs in 10 ml of ASW. Check for the presence of fertilization membranes after 5 and 10 minutes. Discard the ASW containing sperm and replace with 40 ml fresh ASW. For long term cultures, scale up 4 fold and transfer to shallow finger bowl after washing fertilized embryos.