1 2

		Procedure 1a) To inhibit the fast block to polyspermy, resuspend eggs in ASW or in nicotine (Sigma Chemical Company) at 0.25 mM, 0.5 mM and 1 mM, (dilute 1.25 ul, 2.5 ul and 5 ul of a 40% solution of nicotine free base in 10 ml ASW and use immediately). Fertilize eggs with diluted sperm as described in the basic protocol. Be sure to allow sperm to activate for at least 5 minutes before adding to eggs. 1b) To inhibit the slow block to polyspermy, resuspend eggs in ASW or in soybean trypsin inhibitor (SBTI, Sigma Chemical Company,) at 0.4 mg/ml (1/10 volume of 4 mg/ml stock). Fertilize eggs with diluted sperm as described in the basic protocol. Be sure to allow sperm to activate for at least 5 minutes before adding to eggs. 2. Examine eggs after 10 minutes to score for the presence (or absence) of the fertilization envelope and periodically to observe the events associated with the first cleavage division. References Gilbert, S.F., (2000), Developmental Biology, Sinauer Associates, Sunderland, MA Just, E.E., (1919), The fertilization reaction in Echinarachinus parma, Biol. Bull., 36:1-10. Jaffe, L.A., (1980), Electrical polyspermy block in sea urchins: nicotine and low sodium experiments, Develop. Growth and Differ., 22:503-507. Vacquier, V.D., Tegner, M.J., and Epel, D., (1973), Protease release from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. , 80:111-119.