Within about a minute of sperm contacting the egg plasma membrane, a fertilization envelope forms around the egg to prevent polyspermy (penetration by additional sperm). A hyaline layer forms directly on the surface of the egg plasma membrane to help hold the blastomeres in the dividing egg together. I had to remove the fertilization envelope and the hyaline layer in order to separate the blastomeres. To accomplish this I fertilized the eggs in filtered sea water (FSW). Within thirty seconds of fertilization I replaced the FSW with calcium-magnesium-free sea water. This prevents the fertilization envelope and hyaline layer from hardening, and allows one to remove them by sheering the eggs with a pipette. Once the eggs are denuded (their coats removed) they become very sticky, so I coated the culture dishes and glass knives used for surgery with BSA (Bovine Serum Albumin) to prevent the embryos from sticking to things and being damaged. The top two pictures show two 8-cell stage embryos of S. purpuratus - one with the fertilization envelope, and one without (denuded).
Without the fertilization envelope and hyaline layer, I was able to cut the eggs in half using a fine glass capillary, that I pulled over a flame. I cut some eggs in half at the 2-cell, and some at the 4-cell stage. The half-embryos proceeded to develop. This picture shows two embryos - a control (top left) and a half-embryo (bottom right) as they undergo gastrulation, by invagination, to form the gut. The experimental embryo is about half the size of the control.
I am attempting to rear these half-embryos all the way through to metamorphosis, so stay tuned to see the outcome and (hopefully) miniature juvenile urchins! See part two.