Selective Identification of Sperm Fused with the Surface of Echinoderm Eggs by DNA‐Specific Bisbenzimide (Hoechst) Fluorochromes

When unfertilized echinoderm eggs are treated with the DNA‐specific bisbenzimide fluorochrome Hoechst 33342 and then fertilized with unlabeled sperm, a single spermatozoan bound to the egg surface becomes fluorescent. Several lines of evidence, including correlative scanning electron microscopic studies, indicate that the fluorescent sperm is, in fact, the fertilizing sperm which acquires fluorescence as a consequence of membrane fusion between the sperm and egg. Comparative studies show that several fluorochromes structurally related to H33342 can be used to selectively identify the fertilizing sperm at the egg surface and that H33258 possesses a distinct advantage when used to visualize the male and female pronuclei in eggs fixed prior to fluorochrome exposure. Finally, none of the fluorochromes tested in these studies have any discernible effect on development from the first cell division through the pluteus larva stage. These observations suggest that the fluorochrome‐transfer technique for identifying the fertilizing sperm may be useful in a wide variety of studies of gamete interaction as a simple and rapid cytological indicator for sperm‐egg fusion.