Immunocytochemical study of the distribution of a ganglioside in sea urchin eggs

An antibody against M5 ganglioside (NeuGca2-6Glc/91-lCer), the dominant ganglioside in the eggs of the sea urchin, Anthocidaris crassispina, was purified by affinity chromatog-raphy from rabbit antiserum against crude ganglioside of the eggs. The specificity of the antibody was verified by enzyme-linked immunosorbent assay and TLC immunostaining. M5 ganglioside was also the major one in the eggs of another sea urchin, Hemicentrotus pulcherrimus, as judged from TLC analyses including immunostaining. Cryostat-sections of H. pulcherrimus eggs were examined to determine the distribution of M5 ganglioside by indirect immunofluorescence microscopy with the antibody. Before fertilization, the egg cortex was highly stained, while the other part of cytoplasm was uniformly but much more weakly stained. After fertilization, the staining rapidly decreased in the cortex and was restricted to a very thin peripheral layer and to cytoplasmic patches. The immunore-activity was also observed in the esophagus and the somatic cells of the testis, but the spermatozoa were never stained with the antibody. Knowledge of the distribution of glycosphingolipids in cells should provide important clues as to their biological func-tions. They are generally believed to exist mainly in the plasma membrane and are thus anticipated to function in cell-to-cell interactions. Fertilization is an event involving direct cell interactions and seems to be a good system, especially in some invertebrates such as sea urchins, to explore the biological functions of glycosphingolipids. Ohsawa and Nagai (1) first showed in invertebrates that sialoglycosphingolipids reside on the surface of the plasma membrane of sea urchin spermatozoa. Sugita et al. (2) reported that a glucuronic acid-containing ceramide octa-saccharide (Lipid IV) is localized at the apex of the sperm head of the fresh water bivalve, Hyriopsis schlegelii. We have determined the structures of four gangliosides in the gametes of the sea urchin, Anthocidaris crassispina