Immuno-Electron Microscope Analysis of the Surface Layers of the Unfertilised Sea Urchin Egg

Abstract

The response of unfertilised Paracentrotus lividus eggs to T-globulin fractions of antisera against isolated homologous jelly coat substance or homologous homogenates of jellyless eggs has been studied at the ultrastructural level. The antijelly T-globulin caused precipitation of the jelly layer, the density of precipitation varying between different eggs and being proportional to the T-globulin concentration. Agglutination of the .jelly substance of adjacent eggs, which is species specific, occurred frequently with higher T-globulin concentrations. Antiegg T-globulins (from antiserum against total homogenates of jelly-free eggs or the heat-stable fraction thereof) did not produce these effects. Instead, these 3'-globulins caused various structural alterations mostly representing stages in parthenogenetic activation. This species-specific activation was induced by the reaction of antibodies with some heat-stable egg antigens different from those involved in jelly precipitation. Surface alterations included the formation of small papillae, membrane blisters, hyaline layer, and activation membrane, the release of material from the cell surface, and the breakdown of cortical granules. These alterations were dependent on both T-globulin concentration and the variable reactivity among different females. Aster formation, found intracellularly, verified that the surface responses represented real parthenogenetic activation and were not the result of immune lysis. No such alterations appeared in the controls.

Immuno-Electron Microscope Analysis of the Surface Layers of the Unfertilised Sea Urchin Egg

“…The same rabbit antisera against P. lividus eggs or jelly substance described in the previous papcr (5) as well as antisperm serum (19) were used, and the experiments were again performed with chromatographically isolated T-globulin (28). In addition, the isolated T-globulin of a sheep anti-rabbit T-globulin serum was used for indirect staining.…”

“…Absorption of the T-globulin preparations for specific removal of antibody was performed as described in the previous paper (5). The efficiency of absorption was checked in the light microscope (I, 3) and in the electron microscope by addition of ferritin conjugate to eggs treated with absorbed T-globulin.…”

“…The presence of a number of surface antigens in the sea urchin egg has been demonstrated and it has been shown that these antigens have a close link with specific changes taking place in different parts of the egg surface (1)(2)(3)(4)(5). It was therefore considered desirable to localise these antigens within the egg ultrastructure.…”

“…Other methods of making the antibody molecule sufficiently dense to be detected with the electron microscope have been used by coupling antisera to uranium (22) or to mercury (23,24), but ferritin labelling appears to be easier to detect and is preferable in most cases. Unmodified antibody can also be visualised for electron microscopy (5,13,(25)(26)(27).…”

“…Experiments have been made on living sea urchin eggs prepared for electron microscopy with a view to correlating immunochemical (1) and ultrastructural studies (5). In particular, use has been made of two layers of antisera in order to facilitate the analysis of a greater number and variety of antibodies.…”