Agglutination of Jelly Coat and Cortical Granule Components and the Block to Polyspermy in the Amphibian Xenopus laevis

A block to polyspermy in amphibians is established at fertilization by the conversion of the vitelline envelope to the fertilization envelope. In Xenopus laevis a major ultrastructural change in the envelope at fertilization is the appearance of an electron-dense layer, termed the F layer, between the envelope and the inner-most jelly coat layer, J(1). The F layer is derived, at least in part, from materials released from the cortical granules. Further definition of the origin and chemical nature of the F layer was sought by using isolated cortical granule (CG) exudate and jelly coat layer J(1). In double diffusion experiments, the isolated components interacted in an agglutination reaction producing a band of precipitation. The agglutination involved alpha-galactoside residues and metal ions (Ca(++)). Employing chemically modified jelly, we demonstrated that sulfhydryl-disulfide interchanges were not involved in the agglutination and, with (35)S-labeled jelly, that the agglutinating J(1) component possessed sulfate esters. Both the CG exudate and the J(1) components contained carbohydrate, as evidenced by their lectin reactivity. A number of ionic polymers, both natural and synthetic, were tested as chemical analogs of CG exudate and J(1); none gave an agglutination band. Dissolved jelly coat material from eggs of two different species of frogs agglutinated with CG exudate, while jelly from sea urchin eggs and hyaluronic acid from mammalian eggs did not. Thus, the agglutination reaction was chemically and phylogenetically specific. An electron-dense layer, similar to the F layer, formed on the outer of the vitelline envelope when jellied unfertilized eggs were immersed in CG exudate; such eggs were not fertilizable. We suggest that in Xenopus laevis, and perhaps other organisms as well, an agglutination type of reaction between cortical granule components and egg integuments is a participant in the structural and molecular events establishing a block to polyspermy.