The extracellular matrix surrounding Xenopus laevis eggs includes three morphologically distinct jelly layers designated J1, J2, and J3 from the innermost to outermost. Previously, using the quick-freeze, deep-etch, rotary-shadow technique, we found that each layer has a unique fibrillogranular ultrastructure. In this study, we show that the fibrillar network is composed of high-molecular-weight glycoconjugates, while the globular material consists of low-molecular-weight proteins some of which are released into the aqueous medium. Analysis by SDS-PAGE and differential staining of individually dissected jelly layers shows that both J1 and J2 contain three high-molecular-weight, acidic, Alcian blue-straining components (450, 630, and 900 kDa), while J3 contains two high-molecular-weight components that strain with PAS but not with Alcian blue. Each jelly layer also contains low-molecular-weight proteins from 75 to 250 kDa that do not stain with PAS or Alcian blue. Chromatography of whole egg jelly on a Sephacryl 500 column resulted in isolation of the major Alcian blue staining band (630 kDa) which eluted first, and two PAS staining bands which eluted second. Rotary-shadowing demonstrated that these high-molecular-weight glycoconjugates are long and branched, suggesting that they are major constituents of the jelly fiber network. SDS-PAGE analysis shows that these networks are stable for at least 16 hr after eggs are oviposited. In contrast, the low-molecular-weight globular proteins which constitute 30% of the total jelly protein are steadily released into the surrounding medium.

Xenopus laevis eggs are surrounded by an extracellular matrix consisting of a vitelline envelope, and three jelly layers, J1, J2, and J3 (from egg surface outward). The jelly layers vary in thickness (about 150, 15 and 200 μm for J1, J2 and J3 respectively) but all are translucent allowing observation of sperm penetration. Video microscopy demonstrated that sperm are able to penetrate and traverse J3 at velocities approaching 30 μm/s. Sperm swim through jelly in a corkscrew-like manner with their rotational and forward velocities being tightly coupled at about 30°/μm forward travel. They are propelled by whip-like power strokes involving hairpin bends in the flagellum that are generated every 180° of rotation and which are propagated from base to tip. The overall trajectories of individual sperm are quite variable. Many sperm head directly for J2 but some do not, these swimming circumferentially, or even away from the egg surface. Most sperm (over 97%) that enter the jelly do not get to the egg surface but are stopped at a variety of positions within J3 or at the outer surface of J2. Efficient sperm penetration and passage through the jelly layers requires a low electrolyte concentration in the surrounding medium, and is inhibited by the lectin wheat germ agglutin (WGA) in a dose-dependent manner. WGA does not block sperm penetration of J3 but does block further progression towards the egg surface. This observation suggests that sperm motility within the jelly is dependent on the carbohydrate moieties of the large glycoconjugates present, and that their alteration by WGA binding accounts for the inability of sperm to reach the egg surface and fertilise the egg.