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Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, SUNY at Stony Brook,
Stony Brook, New York 11794-5215
A quantitative assay was developed to study
the interaction of Xenopus laevis sperm and eggs. Using
this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of
the fertilized egg. To understand the molecular basis of
sperm binding to the egg vitelline envelope (VE), a
competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding
in a concentration-dependent manner. Individual VE
proteins were then isolated and tested for their ability
to inhibit sperm binding. Of the seven proteins in the
VE, two related glycoproteins, gp69 and gp64, inhibited
sperm-egg binding. Polyclonal antibody was prepared
that specifically recognized gp69 and gp64. This gp69/64
specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm
binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs
with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64
also inhibited sperm-egg binding and this inhibition was
abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the
gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these
glycoproteins may play a critical role in this process.
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