Isolation of sperm bindin from the oyster (Crassostrea gigas)

Abstract

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosyl). The isolated acrosome granule content is ring-shaped and is 84% protein by welght. SDS-polyacrylamide gel electrophoresis of this material yields from 1 t o 4 bands of 65,000; 53,000: 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glycoprotein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm t o the egg during fertilization.

Oyster sperm bindin is a combinatorial fucose lectin with remarkable intra-species diversity

“…The methods presented here are a summary of those described by Brandriff et al (1978) and Moy et al (2008). Sperm are obtained and washed into filtered seawater (containing 10 mM MES, pH 6.0; MES-SW) by sedimentation at 1,000 xg (15 min) and resuspension with a spatula in fresh 25-100 vol MES-SW (all procedures are at 4°C).…”

“…The isolated AR appear as rings when viewed by phase contrast or transmission electron microscopy ( Fig. 2; The preparation for electron microscopy is described in Brandriff et al, 1978).…”

“…Early work showed that isolated AR agglutinate unfertilized oocytes (Brandriff et al, 1978). Digestion of oocytes with trypsin, followed by inhibition of the enzyme with SBTI, and removal of the digested oocytes, yielded a glycopeptide digest of the egg surface.…”

250 Million Years of Bindin Evolution

“…As Vacquier (1998) has pointed out, molecules that mediate fertilization•in contrast to those central to other basic life processes•often differ between taxa. For example, in the molluscan class Bivalvia, completely different proteins are involved in gamete recognition of oysters (Brandriff et al, 1978) and of mussels (Takagi et al, 1994). It is, therefore, not safe to assume without empirical evidence that bindin is present in all orders of echinoids, or that it has the same general structure as in the taxa in which it has already been characterized.…”

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“…6,11,12 Although acrosome of sperm from the oyster, Crassostrea gigas, is small, with a dimension of 0.3 µm in length and 0.8-0.9 µm in diameter, 13,14 it is about twice the size that of the sperm from Paphia. The length of the fully extended AP reported in various bivalves does not seem to correlate with the size of the acrosome.…”