Purification of the sperm-binding factor and identification of a sperm attack molecule from the egg of the sea urchin, Hemicentrotus pulcherrimus.
A sperm-binding factor, which seems to have a primary role in binding sperm to the egg, was isolated from the egg surface of Hemicentrotus pulcherrimus and purified by monitoring the neutralization of the fertilization inhibition exerted by Fab fragments against crude sperm-binding factor. An improved purification for this sperm-binding factor is described in the present paper. The preparation of purified sperm-binding factor revealed one major protein band with an apparent molecular weight of 61,000 after sodium dodecylsulfate-polyacrylamide gel electrophoresis. A substance with the fertilization inhibitory effect on sperm, was isolated by diethylaminoethyl (DEAE)-Sephadex column chromatography in the course of purification of the sperm-binding factor and termed "sperm attack molecule." One precipitin line was formed between the sperm attack molecule and anti-crude sperm-binding factor serum in a double-immunodiffusion test. Fab fragments were prepared against partially purified sperm-binding factor or sperm attack molecule, and the effect of these Fab fragments on eggs was investigated. Anti-sperm-binding factor Fab fragments inhibited the fertilizability of eggs, whereas anti-sperm attack molecule Fab fragments did not. However, anti-sperm attack molecule Fab fragments impaired elevation of the vitelline layer. It is possible that the sperm attack molecule prevents polyspermy. Sperm attack molecule contains 3.7% neutral sugars. Its inhibitory effect was cancelled by trypsin or heat.
Yoshida
M
M
Department of Biology, Faculty of Science, Nagoya University, Japan.
Aketa
K
K
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D005260:Female; D005306:Fertilization / Q000187:drug effects; D005779:Immunodiffusion; D007140:Immunoglobulin Fab Fragments; D008297:Male; D008970:Molecular Weight; D010063:Ovum / Q000032:analysis* / Q000502:physiology; D012617:Sea Urchins; D013094:Spermatozoa / Q000502:physiology*; D014357:Trypsin / Q000494:pharmacology
D007140:Immunoglobulin Fab Fragments; D014357:Trypsin