PURIFICATION AND CHARACTERIZATION OF PROTEIN D E, A PUTATIVE SPERM-BINDING PROTEIN INVOLVED IN FERTILIZATION/

We describe a method for the efficient purification of a 32 Kd glycopr otein from rat epididymal tissue. The glycoprotein was purified by gel filtration, ion-exchange, affinity, and reverse phase high pressure l iquid chromatography. The highly purified glycoprotein was radiolabele d with an iodinatable, cleavable, photoreactive cross-linking agent, 1 -[N-(2-hydrox-5-azidobenzoyl)-2-aminoethyl]- 4-( -hydroxysuccinimidyl) -succinate (HAHS). The soluble radiolabled glycoprotein was bound to w ashed epididymal spermatozoa in a time-dependent, saturable, and rever sible manner. Scatchard analysis demonstrates that there are approxima tely 3,403 binding sites/spermatozoon. The binding efficiency (K-d) fo r spermatozoa was similar to 2.0 x 10(-10) M. The function of this gly coprotein was verified by using an in vivo artificial insemination fer tilization assay. The fertility rate for control spermatozoa was simil ar to 53%, but the rate for spermatozoa exposed to polyclonal anti-gly coprotein antibodies was only 5%. These data suggest that the binding of the glycoprotein to the surface of rat spermatozoa is mediated by a receptor-type mechanism and is involved in the fertilization process.