PURIFICATION OF BOVINE ESTRUS-ASSOCIATED PROTEIN AND LOCALIZATION OF BINDING ON SPERM

An oviduct-specific, estrus-associated glycoprotein (EAP) of 85-95 kDa is detectable in both conditioned medium (CM) from oviductal explants and cannula-derived oviductal fluid (ODF). The objectives of this stu dy were to purify EAP from both ODF and CM, to characterize the glycos ylation of EAP, and to localize binding of EAP on sperm. EAP was purif ied from ODF by ammonium sulfate precipitation and ammonium sulfate ba ck-extraction followed by electroelution from one-dimensional SDS-PAGE gels. EAP was recovered from CM by electroelution from sos-PAGE gels. Purified EAP was used as antigen to produce polyclonal antibodies (an ti-EAP), and the specificity of anti-EAP was demonstrated as a single band in Western blots of ODF. N-linked sugar residues were enzymatical ly removed from EAP purified from ODF. The resulting molecule was 7 kD a smaller and was similar in molecular mass to EAP derived from CM. Sp erm were incubated with S-35-proteins synthesized by oviductal explant cultures. Autoradiographs of solubilized sperm membranes contained a 90-95-kDa protein that was confirmed by Western blotting to be EAP. EA P was localized on permeabilized membranes of sperm incubated in ODF b y immunocytochemistry using polyclonal anti-EAP. EAP was bound to the head and middle piece of 97% of the sperm incubated for 4 h in ODF. Fr om these results, we concluded that N-linked sugars account for approx imately 8% of the molecular mass of ODF-derived EAP and that EAP binds to the head and middle piece of sperm.