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 study were to purify EAP from both ODF and CM, to characterize the glycosylation of EAP, and to localize binding of EAP on sperm. EAP was purified from ODF by ammonium sulfate precipitation and ammonium sulfate back-extraction followed by electroelution from one-dimensional SDS-PAGE gels. EAP was recovered from CM by electroelution from SDS-PAGE gels. Purified EAP was used as antigen to produce polyclonal antibodies (anti-EAP), and the specificity of anti-EAP was demonstrated as a single band in Western blots of ODF. N-linked sugar residues were enzymatically removed from EAP purified from ODF. The resulting molecule was 7 kDa smaller and was similar in molecular mass to EAP derived from CM. Sperm were incubated with 35S-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. EAP was localized on permeabilized membranes of sperm incubated in ODF by 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. From these results, we concluded that N-linked sugars account for approximately 8% of the molecular mass of ODF-derived EAP and that EAP binds to the head and middle piece of sperm.
King
R S
RS
Department of Dairy and Animal Science, Pennsylvania State University, University Park 16802.
Killian
G J
GJ
D000581:Amidohydrolases / Q000378:metabolism; D000645:Ammonium Sulfate; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability; D011232:Chemical Precipitation; D017077:Culture Media, Conditioned; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004971:Estrus; D005187:Fallopian Tubes / Q000737:chemistry; D005260:Female; D006031:Glycosylation; D007150:Immunohistochemistry; D008297:Male; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism*
D017077:Culture Media, Conditioned; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000645:Ammonium Sulfate