Fig. 9. Detection of PECAM-1 in human spermatozoa. The presence of a PECAM-1 homologue in human spermatozoa was confirmed by immunoblotting and immunolocalization using anti-PECAM-1 antibodies. (A) Human sperm and platelets were solubilized in SDS extraction buffer and the proteins resolved by 1D SDS-PAGE before being transferred to nitrocellulose. Blots were successively probed with normal rabbit serum, polyclonal anti-PECAM-1 antibodies and with biotinylated WGA lectin. Cross-reactive bands of approximately 125 kDa were observed in both preparations using the anti-PECAM and WGA probes. (B) A protein band of identical molecular mass was also detected using a panel of three additional anti-PECAM-1 monoclonal antibodies. (C) To confirm the affinity of the sperm PECAM-1 molecule for WGA, this lectin was used to isolate binding partners from a pool of solubilized platelet and sperm proteins. These proteins were resolved by SDS-PAGE, blotted onto nitrocellulose and probed successively with normal rabbit serum and anti-PECAM-1 antibodies. An additional control in which streptavidin beads were incubated directly with solubilized sperm and platelet proteins in the absence of biotinylated WGA was also included to preclude the possibility of non-specific affinity purification. (D) Histochemical staining of live human spermatozoa with anti-PECAM-1 and WGA revealed the respective antigens were present on the majority of the surface of greater than 90% of spermatozoa.