Changes of the major sperm maturation-associated epididymal protein HE5 (CD52) on human ejaculated spermatozoa during incubation in capacitation conditions

HE5 (CD52) is a glycoprotein which is secreted by the epididymis and which becomes inserted onto maturing spermatozoa. We have previously shown that, in cynomolgus monkey spermatozoa, changes occur upon maturation rendering c ryptic the epitope to the monoclonal antibody CAMPATH-1G; the recognition s ite is then re-exposed during incubation under capacitation conditions, The present study investigated human ejaculated spermatozoa during incubation under similar conditions, using monoclonal antibodies that recognize differ ent epitopes of the HE5 molecule comprising parts of the N-glycan (2E5) or peptide segments, including (CAMPATH-1G) or excluding (097) the glycosylpho sphatidylinositol (GPI) anchor, to reveal modifications of sperm surface HE 5, Flow cytometric analysis showed equally high percentages (similar to 90% ) of viable spermatozoa cross-reacting with the antibodies before and after 6 h incubation. However, during incubation, the staining intensity increas ed 57% with CAMPATH-1G, 31% with 097, but remained unchanged with 2E5, The lymphocyte CD52 antibody CF1D12 stained only similar to 10% of spermatozoa either before or after incubation, Western blotting of sperm protein extrac ts using lectins indicated an increase in the exposure of sialic acid resid ues of HE5 after incubation. These results suggest that during incubation i n capacitating conditions, there is an opening up of the HE5 glycoprotein m olecule, increasing accessibility of some sialic acid residues and of the c ore peptide, particularly the GPI anchor.