LIGHT AND ELECTRON-MICROSCOPIC IMMUNOLOCALIZATION OF SPERM PROTEINS IDENTIFIED BY MONOCLONAL-ANTIBODIES FROM THE WORLD-HEALTH-ORGANIZATION-TASK-FORCE-ON-SPERM-ANTIGENS

Sperm antigens recognized by monoclonal antibodies (mAbs), S19, S69, S71, S72 and S77 submitted to the World Health Organization (WHO)-Sponsored Sperm Monoclonal Antibody Workshops were immunolocalized by light (LM) and transmission electron microscopy (TEM). S19 was surface reactive while mAbs S69, S71, S72 and S77 recognized internal antigens. Indirect immunofluorescence staining of permeabilized sperm with the mAbs revealed that S69 recognized an internal tail antigen, while S71, S72 and S77 recognized acrosomal proteins. Preservation of immunoreactivity after fixation in various combinations of glutaraldehyde, paraformaldehyde and tannic acid was evaluated for mAbs S69 to S77 using immunofluorescence microscopy. The epitopes recognized by these mAbs were adversely affected by these fixatives; therefore, pre-embedding immunogold staining was employed, prior to fixation, osmication, dehydration and embedding. Using this approach, the antigen recognized by mAb S19 was found associated with the plasmalemma of the head and tail of intact sperm. Monoclonal antibody S69 localized to the fibrous sheath. The mAbs S71, S72 and S77, which required sperm permeabilization to expose their acrosomal locus by LM, did not immunoreact with the plasmalemma at the TEM level. Ultrastructural examination of acrosome-reacted sperm revealed the localization of S71 and S77 on the inner and outer acrosomal membranes and with acrosomal matrix. The S72 antigen was associated with the inner and outer acrosomal membranes.