Rafaella De Martino - Subcellular Localization of Antisperm-Directed Monoclonal Antibodies

Raffaella De Martino, Jennifer J. Venditti, and Barry Bean
Lehigh University, Bethlehem, PA
Previous investigations in this laboratory generated a collection of monoclonal antibodies (mAbs) directed against superficial antigens of human sperm cells (Tang & Bean, J Androl, 1998). More recent preliminary results suggest that treatment of sperm with some of these antibodies can alter the expected pathway toward acrosomal exocytosis.  These observations raise the possibility that those target antigens may have important functions in events preceding fertilization. Here we report the subcellular distribution of binding sites for two antisperm mAbs in Percoll® washed, capacitated, and acrosome induced cell populations.  Human sperm cells were dried onto slides, fixed with 100% methanol and incubated with primary, mouse anti-human monoclonal antibodies 77 or 37.  Following primary antibody, slides were washed and incubated with RITC-conjugated goat anti-mouse IgG secondary antibody. Staining patterns were evaluated using a Zeiss LSM 510 confocal microscope.  mAb 77 revealed the brightest localization signal in Percoll® washed and capacitated cells, both showing diffuse head staining.  Immunolocalization of acrosome induced cells with mAb 77 showed no head staining.  mAb 37 showed minimal staining of Percoll® washed cells.  Following capacitation, whole head staining was apparent.  Acrosome induced cells had the brightest mAb 37 signal localized in the posterior head region.  These observations confirm that these antibodies recognize different antigens of the sperm cell, both of which experience changes in their location as sperm capacitate and undergo acrosomal exocytosis.