In this work, we have investigated the role of the sperm proteasome during in 
vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome 
activity was measured in extract and intact sperm using a specific substrate. In 
addition, sperm were treated with specific proteasome inhibitors and evaluated 
during IVF, binding to the zona pellucida, and progesterone- and zona 
pellucida-induced acrosome reactions. In other experiments, sperm membrane 
proteins were obtained resuspending them in Triton X-114, shaking vigorously and 
let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous 
phase and sperm membrane proteins in the detergent phase. In both phases, 
proteasome activity was measured. Labeling of cell surface sperm proteins was 
carried out with the cell-impermeable NHS-LC biotin, extracted with Triton 
X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were 
incubated with an anti-proteasome monoclonal antibody and evaluated by indirect 
immunofluorescence. The results indicate that sperm extracts as well as intact 
sperm had proteasome activity; the sperm proteasome was involved in IVF, 
specifically during sperm-zona pellucida binding and the acrosome reaction; 
soluble sperm membrane proteins exhibited proteasome activity; biotin 
experiments indicated the presence of proteasomes on the sperm surface, which 
was corroborated by indirect immunofluorescence experiments. All these 
observations indicate that the mouse sperm proteasome participates in the 
binding to the zona pellucida and the acrosome reaction and that there is a pool 
of proteasomes located on the sperm head.