Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa

Mammalian sperm must undergo an acrosome reaction prior to penetration of t he zona pellucida and subsequent fusion with an oocyte. Sperm gain the capa bility to acrosome react after a period of capacitation, which primarily in volves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cel lular mechanisms that regulate capacitation and the acrosome reaction remai n unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as th e small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the ou ter acrosomal membrane of cynomolgus macaque sperm. After the acrosome reac tion, all of the anti-calpain antibodies labeled the acrosomal shroud, sugg esting that calpains are located throughout the cytoplasmic area between th e 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpai n activity and is also localized within the same cytoplasmic region as calp ains. The antibodies used for ultrastructural localization were also used t o probe Western blots of sperm extracts. Antibodies to either the mu- or m- calpain recognized an 80-kd protein, which is similar to the molecular weig hts of other ubiquitous calpains described. The small subunit (30 kd) was a lso recognized with a specific monoclonal antibody. An antibody to calpasta tin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome re action, or both.