Role of calpain in human sperm activated by progesterone for fertilization

We evaluated the activation of mu-calpain in progesterone-activated human sperm. Semen collected from fertile donors with informed consent was liquefied and subjected to percoll gradient centrifugation. After exposure to different concentrations of progesterone, the samples were used for immunostaining, SDS-PAGE and Western blot analysis. An increase of the intracellular free calcium concentration in the sperm following the addition of progesterone was observed using fura-2 AM. Immunostaining using an antibody against active mu-calpain produced 6 distinct staining patterns: (1) the acrosome, (2) an equatorial segment, (3) the whole head, (4) the neck, (5) the neck and tail or (6) unstained sperm. After addition of progesterone, the predominant type changed from the neck type (90%) to the neck and tail type (79%). Western blot analysis using a pro-mu-calpain and a mu-calpain domain III antibody revealed autodigestion of mu-calpain, indicating activation by progesterone. Using calpain-specific inhibitors it was shown that calpain activation contributes to sperm motility as well as to the acrosome reaction. These results suggest the possibility that activation of mu-calpain in human sperm by progesterone plays an important role in fertilization.