Calcium/calmodulin and calmodulin kinase II stimulate hyperactivation in demembranated bovine sperm

Hyperactivated motility is observed among sperm in the mammalian oviduct near the time of ovulation. It is characterized by high-amplitude, asymmetrical flagellar beating and assists sperm in penetrating the cumulus oophorus and zona pellucida. Elevated intracellular Ca2+ is required for the initiation of hyperactivated motility, suggesting that calmodulin (CALM) and Ca2+/CALM-stimulated pathways are involved. A demembranated sperm model was used to investigate the role of CALM in promoting hyperactivation. Ejaculated bovine sperm were demembranated and immobilized by brief exposure to Triton X-100. Motility was restored by addition of reactivation medium containing MgATP and Ca2+, and hyperactivation was observed as free Ca2+ was increased from 50 nM to 1 mu M. However, when 2.5 mM Ca2+ was added to the demembranation medium to extract flagellar CALM, motility was not reactivated unless exogenous CALM was readded. The inclusion of anti-CALM IgG in the reactivation medium reduced the proportion hyperactivated in 1 mu M Ca2+ to 5%. Neither control IgG, the CALM antagonist W-7, nor a peptide directed against the CALM-binding domain of myosin light chain kinase (MYLK2) inhibited hyperactivation. However, when sperm were reactivated in the presence of CALM kinase II (CAMK2) inhibiting peptides, hyperactivation was reduced by 75%. Furthermore, an inhibitor of CAMK2, KN-93, inhibited hyperactivation without impairing normal motility of intact sperm. CALM and CAMK2 were immunolocalized to the acrosomal region and flagellum. These results indicate that hyperactivation is stimulated by a Ca2+/CALM pathway involving CAMK2.