Cyclic AMP- and calmodulin-dependent phosphorylation of 21 and 26 kDa proteins in axoneme is a prerequisite for SAAF-induced motile activation in ascidian spermatozoa

Sperm activating and -attracting factor (SAAF), derived from the egg of the ascidian Ciona, activates sperm motility through adenosine 3':5'-cyclic mo nophosphate (cAMP)-synthesis. A demembranated preparation of intact immotil e sperm without SAAF was shown to require cAMP for reactivation. However, a demembranated preparation of intact motile sperm treated with SAAF did not require cAMP for reactivation, suggesting that cAMP is a prerequisite fact or for SAAF-dependent activation of sperm motility. Furthermore, a cAMP-dep endent protein kinase (PKA) inhibitor, H-89, was found to inhibit sperm mot ility. During in vivo or in vitro activation of sperm motility by SAAF or c AMP, a 26 kDa axonemal protein and 2.1 kDa dynein light chain were phosphor ylated, respectively, suggesting the involvement of PKA-dependent phosphory lation of these proteins in sperm activation. The calmodulin antagonist, W- 7, and an inhibitor of calmodulin-dependent myosin light chain kinase, ML-7 , also inhibited the activation of sperm motility. Inhibition was reversed by the addition of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Demembranated preparations of immotile sperm in the presence of W-7 or ML- 7 were reactivated by cAMP, suggesting that calmodulin participated in sper m activation and that cAMP synthesis was followed by activation of a calmod ulin-dependent mechanism.