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 monophosphate (cAMP)-synthesis. A demembranated preparation of intact immotile 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 factor for SAAF-dependent activation of sperm motility. Furthermore, a cAMP-dependent protein kinase (PKA) inhibitor, H-89, was found to inhibit sperm motility. During in vivo or in vitro activation of sperm motility by SAAF or cAMP, a 26 kDa axonemal protein and 21 kDa dynein light chain were phosphorylated, respectively, suggesting the involvement of PKA-dependent phosphorylation 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 sperm activation and that cAMP synthesis was followed by activation of a calmodulin-dependent mechanism.
Nomura
M
M
Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, Miura, Kanagawa, Japan. mnomura@mmbs.s.u-tokyo.ac.jp
Inaba
K
K
Morisawa
M
M
D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D004527:Egg Proteins / Q000494:pharmacology* / Q000502:physiology; D008297:Male; D010766:Phosphorylation; D015398:Signal Transduction; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*; D014561:Urochordata
D002147:Calmodulin; D004527:Egg Proteins; D000242:Cyclic AMP