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

Abstract

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.

Effects of a Protein Kinase Inhibitor on Sperm Motility in the Japanese Quail

“…For example, sperm -attracting and -activating factor (SAAF) in Ciona induces Ca 2+ influx, membrane hyperpolarization and activation of adenylyl cyclase to produce cAMP for sperm motility activation (Nomura et al, 2004). A transient increase in intracellular cAMP and subsequent cAMP-dependent phosphorylation of dynein subunits were observed in response to the addition of SAAF (Shiba and Inaba, 2014;Nomura et al, 2000). Thus, cAMP is one of the important factors in SAAF-induced sperm motility activation in Ciona.…”

A chemoattractant for ascidian spermatozoa is a sulfated steroid

“…We showed in a previous study that the eggs probably release a sperm-activating and -attracting factor (SAAF) from their vegetal pole (14,15). SAAF induces entry of extracellular Ca 2ϩ and an increase in intracellular cAMP in the sperm (15,16), which induces protein kinase A-dependent phosphorylation of 21-and 26-kDa axonemal proteins and activation of sperm motility (17). On the other hand, the chemotactic behavior of the ascidian sperm also requires extracellular Ca 2ϩ , but theophyllineactivated sperm, in which the drug induces increase in the [cAMP] i by virtue of being a phosphodiesterase inhibitor, show similar chemotactic behavior to that of normal sperm (14,15).…”

Acclimation of sperm motility apparatus in seawater-acclimated euryhaline tilapiaOreochromis mossambicus

“…In sperm of puffer fish (Oda and Morisawa, 1993), lancelet (Tanaka et al, 2002), ascidians (Nomura et al, 2000;Yoshida et al, 1994Yoshida et al, , 2003, carp (Krasznai et al, 2000) and salmonidae (Boitano and Omoto, 1992;Tanimoto and Morisawa, 1998;Kho et al, 2001), increased [Ca 2+ ]i is known to play an important role in activation of the sperm motility. Demembranated sperm is assumed to simulate a regulatory mechanism of motility in intact sperm.…”