Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa

Abstract

SUMMARY Numerous kinases and phosphatases are most likely implicated in sperm motility initiation and maintenance. Data on these signaling molecules were mostly obtained from studies conducted on in vitrodemembranated–reactivated sperm models but are not necessarily representative of the in vivo situation. We therefore investigated the effect of a variety of cell-permeable chemicals, mostly kinase inhibitors,on the motility initiation and maintenance of intact sea urchin spermatozoa. Among the 20 substances tested, the protein kinase C (PKC) inhibitor chelerythrine was the most potent, arresting motility at concentrations starting from 1.5–2 μmol l–1. Motility was also inhibited by two other PKC inhibitors as well as staurosporine. Furthermore,these inhibitors prevented the motility-associated increase in phosphorylation of at least four PKC substrates. These phospho-PKC target proteins, as assessed with an antibody specific to phosphorylated motifs of PKC substrates,were found to be associated with the flagellum, either in the Triton X-100 soluble portion or the axoneme (Triton X-100 insoluble). A phosphorylated PKC-like enzyme was also detected by immunoblotting in the flagellum, as well as a significant 50 kDa PKC cleavage product. Taken together, the data strongly indicate for the first time that, in vivo, which means on intact spermatozoa, PKC is a key signaling mediator associated with the maintenance of sea urchin sperm motility.