Possible role of protein kinase C in regulation of flagellar motility and intracellular free Ca2+ concentration of fowl spermatozoa

Abstract

The presence and possible role of protein kinase C in the regulation of fowl sperm functions were investigated. Immunoblot analysis of sperm extract using antibody to protein kinase C revealed a crossreacting protein of approximately 80 kDa. As the concentration of the protein kinase C activators N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) or 1-oleoyl-2-acetylglycerol (OAG) was increased, the motility of intact spermatozoa at 30 degrees C was reduced. However, this inhibition of motility was reversed by reducing the concentrations of activators. Even in the presence of 1 mmol CaCl2 l-1, the addition of SC-9 and OAG inhibited the motility of intact spermatozoa. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of SC-9 or OAG at 30 degrees C. However, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C inhibitor, did not appreciably affect the motility of either intact or demembranated spermatozoa at 30 degrees C. At 40 degrees C, both intact and demembranated spermatozoa were almost immotile in the presence or absence of the activators or inhibitor. Intracellular free Ca2+ concentrations, measured by means of a fluorescent Ca2+ indicator, fura-2, gradually increased after the addition of SC-9 and OAG, but no changes were observed in H-7-treated spermatozoa. These results suggest that endogenous protein kinase C is present in the cytoplasmic matrix or the membrane, but is not retained in the axoneme, and that the activation of this enzyme may contribute to a decrease in the flagellar movement of fowl spermatozoa.