Regulation of flagellar motility of fowl spermatozoa: Evidence for the involvement of intracellular free Ca2+ and calmodulin

Abstract

The possible role of intracellular free Ca2+ and calmodulin in the regulation of fowl sperm motility was investigated using an intracellular Ca2+ chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) and calmodulin antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and trifluoperazine. Intact fowl spermatozoa maintained vigorous movement in a Ca2+-free medium at 30°C. In contrast, the motility of spermatozoa loaded with BAPTA/AM was negligible at 30°C, but could be instantly restored by the addition of 2 mmol CaCl2 l-1. At this time, the intracellular free Ca2+ concentrations increased from 0 to about 100 nmol l-1, as measured by the fluorescent Ca2+ indicator fura-2. At 40°C, neither control nor BAPTA/AM-treated spermatozoa were motile, but the motility of both kinds of spermatozoa was restored by the subsequent addition of 2 mmol CaCl2 l-1. Even in the presence of 2 mmol CaCl2 l-1, the addition of W-7 and trifluoperazine inhibited the motility of intact spermatozoa at 30°C and 40°C, and induced a concomitant decrease in the rate of oxygen consumption and ATP concentration, suggesting that energy depletion might be involved in the inhibition of motility. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of W-7 and trifluoperazine at 30°C. The addition of W-5, a weaker antagonist, did not appreciably affect the motility of either intact or demembranated spermatozoa. These results suggest that intracellular free Ca2+ is indispensable for the maintenance of fowl sperm motility. Calmodulin, which occurs in the cytoplasm or mitochondria but not in the axoneme, is a prominent candidate for the signal transducer in Ca2+-stimulated motility.