cAMP and calcium are two important regulators of sperm flagellar motility. cAMP stimulates sperm motility by activating cAMP-dependent protein kinase and catalyzing the phosphorylation of sperm proteins. The stimulation of sperm motility by cAMP appears to be at two different levels. Evidence has been presented to suggest that cAMP-dependent phosphorylations may be required in order for motility to be initiated. In addition, cAMP-dependent phosphorylation appears to modulate specific parameters of motility resulting in higher beat frequency or greater wave amplitude. Calcium, on the other hand, when elevated intracellularly to 10(-6) M or higher, inhibits flagellar motility. The calcium-binding protein, calmodulin, appears to mediate a large number of effects of calcium on motility. Evidence suggests that calcium-calmodulin may be involved at the level of the membrane to pump calcium out of the flagellum. In addition, calcium-calmodulin may be involved in the control of axonemal function by regulating dynein ATPase and myosin light chain kinase activities. The identification of cAMP-dependent protein kinase, calmodulin and myosin light chain kinase in the sperm head suggests that cAMP and calcium-dependent phosphorylations are also involved in the control of the fertilization process, i.e., the acrosome reaction, in a manner similar to that known for the control of stimulus/secretion coupling. Finally, the effects of cAMP on flagellar motility are mediated by protein phosphorylation while the effects of calcium on motility are also in part, mediated by effects on protein phosphorylation.

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Motility and protein phosphorylation have been measured under identical experimental conditions in ejaculated dog sperm lysed with low concentrations of Triton X-100 and reactivated with [gamma-32P]ATP. Cyclic AMP stimulates motility and protein phosphorylation while calcium inhibits motility and the overall incorporation of phosphate into endogenous proteins. Analysis of 32P-labeled sperm proteins on 1- and 2-dimensional polyacrylamide gels demonstrates that an enhanced phosphorylation of a defined number of specific proteins is associated with cAMP-stimulated motility. A major axonemal proteins, namely tubulin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP. The phosphorylation of tubulin is almost completely dependent upon cAMP and is not affected by microM calcium. On the other hand, the cAMP-dependent stimulated phosphorylation of the other sperm proteins still occurs, but in most instances at a reduced rate in the presence of calcium. Two high molecular weight (Mr) phosphoproteins (350,000 and 260,000 daltons) whose phosphorylation states are modified by cAMP and calcium also were identified. It is suggested that 1 or both these proteins may be high Mr subunits of dynein. The phosphorylation of 1 of these proteins is stimulated by cAMP, but not affected by calcium; the other is stimulated by cAMP and inhibited by calcium. Three major cAMP-independent phosphoproteins of Mr 98,000, 43,000 and 26,000 have been identified. The phosphorylation of the 98,000 Mr protein is markedly reduced by micromolar calcium and not restored by cAMP. Using anticalmodulin drugs to inhibit motility, we suggest that the inhibitory effects of calcium on flagellar motility may be mediated in part by calmodulin. We conclude that the regulation of flagellar motility in cAMP and calcium includes mechanisms involving the control of the phosphorylation state of sperm proteins, some of which may be axonemal components.

Abstract. Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca 2÷ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KC1 from isolated flagella from dog, pig, and sea urchin sperm. All saltextractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.T rlE apparent requirement of cAMP-dependent protein phosphorylation for flagellar motility (Tash et al., 1984(Tash et al., , 1986) is contrasted by the inhibitory and waveform modulating effects of Ca 2+ (Hyams and Borisy, 1978;Gibbons, 1980). Considerable effort has been exerted by a number of laboratories concerning possible mechanisms of action of Ca 2÷ on the regulation of flagellar movement (Satir, 1985;Gibbons, 1982;Brokaw et al., 1974;Bessen et al., 1980). More recent attention has concentrated on calmodulin as the potential primary signal transducer in Ca 2+-regulated flagellar motility. However, results have been contradictory, which may result from differences in species as well as the techniques used (Gitelman and Witman, 1980;Blum et al., 1980). Perhaps the most compelling evidence to date to suggest an involvement of calmodulin in this regulation comes from the observations of Brokaw and Nagayama (1985). The asymmetry-inducing effects of calmodulin-conraining flagellar extracts were eliminated by passing the extracts through trifluoperazine columns. Furthermore, they found that purified calmodulin reverses the symmetric swimming patterns of sperm demembranated in the presence of Ca 2+ to produce asymmetric flagellar bending. While evidence suggesting an involvement of calmodulin in the regulation of flagellar motility is increasing, a defined calmodulindependent biochemical pathway, which could explain the effects of Ca2+-calmodulin on motility, remains to be elucidated and characterized, The present study reports the identification of calmodulindependent protein phosp...

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