The motility of the spermatozoa of freshwater fish is usually of short duration, but a precise description has rarely been provided. Motility requires a high dilution (more than 1,000-fold) for initiation of synchronous motility in 100% of spermatozoa; a two-step procedure is necessary, with a n initial dilution of 1 to 100 in a medium that keeps the spermatozoa immotile and allows good mixing of the viscous semen. The second dilution (1 to 20) in the activating solution can be made directly under the microscope. Studies of carp sperm indicate that movement of live sperm is influenced by the ionic environment. The inhibition of motility in semen is mainly due to K + ions in trout and osmotic pressure in carp, but other ions such as N a + , H', and Mg2+ also interfere.Initiation of motility in trout requires external divalent cations. Immediately after dilution a t 20"C, spermatozoa exhibit large circular trajectories (>400 pm in diameter), high beat frequencies (60 Hz), and velocities of 250 pm/sec. These values decrease rapidly. Within 20 sec after dilution, most spermatozoa stop moving, although some of them show some agitation with low beat frequency (< 10 Hz) and with very limited displacement during the next few minutes. A similar pattern is observed in carp, with active motility lasting 40 sec. Under certain ionic conditions, intratesticular spermatozoa are motile and have some fertilizing capacity.Studies on motility of fish sperm have been restricted to a very limited number of groups. At most, 20 different species have been studied, even though more than 20,000 species of fish are known. Our knowledge of sperm motility is limited, given the diversity of modes of reproduction found in fish (Breder and Rosen, '66). Most recent studies involve freshwater fish with external fertilization and are focussed on conservation, preservation, and appropriate dilution conditions for artificial insemination (see reviews by Scott and Baynes, '80; Scott, '81; Stoss, '83; Billard et al., '86). Several independent investigators have carried out research on salmonids, in particular the rainbow trout, Oncorhynchus mykiss, generating data on the ionic conditions that regulate sperm movement (Billard et al., '74; Baynes et al., '81; Morisawa et al., '83). Information has also been obtained on the dynamics and on the molecular mechanisms that accompany flagellar movement at the axonemal level in trout (Gibbons et al., '83, '85; Morisawa, '85). Since large quantities of spermatozoa are available, purification of flagellar dynein adenosine triphosphatase (ATPase) has been possible (Ogawa et al., '80; Gatti et al., '88).Studies on sperm biology started in the nineteenth century. Several characteristics of the biology of fish sperm were quickly identified: the immobility of the spermatozoa in the semen, the short duration of movement once motility has been initiated, and the necessity of dilution in water for initiation of sperm movement. Spallanzani (quoted by De Quatrefages, 1853) mentioned that the duration of motilit...

The motility of demembranated sea urchin sperm flagella and that of embryo cilia reactivated with 0.1 mM ATP are completely inhibited by 4 AM and 0.5 AM vanadium (V) [V(V), in vanadate], respectively. The Mg2+-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) of the latent form of dynein 1 is inhibited 50% by 0.5-1 AM V(V), while the Ca2+-activated ATPase activity is much less sensitive. The inhibition of flagellar beat frequency and of dynein 1 ATPase activity by V(V) appears not to be competitive with ATP. In agreement with other reports, the inhibition of (NaK)ATPase by V(V) shows a slow onset in the presence of ATP and is relatively rapid in its absence. With dynein, however, the inhibition occurs at a rapid rate whether or not ATP is present. Catechol at a concentration of 1 mM reverses the V(V) inhibition of reactivated sperm motility, dynein ATPase, and (NaK)ATPase. (5).In this paper we report that vanadium(V), V(V), is a potent inhibitor of dynein 1 and of the motility of reactivated sea urchin sperm flagella and embryo cilia. We have also examined the effect of V(V) on myosin and (Na,K)-ATPase. MATERIALS AND METHODSMaterials. Sodium metavanadate (NaVO3) and sodium orthovanadate (Na3VO4) were obtained from Fisher ScientificCo. Stock solutions of sodium metavanadate that had been recrystallized from methanol/water were prepared in 10 mM Tris.HCl buffer, pH 8.1. Identical results were obtained with sodium orthovanadate in 0.1 M NaOH. Cateohol, norepinephrine, ouabain, NADH, phosphoenolpyruvate, lactate dehydrogenase, and pyruvate kinase were obtained from Sigma. ATP was obtained from Boehringer Mannheim Corporation. Stock solutions of 0.25 M catechol and norepinephrine were prepared freshly in 1 mM HCl.Sperm and eggs were obtained from the sea urchin Tripneustes gratilla by injection with 0.5 M KCl.Reactivated Sperm and Cilia. Sea urchin sperm were demembranated with Triton X-100 and reactivated with 0.1 mM ATP as described previously (7,8). Cilia were obtained from sea urchin embryos grown in Ca2+_free artificial sea water, disrupted mechanically into individual blastomeres, and treated with demembranating solution (7) at pH 7.5. They were then transferred to reactivating solution, pH 7.5, containing 0.1 mM ATP.Preparation of ATPases. LAD-1 was extracted from freshly Before their Mg2+-and Ca2+-activated ATPase activities were compared, preparations of LAD-1 were dialyzed against 0.5 mM EDTA/7 mM 2-mercaptoethanol/5 mM Tris-HCl, pH 8.0, to remove all divalent cations. Activated dynein 1 was obtained by incubating LAD-1 with 0.1% (wt/vol) Triton X-100 for 10 min at room temperature (5). Myosin and actin were prepared from rabbit muscle by the procedures of Perry (10) 2220The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Villefranche-sur-mer (M. P. C.), Laboratoire d'lchtyologie, Museum d'Histoire Naturelle, Paris (R.B.) The initiation of motility of trout spermatozoa is inhibited by the presence of millimolar concentrations of external K + , but external Ca2+ might also be implicated in this control as it has been shown to antagonize the K + inhibition of motility [S.M. Baynes et al.: J. Fish. Biol., 19:259-267, 19811. The present work aimed to investigate internal Ca2+ levels during the motility phase of trout spermatozoa. Internal Ca2+ concentrations were monitored by the fluorescent quinoline Ca2+-indicator, "Quin-2" [R.Y. Tsien: Nature 290527-529, 19811. Trout spermatozoa were loaded with Quin-2IAM under conditions that gave efficient intracellular hydrolysis of Quin-2 and that did not impair the ability of loaded spermatozoa to initiate movement. The beat frequencies, cell velocities, and flagellar asymmetries of sperm movement were not significantly modified by the presence of the internal dye. Upon initiation of flagellar movement, an increase of the internal Quin-2 fluorescence was observed that reflected a sixfold increase of the free Ca2+ concentration. The free Ca2+ remained elevated after the cessation of movement. The variation of fluorescence was completed within 40 seconds, whereas the initiation of motility was nearly instantaneous, and the total duration of flagellar beating lasted for about 80-100 seconds (measurements at 11°C). The increase in the internal free Ca2+ concentration is completed after the initiation of flagellar beating but its occurrence correlates with that of sperm movement. Fluorescence increase was not observed in the presence of 40 mM K + , a condition in which spermatozoa did not initiate flagellar beating. In the presence of the Ca2 + channel blocker desmethoxyverapamil, neither sperm motility nor fluorescence increases were observed, which suggested that the increase of internal free Ca2+ was produced by a flux of external Ca2+ into the cell rather than by a mobilization of internal Ca2+ stores.