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1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.

The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a t tic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement. Flagellar movement is a particular, rhythmic, propagating bending. The sliding-microtubule model, principally based on the sliding of the arms against the neighboring outer doublet microtubules, has been presented for interpreting the mechanisms that are responsible for producing this bending (1-4). Experimental evidence for such a sliding process was reported by Summers and Gibbons (5) vealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles and that the enzymatically active site is located in the distal part of the outer arm. MATERIALS AND METHODSSpermatozoa of the sea urchin Anthocidaris crassispina were used in this study. Some points were corroborated with other species, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Shedding was induced by introducing a few drops of 0.5 M KCI into the body cavity.The spermatozoan plasma membrane-was removed by adding 20 volumes of 0.025% (vol/vol) Triton X-100 in 0.15 M KCI/4 mM MgSO4/1 mM CaCl2/2 mM EDTA/5 mM 2-mercaptoethanol/2 mM Tris-HCl, pH 8.2. After 1 min at room temperature, the sperm suspension was diluted 6-fold with the above medium without Triton X-100, and then centrifuged at 3000 rpm for 5 min. (Centrifugations were in a Hitachi instrument.) The sedimented spermatozoa were washed three times with the same medium and finally suspended in a small volume of 0.1 M sodium phosphate buffer, pH 7.5. In order to remove the outer arms from the flagellar axoneme, the spermatozoa were first suspended in 0.04% Triton X-100/0.15 M NaCl/4 mM MgSO4/0.5 mM EDTA/1 mM dithiothreitol/2 mM Tris-HCI, pH 8.0, and homogenized to detach the fla...

Detection and localization of dynein in cleaving sea urchin eggs were attempted using antidynein serum (prepared against a tryptic fragment of dynein, Fragment A, of sea urchin sperm flagella) and fluorescein conjugated goat antiserum to rabbit y-globulin. In both unfertilized and newly fertilized eggs, fluorescence was distributed rather uniformly within the cells but was absent from the nuclei. At prophase, intense fluorescence was observed on both sides of nucleus, suggesting accumulation of dynein in developing asters. From metaphase to anaphase, the whole mitotic apparatus (MA) was stained with the exceptions of the chromosomes and pole areas. Fluorescence then again became dispersed within the eggs. Throughout the mitotic process and cytokinesis, the egg cortex including the cleavage furrow was stained intensely, presumably reflecting the presence of dynein in this region. Similar distributions of fluorescence were obtained with the isolated MAS. Neither non-immune serum nor the antiserum to which Fragment A was absorbed stained the eggs. Little staining was obtained with the antiserum against starfish egg myosin. The results, together with the finding that the chromosome motion in the isolated MAS was completely inhibited by anti-dynein serum, but not with the anti-myosin serum, suggest an active role played by a tubulindynein system in mitosis.Several hypotheses such as a dynamic equilibrium between microtubules and tubulin dimers (1 j, sliding between continuous (pole-to-pole) and chromosomal microtubules by means of arms or bridges (2), or zippering between microtubules (3), have been presented to explain the mechanism of chromosome movement during mitosis. However, the question as to what provides the motive force for the mitotic machinery remains unsolved.One of the possible candidates for the energy transducing molecule involved in chromosome motion is dynein ATPase, which constitutes the arms attached t o the outer doublet microtubules and is believed to be responsible for local sliding between the adjacent doublets in flagellar and ciliary movement (see (4) for review). Although projections similar to the arms found in flagella and cilia have been observed associated with spindle microtubules (5, 6,7) and the isolation of dynein-like ATPase has been reported both from the mitotic apparatus (8) and from the cortex of sea urchin eggs (9), convincing evidence for the presence or participation of dynein in dividing cells, especially in the mitotic apparatus (MA) is still lacking.Another candidate for this role is the actin-myosin couple operating as in muscle contraction. This possibility has emerged from the recent findings that actin-like fibers are present in the mitotic spindle (1 0, 1 1 ,12) and that myosin occurs in the polar area as well as in the cleavage furrow (1 3 j. Up to the present, however, no conclusive data have been presented t o indicate * This paper is dedicated to Prof. Emer. Katsuma Dan for the celebration of his 70th birthday.

Since starfish spermatozoa have spherical heads, it is not easy to determine the topographical relationship of the axoneme to the directions of the flagellar bends, the principal, and the reverse bends as defined by Gibbons and Gibbons [J. Cell. Biol. 1972, 63:970-985]. The demembranated spermatozoa are known to take the quiescent "cane" shape with a sharp principal bend at the proximal region of the flagellum in the presence of high concentration of Ca2+. When such spermatozoa were placed on a grid for electron microscopy, fixed with osmic acid vapor, washed with distilled water, and negatively stained with uranyl acetate, the head of the spermatozoon was disrupted and dispersed disclosing the proximal centriole at the proximal end of the flagellum. The proximal centriole was always found on the concave side of the "cane"-shaped flagella. Electron microscopy of the serial thin sections of intact and demembranated spermatozoa revealed that the doublet microtubules numbers 5 and 6 were contained in the convex edge of the principal bend.