Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.     

Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella

The relaxation (straightening) of flagellar rigor waves, which is known to be induced by micromolar ATP concentrations was investigated with respect to its dependence on the binding and hydrolysis of ATP. Flagellar rigor waves were formed by the dilution of demembranated, reactivated sea urchin (Lytechinus pictus) spermatozoa into ATP-free buffer. Relaxation in response to nucleotide was quantitated by measuring theta, the mean flagellar bend angle per sperm; this novel assay permitted determination of the rate of relaxation. It was found that (a) the rate of flagellar relaxation induced by 4 X 10(-6) M ATP was inhibited 80% by vanadate concentrations of 3 X 10(-6) M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and epsilon-ATP) were also capable of effecting relaxation; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP relaxation depends on ATP hydrolysis; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon. A general model of relaxation is proposed based on active microtubule sliding.     

Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm

A nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), has been used to study the role of ATP binding in flagellar motility. Sea urchin sperm of Lytechinus pictus were demembranated, reactivated, and locked in "rigor waves" by a modification of the method of Gibbons and Gibbons (11). Rigor wave sperm relaxed within 2 min after addition of 4 micrometer ATP, and reactivated upon addition of 10-12 micrometer ATP. The beat frequency of the reactivated sperm varied with ATP concentration according to Michaelis-Menten kinetics ("Km" = 0.24 mM; "Vmax" = 44 Hz) and was competitively inhibited by AMP-PNP (Ki" approximately to 8.1 mM). Rigor wave sperm were completely relaxed (straightened) within 2 min by AMP-PNP at concentrations of 2-4 mM. The possibilities that relaxation in AMP-PNP was a result of ATP contamination, AMP-PNP hydrolysis, or lowering of the free Mg++ concentration were conclusively ruled out. The results suggest that dynein cross-bridge release is dependent upon ATP binding but not hydrolysis.     

Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with KC1.

Demembranated sea urchin sperm were extracted with 0.5 M KCl as described earlier and reactivated in a solution containing 1 mM ATP. Their flagellar beat frequency was approximately 13 Hz, while that of standard reactivated sperm which had not been extracted with KCl was approximately 31 Hz at 23°C. Addition of soluble dynein 1 caused a gradual increase in the flagellar beat frequency to approximately 25 Hz after 10 min at room temperature. This restoration of frequency occurred in the absence or presence of ATP. Examination by electron microscopy showed that, whereas KCl-extracted sperm were lacking the majority of the outer arms on the doublet tubules, they had regained most of their outer arms following incubation with soluble dynein 1.     

The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm

Rabbit antiserum prepared against an ATPase-containing tryptic fragment of dynein by Ogawa and Mohri (J. Biol. Chem. 250: 6476-6483) specifically inhibited the ATPase activity of dynein 1 and not that of dynein 2. Varying amounts of this antidynein 1 serum were added to demembranated sperm while they were swimming in reactivating solution containing 1 mM ATP. The sperm continued to form regularly propagated flagellar bending waves, but the beat frequency decreased gradually with time, the greater part of the change occurring in the first 15 min. The beat frequency after 1 h was a function of the amount of antiserum used, and could be as low as 1 Hz. The waveforms of the treated sperm resembled those of normal reactivated sperm except that the bend angles of both the principal and reverse bends were larger in the proximal portion of flagellum. The ATPase activity and corresponding beat frequency of sperm which had been pretreated with varying amounts of antidynein 1 serum for 15 min at 0 degrees C and then diluted were both decreased as a function of the amount of antiserum added, the ATPase activity of homogenized, nonmotile sperm also decreased upon pretreatment with antiserum, but the percentage decrease was less than for motile sperm. For moderate to low concentrations of antiserum, the rates of reaction with motile and with rigor sperm were almost identical. The overall results suggest that antidynein 1 inhibits the functioning of the dynein arms, probably by blocking the ATPase sites of the dynein 1.     

Calcium-induced quiescence in reactivated sea urchin sperm

Sperm flagella of the sea urchin Tripneustes gratilla beat with asymmetrical bending waves after demembranation with Triton X-100 in the presence of EGTA and reactivation at pH 8.1 with 1 mM ATP in the presence of 2 mM MgSO4. Addition of 0.1--0.2 mM free Ca2+ to these reactivated sperm induces 70--95% of them to become quiescent. This quiescence can be reversed by reduction of the free Ca2% concentration with EGTA, or by dilution to reduce the MgATP2- concentration below 0.3 mM. The quiescent waveform is characterized by a sharp principal bend of approximately 5.6 rad in the proximal region of the flagellum, a slight reverse bend in the midregion that averages approximately 0.3 rad, and a principal bend of approximately 1.1 rad in the tip. The quiescent sperm are highly fragile mechanically, and disruption, including microtubule sliding, occurs spontaneously at a slow rate upon standing or immediately upon gentle agitation. Mild digestion by trypsin causes a gradual appearance of normal, symmetrical flagellar beating. Addition of increasing concentrations of vanadate to quiescent sperm causes a graded decrease in the proximal bend angle, with 50 micrometers vanadate reducing it to approximately 2.6 rad. In the presence of 0.1 mM free Ca2% and 10 micrometers vanadate, a characteristic, crescented stationary bend is induced in the demembranated sperm, without intermediate oscillatory beating, by the addition of either 0.1 or 1 mM ATP. In the absence of vanadate, these two concentrations of ATP produce asymmetric beating and quiescence, respectively. The results support the hypothesis that quiescence in live sperm is induced by an elevated concentration of intracellular Ca2%. In addition, they demonstrate that bending can occur in flagella in which oscillatory beating is inhibited and emphasize the close relationship between asymmetric beating and quiescence.     

Calcium regulation of flagellar curvature and swimming pattern in triton X-100--extracted rat sperm

Free Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10(-7) M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 x 10(-6) M Ca2+ to assume a tight, hook-like bend at concentrations of 10(-5) to 10(-4) M free Ca2+. Sodium vanadate at 2 x 10(-6) M blocked flagellar motility, but did not inhibit the Ca2+-mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 microM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+-dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+-treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1-5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+-induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.     

The equation of motion for sperm flagella.

The equation of motion for sperm flagella, in which the elastic bending moment and the active contractile moment are balanced by the moment from the viscous resistance of the surrounding fluid, is solved for a wave solution that superimposes partial solutions. Substitution of the expression for the wave solution into the equation leads to an expression for the active contractile moment. This active moment can be decomposed into two parts. The first part describes an active moment that travels over the flagellum with the mechanical flagellar wave, the second part represents a moment in phase over the entire length of the flagellum, which decreases linearly towards the distal tip. The linear synchronous moment, to which an amount of traveling moment has been added as a perturbation, leads to wave solutions that closely resemble flagellar waves. Properties such as wavelength and wave amplitudes and also the shape of the waves in sea urchin sperm flagella at different frequencies are accurately described by the theory. The change in wave shape in sea urchin sperm flagella at raised viscosity is predicted well by the theory. The different wave properties caused in bull sperm flagella by different boundary conditions at the proximal junction are explained. When only a traveling active moment is present in a flagellum, the wave solutions describe waves of a small wave length in a long flagellum. Some properties of the wave motion of sperm flagella are derived from the theory and verified experimentally.     

Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with triton X-100

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25°C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 µm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 µm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 µmole P_i/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 µmole P_i/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg⁺⁺, although some motility was also obtained with Mn⁺⁺ and Ca⁺⁺. The coupled ATPase activity had a Michaelis constant (K_m) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "K_m" of 0.2 mM.     

Mechanisms of Bactericidal Action of Cinnamaldehyde against Listeria monocytogenes and of Eugenol against L. monocytogenes and Lactobacillus sakei

The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20°C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20°C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 μM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 μM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Effects of calcium on the longituindal flagellum of Oxyrrhis marina

Summary— 2–4 nm filaments represent a new class of cytoskeletal components. They are found in ciliary and flagellar roots and centrosomes of all eucaryotes. They are also the major components of paraflagellar rods (PFR) in Euglena, trypanosomes and dinoflagellates. Oxyrrhis marina, a marine dinoflagellate, possesses a transverse and a longitudinal flagellum. Only the longitudinal flagellum carries the PFR along the proximal two-thirds of its length. This flagellum is not only capable of the classic flagellar beat but is also able to retract and bend, a property mediated by external calcium. To determine if calcium has a direct role in the bending, experimental conditions were established to permeabilization and reactivation. Our conditions to reactivate the axoneme function (wave propagation) appear similar to those observed in the case of the sea urchin sperm. The results show that in vitro, an increase in calcium concentration induces a conformational change of the longitudinal flagellum in the absence of ATP with a half maximum effect at 0.1 μM. In the presence of ATP, this morphology modification causes a total inhibition of the wave propagation which is replaced by non-propulsive contractions of low amplitude. As these properties are not shared by reactivated sea urchin sperm flagella or the transverse flagellum of O marina devoid of PFR, we propose that PFR are responsible for the bending phenomenon. A calcium shock also induces flagellar excision with a half maximum effect at 0.3 μM, and immunofluorescence results suggest that a centrin-like protein is present in O marina and is responsible for this excision.     

Calcium Efflux from Internally Dialyzed Squid Giant Axons

Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm²·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]_i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]_o and [Ca]_o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]_i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10^-7 mol Ca⁺⁺/mg of protein with an initial rate of 2.6 x 10^-8 mol Ca⁺⁺/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources.     

Nanoparticle Incorporation of Melittin Reduces Sperm and Vaginal Epithelium Cytotoxicity

Melittin is a cytolytic peptide component of bee venom which rapidly integrates into lipid bilayers and forms pores resulting in osmotic lysis. While the therapeutic utility of free melittin is limited by its cytotoxicity, incorporation of melittin into the lipid shell of a perfluorocarbon nanoparticle has been shown to reduce its toxicity in vivo. Our group has previously demonstrated that perfluorocarbon nanoparticles containing melittin at concentrations <10 µM inhibit HIV infectivity in vitro. In the current study, we assessed the impact of blank and melittin-containing perfluorocarbon nanoparticles on sperm motility and the viability of both sperm and vaginal epithelial cells. We found that free melittin was toxic to sperm and vaginal epithelium at concentrations greater than 2 µM (p<0.001). However, melittin nanoparticles were not cytotoxic to sperm (p = 0.42) or vaginal epithelium (p = 0.48) at an equivalent melittin concentration of 10 µM. Thus, nanoparticle formulation of melittin reduced melittin cytotoxicity fivefold and prevented melittin toxicity at concentrations previously shown to inhibit HIV infectivity. Melittin nanoparticles were toxic to vaginal epithelium at equivalent melittin concentrations ≥20 µM (p<0.001) and were toxic to sperm at equivalent melittin concentrations ≥40 µM (p<0.001). Sperm cytotoxicity was enhanced by targeting of the nanoparticles to the sperm surface antigen sperm adhesion molecule 1. While further testing is needed to determine the extent of cytotoxicity in a more physiologically relevant model system, these results suggest that melittin-containing nanoparticles could form the basis of a virucide that is not toxic to sperm and vaginal epithelium. This virucide would be beneficial for HIV serodiscordant couples seeking to achieve natural pregnancy.     

Inhibition of the ATP-induced reactivation of demembranated hamster spermatozoa by the action of free ATP4- and MgATP2-

Hamster spermatozoa collected from the caput and cauda epididymidis were washed, diluted in a medium containing Triton X-100 to dissolve the cell membrane and reactivated with various concentrations of MgSO4 and ATP. Stepwise increase in the concentrations of free ATP4- from 0.08 to 1.1 mM at constant concentrations of MgATP2- caused a dose-dependent delay of reactivation but the maximal percentage of motile spermatozoa was inhibited only at 1.1 mM. The inhibitory effect on caput spermatozoa was greater than that on cauda spermatozoa. When concentrations of ATP4- were fixed at 0.2 mM, 2.9 mM-MgATP2- suppressed the reactivation of cauda spermatozoa. When compared to 0.9 mM-MgATP2-, reactivation of caput spermatozoa was delayed at 1.9 mM and almost completely blocked by 2.9 mM-MgATP2-. Inhibition of cauda sperm reactivation by ATP4- and MgATP2- were both prevented by the presence of trypsin (50 ng/ml). Incubation of cauda spermatozoa in the reactivation medium for 1 and 2 min before the addition of ATP progressively reduced the inhibitory effect of ATP4-; inhibition by MgATP2- was reduced to a lesser extent. Addition of 100 microM-cyclic AMP to the medium abolished the delay of reactivation by ATP4- but not that by MgATP2-. Before reactivation occurred, inhibitory concentrations of ATP4- and MgATP2- both induced large-angle coiling of sperm tails but in opposite direction to each other with reference to the asymmetric sperm head. The results suggest that free-ATP4- and superoptimal concentrations of MgATP2- inhibit flagellar movement by different mechanisms.     

THE CONTRACTILE BASIS OF AMOEBOID MOVEMENT : I. The Chemical Control of Motility in Isolated Cytoplasm

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle. Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca. 7.0 x 10^-7 M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations. The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched. When the stabilized cytoplasm was stretched, birefringent fibrils were observed. Thin sections of those fibrils showed thick (150–250 Å) and thin (70 Å) filaments aligned parallel to the long axis of fibrils visible with the light microscope. Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle. In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane-less pseudopodia to extend into the solution from the cytoplasmic mass. These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba.     

Flagellar movement of intact and demembranated, reactivated ram spermatozoa

The flagellar movement of intact ejaculated ram sperm, and of demembranated models reactivated with ATP, has been studied using high-speed, high-resolution video microscopy. Intact sperm attached to the coverslip by their heads had an average beat frequency of 20.9 Hz and an average wave amplitude of 20.2 micron. There was little difference in the beat frequency or waveform of these sperm and sperm swimming freely near the coverslip or captured by their heads with a micropipette and held far from the coverslip, indicating that the flagellar waveform of ram sperm is relatively resistant to distortion as a result of immobilization of the head or proximity to a surface. The beat envelope was nearly planar as determined by observations of free-swimming sperm and sperm captured by their head and oriented so they were beating either parallel or perpendicular to the plane of focus. The effect of various conditions for demembranation and reactivation of the sperm were examined. Treatment of sperm with 0.2% Triton X-100 removed most of their plasma membrane. Under optimal conditions, nearly 100% of the demembranated sperm reactivated at MgATP2- concentrations ranging from approximately 4 microM to approximately 20 mM. From approximately 1 mM to approximately 10 mM MgATP2-, their beat pattern closely resembled that of intact sperm; beat frequency depended on MgATP2- concentration. Percent motility was maximal between pH 7.5 and 8.0 and decreased sharply below pH 7.0 and above pH 8.5. The addition of 50 microM cAMP to the reactivation medium had no effect on percent motility or the beat pattern and did not accelerate the initiation of movement.     

Effects of ATP, cAMP and pH on the initiation of flagellar movement in demembranated models of rat epididymal spermatozoa

Demembranated model of rat epididymal spermatozoa was employed to establish the conditions for the initiation of flagellar movement. Extensive initiation of the flagellar movement required 0.5 mM ATP, 1 μM cAMP and pH 7.9. The requirement for ATP was highly specific and can partially be replaced by 2′-deoxy-ATP only, but not by analogs of ATP or other nucleoside triphosphates. In contrast, the cAMP requirement was less specific and can partially be replaced by other cyclic nucleotides and cAMP-analogs except 2′-deoxy-cAMP and 2′,3′-cAMP. The data implied that the intracellular pH rise, not the cAMP increase, was the probable trigger for the initiation of sperm motility after ejaculation. During sperm maturation, the sperm motile apparatus appeared unchanged with respect to the above conditions of reactivation.