scholarly journals Calcium-induced quiescence in reactivated sea urchin sperm.

1980 ◽  
Vol 84 (1) ◽  
pp. 13-27 ◽  
Author(s):  
B H Gibbons ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Proximal Region ◽  
Bend Angle ◽  
Elevated Concentration ◽  
Bending Waves ◽  
Close Relationship ◽  
Gentle Agitation ◽  
Live Sperm ◽  
Triton X ◽  
Asymmetrical Bending

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.

scholarly journals Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella.

1979 ◽  
Vol 82 (2) ◽  
pp. 401-411 ◽  
Author(s):  
C J Brokaw
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Portion ◽  
Bend Angle ◽  
Bending Waves ◽  
New Information ◽  
Unequal Rates ◽  
Rates Of Growth ◽  
Change In Mean ◽  
Asymmetrical Bending

Asymmetrical bending waves can be obtained by reactivating demembranated sea urchin spermatozoa at high Ca2+ concentrations. Moving-film flash photography shows that asymmetrical flagellar bending waves are associated with premature termination of the growth of the bends in one direction (the reverse bends) while the bends in the opposite direction (the principal bends) grow for one full beat cycle, and with unequal rates of growth of principal and reverse bends. The relative proportions of these two components of asymmetry are highly variable. The increased angle in the principal bend is compensated by a decreased angle in the reverse bend, so that there is no change in mean bend angle; the wavelength and beat frequency are also independent of the degree of asymmetry. This new information is still insufficient to identify a particular mechanism for Ca2+-induced asymmetry. When a developing bend stops growing before initiation of growth of a new bend in the same direction, a modification of the sliding between tubules in the distal portion of the flagellum is required. This modification can be described as a superposition of synchronous sliding on the metachronous sliding associated with propagating bending waves. Synchronous sliding is particularly evident in highly asymmetrical flagella, but is probably not the cause of asymmetry. The control of metachronous sliding appears to be unaffected by the superposition of synchronous sliding.

Effect of imposed head vibration on the stability and waveform of flagellar beating in sea urchin spermatozoa

1991 ◽  
Vol 156 (1) ◽  
pp. 63-80 ◽  
Author(s):  
C. Shingyoji ◽  
I. R. Gibbons ◽  
A. Murakami ◽  
K. Takahashi
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Region ◽  
Proximal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Additional Energy ◽  
The Stability ◽  
Cyclic Changes

The heads of live spermatozoa of the sea urchin Hemicentrotus pulcherrimus were held by suction in the tip of a micropipette mounted on a piezoelectric device and vibrated either laterally or axially with respect to the head axis. Within certain ranges of frequency and amplitude, lateral vibration of the pipette brought about a stable rhythmic beating of the flagella in the plane of vibration, with the beat frequency synchronized to the frequency of vibration [Gibbons et al. (1987), Nature 325, 351–352]. The sperm flagella, with an average natural beat frequency of 48 Hz, showed stable beating synchronized to the pipette vibration over a range of 35–90 Hz when the amplitude of vibration was about 20 microns or greater. Vibration frequencies below this range caused instability of the beat plane, often associated with irregularities in beat frequency. Frequencies above about 90 Hz caused irregular asymmetrical flagellar beating with a marked decrease in amplitude of the propagated bends and a skewing of the flagellar axis towards one side; the flagella often stopped in a cane shape. In flagella that were beating stably under imposed vibration, the wavelength was reduced at higher frequencies and increased at lower frequencies. When the beat frequency was equal to or lower than the natural beat frequency, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with beat frequency in both the proximal and distal regions of the flagella. However, at vibration frequencies above the natural beat frequency, the sliding velocity increased with frequency only in the proximal region of the flagellum and remained essentially unchanged in more distal regions. This apparent limit to the velocity of sliding in the distal region may represent an inherent limit in the intrinsic velocity of active sliding, while the faster sliding observed in the proximal region may be a result of passive sliding or elastic distortion of the microtubules induced by the additional energy supplied by the vibrating pipette. Axial vibration with frequencies either close to or twice the natural beat frequency induced cyclic changes in the waveform, compressing and expanding the bends in the proximal region, but did not affect bends in the distal region or alter the beat frequency.

scholarly journals Maintenance of Constant Wave Parameters by Sperm Flagella at Reduced Frequencies of Beat

1973 ◽  
Vol 59 (3) ◽  
pp. 617-629
Author(s):  
C. J. BROKAW ◽  
R. JOSSLIN
Keyword(s):  
Bend Angle ◽  
Satisfactory Explanation ◽  
Low Frequencies ◽  
Bending Waves ◽  
Wave Parameters ◽  
Low Concentrations ◽  
Bending Resistance ◽  
Wide Range ◽  
Triton X ◽  
Lower Frequencies

1. Treatment of Ciona spermatozoa with low concentrations of Triton X-100 (less than 0·01 %) causes them to beat at lower than normal frequencies. The wavelength of the flagellar bending waves remains constant over the range from 10 to 40 Hz. There is a small increase in wavelength at lower frequencies; in the range of 1·5-6·2 Hz, the wavelength averaged 114% of the normal value for Ciona spermatozoa. The angle of bend of the bent regions of the flagellar bending waves remained constant within ± 10% over this range of frequencies. 2. Decapitated sperm flagella from Lytechinus beat at a continually declining frequency as they exhaust their content of ATP. Both wavelength and bend angle retain normal values until the frequency falls below about 8 Hz. Both parameters increase at lower frequencies, with a sharp increase below 3 Hz. 3. ATP-reactivated spermatozoa from Lytechinus show relatively small changes in wavelength and bend angle as the frequency is varied over the range from 5 to 25 Hz by varying the ATP concentration. 4. Constancy of wavelength over a wide range of frequencies is consistent with the hypothesis that wavelength is determined by the relative values of viscous bending resistance within a flagellum and external viscosity. 5. No satisfactory explanation is available at present for the constancy of bend angle over a wide range of frequencies nor for the changes in wave parameters which are observed at low frequencies.

The Effect of Partial Extraction of Dynein Arms on the Movement of Reactivated Sea-Urchin Sperm

1973 ◽  
Vol 13 (2) ◽  
pp. 337-357 ◽  
Author(s):  
BARBARA H. GIBBONS ◽  
I. R. GIBBONS
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Beat Frequency ◽  
Wave Form ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Elastic Forces ◽  
Dynein Arms ◽  
Triton X ◽  
Sea Urchin Sperm

Sea-urchin sperm were extracted with o.5 M KCl for 45 s at room temperature in the presence of Triton X-100, and then transferred to reactivating solution containing 1 mM ATP. The flagellar beat frequency of these KCl-extracted sperm (16 beats/s) was only about half that of control Triton-extracted sperm that had not been exposed to 0.5 M KCl (31 beats/s), although the form of their bending waves was not significantly altered. Examination by electron microscopy showed that the extraction with 0.5 M KCl removed the majority of the outer arms from the doublet tubules, leaving the inner arms apparently intact. By varying the duration of the KCl-extraction, it was shown that the rate of decrease in beat frequency paralleled the rate of disappearance of the arms. Prolonging the extraction time beyond 45 s at room temperature, or 4 min at o °C, had little further effect on beat frequency. ATPase measurements suggested that 6o-65% of the dynein in the original axonemes had been solubilized when the extraction with KCl was permitted to go to completion. These results indicate that the generation and propagation of flagellar bending waves of essentially typical form are not prevented by the removal of the outer row of dynein arms from the doublet tubules. In terms of the sliding filament model of flagellar bending, the results suggest that the rate of sliding between tubules under these conditions is proportional to the number of dynein arms present. The lack of significant change in wave form implies that the total amount of sliding that occurs during each bending cycle is not affected by the reduced number of dynein arms, but is regulated independently in some manner by the elastic forces generated by other structures in the bent axoneme.

scholarly journals Intermittent swimming in live sea urchin sperm.

1980 ◽  
Vol 84 (1) ◽  
pp. 1-12 ◽  
Author(s):  
B H Gibbons
Keyword(s):  
Sea Urchin ◽  
Divalent Cations ◽  
Dark Field ◽  
Proximal Region ◽  
Ionophore A23187 ◽  
Quiescent State ◽  
Sodium Barbital ◽  
Dark Field Microscopy ◽  
Short Period ◽  
Live Sperm

Sperm of the sea urchin Tripneustes gratilla repeatedly start and stop swimming when suspended in seawater and observed by dark-field microscopy. While in the quiescent state, which usually lasts about a second, the sperm assume s shape resembling a cane, with a sharp bend of approximately 3.4 rad in the proximal region of the flagellum and very little curvature in the rest of the flagellum except for a slight curve near the tip. The occurrence of quiescence requires the presence of at least 2 mM Ca2+ in the seawater, and the percentage of sperm quiescent at any one time increases substantially when the sperm are illuminated with blue light. With intense illumination, close to 100% of the sperm become quiescent, and this percentage decreases gradually to approximately 0.3% over a 10(4)-fold decrease in light intensity. An increased concentration of K+ in the seawater also increases the percentage of quiescence, with a majority of the sperm being quiescent in seawater containing 80 mM KCl. The induction of quiescence by light or by increased KCl is completely inhibited by 10 micrometers chlorpromazine, and approximately 90% inhibited by 1 mM procaine or sodium barbital. Sperm treated with the divalent-cation ionophore A23187 swim quite normally, although for a relatively short period, in artificial seawater lacking divalent cations, but are abruptly arrested upon addition of 0.04--0.2 mM free Ca2%. The flagellar waveform of these arrested sperm is almost identical to that of light-induced quiescence in the live sperm. The results support the hypothesis that quiescence is induced by a rise in intracellular Ca2%, perhaps as a consequence of a membrane depolarization, and that it is similar to the arrest response in cilia.

scholarly journals Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin.

1985 ◽  
Vol 100 (6) ◽  
pp. 1875-1883 ◽  
Author(s):  
C J Brokaw ◽  
S M Nagayama
Keyword(s):  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Binding Activity ◽  
Calmodulin Binding ◽  
Bending Waves ◽  
Affinity Resin ◽  
The Asymmetry ◽  
Triton X ◽  
Asymmetric Bending ◽  
Sea Urchin Sperm

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.

scholarly journals FLAGELLAR MOVEMENT AND ADENOSINE TRIPHOSPHATASE ACTIVITY IN SEA URCHIN SPERM EXTRACTED WITH TRITON X-100

1972 ◽  
Vol 54 (1) ◽  
pp. 75-97 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Adenosine Triphosphatase ◽  
Beat Frequency ◽  
Average Frequency ◽  
Mitochondrial Atpase ◽  
The Difference ◽  
Live Sperm ◽  
Triton X ◽  
Sea Urchin Sperm

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 Pi/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 µmole Pi/(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 (Km) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "Km" of 0.2 mM.

scholarly journals Localized Activation of Bending in Proximal, Medial and Distal Regions of Sea-Urchin Sperm Flagella

1973 ◽  
Vol 13 (1) ◽  
pp. 1-10
Author(s):  
C. J. BROKAW ◽  
I. R. GIBBONS
Keyword(s):  
Sea Urchin ◽  
Adenylate Kinase ◽  
Distal Portion ◽  
Proximal Region ◽  
Triton X ◽  
Sea Urchin Sperm

Spermatozoa from the sea urchin, Colobocentrotus atratus, were partially demembranated by extraction with solutions containing Triton X-100 at a concentration which was insufficient to solubilize the membranes completely. The resulting suspension was a mixture containing some spermatozoa in which a proximal, medial, or distal portion of the flagellum was membrane-covered, while the remaining portion was naked axoneme. In reactivating solutions containing 12 µM ATP, only the naked portions of the flagellum became motile. In reactivating solutions containing 0.8 mM ADP, the membrane-covered regions became motile and beat at 6-10 beats/s, while the naked regions remained immobile, or beat very slowly at about 0.3 beat/s. Activation of membrane-covered regions in ADP solutions probably results from the membrane restricting the diffusion of ATP which is formed from ADP by the axonemal adenylate kinase. The results indicate that any region of the flagellum has the capacity for autonomous beating, and that special properties of the basal end of the flagellum are not required for bend initiation. However, the beating of different regions of the flagellum is not completely independent, for in a fair number of spermatozoa the beating of the distal, membrane-covered region in 0.8 mM ADP was intermittent, and was turned on and off in phase with the much slower bending cycle in the proximal region of naked axoneme.

scholarly journals Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.

1975 ◽  
Vol 66 (1) ◽  
pp. 198-200 ◽  
Author(s):  
D Mazia ◽  
G Schatten ◽  
W Sale
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Experimental Treatment ◽  
The Body ◽  
Transmission Electron ◽  
Coated Plate ◽  
Living Material ◽  
Coated Surfaces ◽  
Triton X

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine-coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).

scholarly journals Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.

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