Polarity in spontaneous unwinding after prior rotation of the flagellar beat plane in sea-urchin spermatozoa

As reported previously, the beat plane of live and reactivated sea-urchin sperm flagella held by their heads at the tip of a vibrating micropipette can be forced to rotate relative to the sperm head by rotating the plane of the imposed vibration (Gibbons et al. 1987; Shingyoji et al. 1991a,b). This rotation of the flagellar beat plane appears to result from a rotation of the pattern of coordinated sliding among the nine outer doublet tubules of the axoneme, with no twisting of the axoneme as a whole. The fact that flagellar bending waves can be generated in any plane around the longitudinal axis of the axoneme indicates that the determination of the beat plane is not primarily the result of a chemical or structural specialization of particular members of the nine doublet tubules, but is the result of some regulatory factor that can be forced to rotate relative to the cylinder of doublet tubules. Indirect evidence suggests that this regulatory factor may be the central pair of microtubules that possibly modulates the sliding activity of individual doublets through the radial spokes that connect the central sheath to each doublet (Shingyoji et al. 1991b).

Spontaneous rotation of the central pair of microtubules has been reported to occur in cilia and flagella of several cell types (Omoto and Kung, 1980; Omoto and Witman, 1981; Kamiya, 1982; Hosokawa and Miki-Noumura, 1987). Therefore, if the central pair is involved in the forced rotation of the beat plane under our conditions, we anticipate that the properties of imposed rotation of the beat plane may depend on whether the rotation is in a clockwise or counterclockwise direction. In this paper, we describe such a polarity in the spontaneous unwinding of the beat plane that occurs upon cessation of the imposed vibration after several cycles of prior winding. The possible influence of the central pair microtubules on the orientation of the flagellar beat plane is discussed.

The motility of live and reactivated sperm of the sea-urchin Hemicentrotus pulcherrimus was observed as described previously (Shingyoji et al. 1991a).

The suction pipettes used to vibrate the sperm heads were prepared as described earlier (Shingyoji et al. 1991a). Sperm were caught by bringing the tip of the micropipette close to the sperm head and gently sucking the anterior quarter to third of the head into the micropipette.

The micropipette was oscillated by means of a piezoelectric driver consisting of two orthogonal piezo bimorphs capable of vibrating at frequencies up to 60 Hz, with the direction being determined by the relative amplitudes of sinusoidal voltage applied to each bimorph (Corey and Hudspeth, 1980; Gibbons et al. 1987; Eshel et al. 1990; Shingyoji et al. 1991a,b). The voltages supplied to the bimorphs were generated by a computer program and a D/A converter (ADALAB-PC) as described previously (Eshel and Gibbons, 1989). In order to avoid undesired transients, cessation of vibration was timed to occur as the micropipette was passing through its resting position.

The recording and analysis of flagellar movement were performed using a high speed video system (200 frames s⁻¹) as described previously (Shingyoji et al. 19916). In cases where fractional rotations needed to be quantitated (as in Fig. 3), we determined the number of frames on the CRT monitor compared to the number of frames required to complete one quarter-rotation.

When the head of a live sperm is held with a vibrating micropipette and the plane of the vibration is gradually rotated, the beat plane of the flagellum rotates along with the plane of vibration. In most live and reactivated sperm, the beat plane can be rotated for about nine revolutions (Gibbons et al. 1987; Shingyoji et al. 1991b). The clockwise or counterclockwise direction of rotation appeared to make no clear difference in the number of revolutions that could be imposed before retrograde slippage occurred. The maximum numbers of revolutions observed in live spermatozoa under standard conditions were 11.5 in the counterclockwise direction and 10 in the clockwise direction.

When the vibration of the micropipette was stopped after several cycles of winding had been imposed on the flagellum, the flagellar beat plane unwound spontaneously as reported by Gibbons et al. (1987). Fig. 1 shows flagellar waveforms during spontaneous unwinding for nine revolutions, after nine revolutions of prior winding of the beat plane in the counterclockwise direction. During the period of unwinding, the flagellum continues to beat stably and the asymmetry of the bending wave rotates together with the plane of beat (Fig. 1). No twisting or other distortion of the flagellar waveform was observed. Changes in the speed of the imposed winding did not appear to affect the subsequent spontaneous unwinding.

In order to determine whether the tip region of the flagellum is required for unwinding to occur, a limited number of observations were made with live sperm whose flagella had been shortened by mild homogenization. The beat plane of sperm with flagella of about half the normal length could be made to rotate for up to three revolutions, and upon cessation of the pipette vibration their beat plane unwound toward its original orientation.

The number of spontaneous unwinding revolutions of the beat plane depended on the direction of the prior imposed winding. As shown in Fig 2A and C for live and reactivated sperm, respectively, when vibration was stopped after counterclockwise winding the number of revolutions of spontaneous winding was nearly the same as the number of revolutions of prior winding, so that the flagellar beat plane came almost completely back to its original orientation. On the other hand, after prior clockwise winding, the flagellar beat plane usually went through fewer than two revolutions of spontaneous unwinding, regardless of the number of revolutions of prior imposed winding (Fig 2B,D). The sperm flagella show a notable tendency to stop unwinding when their beat plane is close to the natural beat plane, as observed before the start of vibration, even when the latter differs from the vibration plane of the pipette. This tendency probably contributes to the scatter of the points from the lines in Fig. 2.

The sperm with shortened flagella showed essentially the same polarity of unwinding after clockwise and counterclockwise winding.

Fig. 3 shows the initial angular velocity of unwinding as a function of the number of prior imposed revolutions. The initial velocity increases with the number of imposed revolutions after counterclockwise or clockwise winding, in both live and reactivated sperm. There was no significant difference between the velocity of unwinding after either counterclockwise or clockwise winding. The maximum angular velocity observed in live sperm was approximately 27 rad s⁻¹, after nine revolutions of winding. The corresponding maximum velocity for reactivated sperm was approximately 20rad s⁻¹, after seven revolutions of winding.

The variation among sperm that results in the scatter of the data shown in Fig. 3, makes it difficult to analyze the time course of unwinding. Therefore, we have performed more detailed kinetic studies on a small number of live sperm. Unwinding of the beat plane began within 5 ms after the stoppage of pipette vibration (Fig. 4). Fig. 4A presents the time course of unwinding after three, six and nine imposed revolutions of counterclockwise winding and Fig. 4B shows the corresponding course after clockwise winding. Since the simplest model of a linear elastic force being responsible for unwinding predicts that the time course of unwinding should be exponential, we fit exponential curves of the form R=∈+R_o e^−t/τ to each of the data sets in Fig. 4A by using the Simplex procedure to vary the coefficients for minimum root-mean-square (r.m.s.) deviation. If this simple elastic model is valid, then ∈ should equal zero, within experimental error, in all experiments (for the system will always return to its initial state of lowest energy), R_o should equal the number of imposed revolutions and τ should be a constant, depending on the elastic modulus and the viscous resistance, that is independent of the number of imposed revolutions; t is time of unwinding. The values obtained from such fitting are presented in Table 1. As can be seen, ∈ averages to zero over the three experiments and R_o approximately equals the number of initial winding revolutions in each case. The values of τ show up to 20 % deviation from their mean value of 3.5, but the irregular manner in which this deviation depends on the number of imposed revolutions suggests that it is due to experimental error. The calculated value of the initial angular velocity is approximately proportional to R_o, with the deviations arising from the scatter in the values of τ. Alternative models, such as fitting a straight line to the first portion of each of the data set, cannot be excluded. However, since we were unable to provide a physical rationale for other such models we have avoided further attempts in this direction.

Following clockwise winding (Fig. 4B), the beat plane unwound rapidly for 0.5–1 revolution immediately upon stoppage of pipette vibration but then rapidly slowed down and stopped. Although the initial velocity of spontaneous unwinding was a function of the number of revolutions of the prior clockwise winding (Fig. 3), the total extent of unwinding in this case did not exceed one to two revolutions, regardless of the number of prior winding cycles (Fig. 4). The time course of unwinding in reactivated sperm was similar to that in live sperm (data not shown).

Since flagella that had undergone incomplete unwinding after clockwise winding appeared to beat stably with normal waveforms, it seemed unlikely that they had been physically damaged during the process of clockwise winding. This is confirmed by the fact that after imposition of a second set of winding revolutions, this time in the counterclockwise direction through the original state and beyond, such sperm flagella unwound spontaneously back only to their original state, and not to the state in which the second set of imposed (counterclockwise) winding had begun. These observations suggest that the sperm flagellum in some way retains information regarding the number of revolutions of prior imposed winding, even when complete spontaneous unwinding does not occur.

In order to test this hypothesis, we applied a series of various numbers of imposed winding and unwinding cycles to live and reactivated sperm and observed the subsequent number of spontaneous revolutions of unwinding upon cessation of vibration. Fig. 5A shows a sperm in which cessation of vibration after four turns of imposed clockwise winding was followed by one turn of spontaneous counterclockwise unwinding. After resumption of vibration and imposition of five turns of counterclockwise winding to the same sperm, cessation of vibration was followed by only two turns of spontaneous clockwise unwinding. The next 3.5 turns of imposed counterclockwise winding applied to the same sperm, however, were followed by a complete 3.5 turns of clockwise unwinding. Analogous results were obtained for the other combinations of winding cycles applied to the two live sperm shown in Fig. 5B,C, as well as in 23 other live sperm examined in this manner. In all cases, spontaneous unwinding from a net counterclockwise orientation was almost or fully complete, whereas the comparable unwinding from a net clockwise orientation underwent only 0.5–1.5 turn.

Similar results were obtained with reactivated sperm (Fig. 5D and E). However, the flagella of reactivated sperm were more fragile than those of live sperm and in most cases we could not apply more than one or two cycles of winding and unwinding to each flagellum.

These results strongly suggest that the sperm flagella retain information regarding the original orientation of their beat plane and asymmetry throughout cycles of clockwise or counterclockwise winding and unwinding. Winding in the counterclockwise direction is always followed by spontaneous unwinding, usually through the same number of revolutions. Although the spontaneous unwinding of the beat plane after imposed clockwise rotation is usually incomplete, information regarding the extent of incomplete unwinding is nevertheless retained within the flagellum and can be detected by subsequently imposing a set of counterclockwise revolutions.

Live and reactivated sperm from the Hawaiian sea-urchin Tripneustes gratilla and the Japanese sea-urchin Strongylocentrotus intermedias showed essentially the same results as those reported above for Hemicentrotus pulcherrimus sperm. However, we were unable to induce a similar rotation of the beat plane with sperm from Pseudocentrotus depressus (Japanese sea-urchin) or Colobocentrotus atratus (Hawaiian sea-urchin). In sperm of these species, the frequency of the flagellar waves became synchronized with that of the pipette vibration, but when the plane of vibration was rotated the plane of flagellar beat always reverted spontaneously to its original orientation after less than one half-cycle of rotation had been imposed.

In the accompanying paper (Shingyoji et al. 1991b), we have demonstrated that the rotation of the beat plane and asymmetry of the flagellar waveform imposed by rotating the plane of external vibration applied to the sperm head appears to be the result of a rotation of the pattern of sliding among the doublet tubules of the axoneme and that it is not accompanied by a twisting of the entire axonemal structure.

After about 10 complete revolutions of winding have been imposed, the magnitude of the elastic restorative force appears to have increased to the point where it begins to exceed the maximum winding force that can be applied through the viscous interaction between the flagellum and the fluid medium. Thus the observed upper limit on the number of imposed revolutions may well be due to the magnitude of this viscous interaction rather than to an intrinsic limitation of the flagellar structure. This hypothesis is supported by the fact that it has been possible to impose only a smaller number of revolutions in sperm with short flagella.

When the external vibration is stopped after the beat plane of the flagellum has been forced to rotate, the beat plane and direction of asymmetry immediately and spontaneously rotate back (Fig. 1). The stable flagellar beating observed during this spontaneous unwinding indicates that the unwinding is probably caused by a similar, but reverse, rotation of the pattern of sliding among the doublet tubules. The approximately exponential behaviour of the time course of unwinding suggests that elastic distortion of some structural component of the axoneme makes a major contribution to the restorative force responsible for unwinding, although the exact origin of this force is unclear. However, the difference in completeness of unwinding after counterclockwise or clockwise winding indicates that other more complex factors are also involved.

The initial angular velocity of unwinding appears to depend only on the number of revolutions and to be independent of their direction, although the total extent of spontaneous unwinding differs substantially, depending on the direction. This result suggests that the magnitude of the force responsible for spontaneous unwinding is essentially the same, regardless of the direction of prior imposed winding, and that the incomplete unwinding observed after clockwise winding is due to the intervention of some other factor that constrains the unwinding process. The information regarding the original orientation of the beat plane that is still present in incompletely unwound flagella is presumably associated with residual distortion of the elastic structure responsible for unwinding.

The seeming block to complete unwinding in the counterclockwise direction (i.e. after clockwise winding) is presumably derived in some manner from an asymmetric aspect of the axonemal structure. Examples are the handedness of the dynein arm arrangement on the doublet tubules (Gibbons, 1961), the radial spokes arranged in a three-start right-handed helix (Goodenough and Heuser, 1985), and the helical lattice of tubulin subunits comprising the wall of the doublet tubules and the central pair (Amos and Klug, 1974; Linck and Amos, 1974). Moreover, spontaneous rotation of the central pair has been reported in cilia of Paramecium (Omoto and Kung, 1980), in a uniflagellate marine alga (Omoto and Witman, 1981) and in Chlamydomonas flagella (Kamiya, 1982; Hosokawa and Miki-Noumura, 1987). Although the central pair in seaurchin sperm flagella are believed not to rotate during normal flagellar beating, it is possible that the handedness of the axonemal structure preferentially facilitates rotation of the central pair in a clockwise direction after it has been forcibly rotated from its normal orientation.

The identity of the elastic axonemal structure responsible for beat plane unwinding remains unknown, but since spontaneous unwinding can occur in short flagella with their distal ends removed, the structure is presumably located either in the basal region of the flagellum or distributed along its length. One possibility is that, as winding proceeds, the central pair complex is displaced longitudinally as a result of rotating along the helical array of radial spokes (Goodenough and Heuser, 1985; Gibbons et al. 1987). A second possibility is that the central pair are tightly attached to the centriole at their basal ends and become twisted in their basal region. Although in a previous electron microscopic study of sea-urchin sperm flagella there was no report of any particular attachment at the basal end of the central pair (Afzelius, 1955), it will be interesting to see if different sea-urchin species show any structural differences that can be correlated with the relative ease with which their beat plane can be rotated by imposed vibration.

Vale and Toyoshima (1988) reported that 14 S dynein from Tetrahymena both translocates microtubules over glass and rotates them clockwise around their longitudinal axis (as viewed from the minus end of the microtubule, which corresponds to the base of the axoneme). On the basis of these observations, they suggested that a 14S dynein-induced torque may induce the rotation of the central pair microtubules within the axoneme of Tetrahymena cilia. More recently, Toyoshima (1989) has also isolated a 13S dynein from sperm flagella of the seaurchin, Hemicentrotus pulcherrimus, that also rotates microtubules on glass.

It is possible that a rotatory activity of a specialized axonemal dynein could underlie the preferential clockwise unwinding that we have observed in sea-urchin sperm flagella under our conditions. While we have no direct evidence that ATP-dependent energy is involved in the unwinding process, we have observed several times that the process of unwinding is interrupted if flagellar beating happens to become arrested while unwinding is in progress, and it continues only when beating resumes. This implies that the relaxation of the elastic distortion postulated to underlie the tendency to restore the natural beat plane can only occur in a beating flagellum. Future observation on the effect of ATP concentration on the unwinding velocity of reactivated sperm flagella may be expected to clarify the possible role of ATP hydrolysis in the unwinding process.

We thank Dr Kenjiro Yoshimura for his help with modifying the computer program to operate the pipette vibration and Dr Barbara Gibbons for assistance with the preparation of the manuscript. This investigation was supported in part by funds provided to K.T. and I.R.G. through the US-Japan Cooperative Science Program sponsored by the Japan Society for the Promotion of Science and the National Science Foundation, USA, the Grant-in-Aid for Scientific Research of the Ministry of Education, Science and Culture of Japan, no. 62480016 to K.T., and N.I.H. grant HD06565 to Dr Barbara Gibbons.