Functional state of the axonemal dyneins during flagellar bend propagation.

When mouse spermatozoa swim in media of high viscosity, additional waves of bending are superimposed on the primary traveling wave. The additional (secondary) waves are relatively small in scale and high in frequency. They originate in the proximal part of the interbend regions. The initiation of secondary bending happens only in distal parts of the flagellum. The secondary waves propagate along the interbends and then tend to die out as they encounter the next-most-distal bend of the primary wave, if that bend exceeds a certain angle. The principal bends of the primary wave, being of greater angle than the reverse bends, strongly resist invasion by the secondary waves; when a principal bend of the primary wave propagates off the flagellar tip, the secondary wave behind it suddenly increases in amplitude. We claim that the functional state of the dynein motors in relation to the primary wave can be deduced from their availability for recruitment into secondary wave activity. Therefore, only the dyneins in bends are committed functionally to the maintenance and propagation of the flagellar wave; dyneins in interbend regions are not functionally committed in this way. We equate functional commitment with tension-generating activity, although we argue that the regions of dynein thus engaged nevertheless permit sliding displacements between the doublets.

A study of helical and planar waves on sea urchin sperm flagella, with a theory of how they are generated.

When the spermatozoon of Echinus esculentus swims in sea water containing methyl cellulose (viscosity 1.5--4 Pa s), its flagellum may generate either a helical or a planar waveform, each type being stable. The helical wave, which is dextral, is complicated by the concurrent passage of miniature waves along it. These miniature waves have a pulsatile origin in the neck region of the spermatozoon. Our videotape analysis indicates that there are two pulses of mechanical activity for each true cycle of the helical wave. (The true helical frequency was obtained from the apparent wave frequency and the roll frequency of the sperm head, the latter being detectable in some sperm when lit stroboscopically.) The planar wave has a meander shape. During the propagation of planar waves, the sliding displacements are adjustable in either direction; moribund flagella can undergo unrestricted sliding. The planar waves are, in fact, exactly planar only at interfaces. Otherwise, there tend to be torsions in the interbend segments between planar bends. Mechanical stimulation of the flagellum can cause a sudden transition from the helical to the planar waveform. To account for the two modes of beating, we advance the hypothesis that circumferential linkages yield beyond a threshold strain. Whether this yield point is exceeded, we suggest, depends upon the balance between the active shear force and the external viscosity (among other factors). We propose that a subthreshold force originates in one array and then triggers the other dynein arrays circumferentially, but unidirectionally, around the base of the flagellum; whereas a suprathreshold force provokes bi-directional circumferential triggering. These may be the two patterns of activation that result in helical and planar waveforms, respectively. The transition from helical to planar bending may result from an increment in the force produced by the dynein motors. The pulsatile origin of the helical wave resembles behaviour described previously for spermatozoa of Ciona intestinalis and of the quail Coturnix coturnix.

Three-dimensional motion of avian spermatozoa.

Observations have been made on spermatozoa from the domestic fowl, quail and pigeon (non-passerine birds) and also from the starling and zebra finch (passerine birds). In free motion, all these spermatozoa roll (spin) continuously about the progression axis, whether or not they are close to a plane surface. Furthermore, the direction of roll is consistently clockwise (as seen from ahead). The flagellar wave has been shown to be helical and dextral (as predicted) for domestic fowl sperm when they swim rapidly in low viscosity salines. Calculations have shown that their forward velocity is consistent with their induced angular velocity but that the size of the sperm head is suboptimal for progression speed under these conditions. Dextrally helical waves also occur on the distal flagellum of fowl, quail and pigeon sperm in high viscosity solutions. But in other cases, the mechanism of torque-generation is more problematical. The problem is most profound for passerine sperm, in that typically these cells spin rapidly while seeming to remain virtually straight. Because there is no evidence for a helical wave on these flagella, we have considered other possible means whereby rotation about the local flagellar axis (self-spin) might be achieved. Sometimes, passerine sperm, while maintaining their spinning motion, adopt a fixed curvature; this must be an instance of bend-transfer circumferentially around the axonemal cylinder-though the mechanism is obscure. It is suggested that the self-spin phenomenon may be occurring in non-passerine sperm that in some circumstances spin persistently, yet without expressing regular helical waves. More complex waves are apparent in non-passerine sperm swimming in high viscosity solutions: added to the small scale bends is a large scale, sinistrally helical curvature of the flagellum. It is argued that the flagellum follows this sinistrally helical path (i.e. "screws" though the fluid) because of the shape of the sperm head and the angle at which the flagellum is inserted into it. These conclusions concerning avian sperm motility are thought to have relevance to other animal groups. Also reported are relevant aspects of flagellar ultrastructure for pigeon and starling sperm.

Real-time visual detection of Pi released by flagellar dynein ATPase.

Using a novel fluorescent probe for Pi, a method for the direct visualization of Pi release from reactivated flagellar dynein ATPase has been developed. The probe undergoes a fluorescence increase when it binds Pi. The technique involves simultaneous imaging of demembranated sperm tails by epi-fluorescence and dark-field microscopy, and the use of the caged ATP technique for axoneme reactivation. To limit diffusion and thus maintain the released Pi within the observed field of view, the assay is carried out within a minute droplet under oil (volume 5-15 pl). The video output of a recursively filtered ICCD camera is used to visualize the fluorescence signal, which is subsequently digitized and automatically analysed on a PC. A major advantage of this technique is that it enables simultaneous analysis of the ATP-utilization rate and the motility of the reactivated axonemes.

The propagation of a zone of activation along groups of flagellar doublet microtubules.

The complexity of the 9 + 2 flagellar axoneme has made it difficult to discover the mechanism of bend propagation. We have studied a simplified preparation of mechanically "opened-out" groups of doublet microtubules that adopt a helical, ribbon-like form. From the very long sperm of the quail, ribbons of doublets up to 130 microns long have been obtained. We reactivated them photolytically by releasing ATP from caged ATP, thus observing the reactivation from the beginning. The response to ATP was a reduction in the pitch and diameter of the helix, in what we refer to as an "active zone" (AZ). Ahead of the AZ was a short region of increase in helical pitch and diameter, the pre-AZ. We ascribe these two altered geometries to the development of tension between the doublets, actively (in the AZ) and passively (in the pre-AZ). The AZ/pre-AZ complex established itself at one end of a helix--almost certainly the proximal end--then it propagated toward the other end of the helix at a mean velocity of 15 microns s-1 (using 1 mM caged ATP), maintaining or increasing its length as it traveled. This is the same velocity as that for bend propagation on cylindrical axonemes detached from their basal structures. Successive propagations on the same helix were seen. Thus, active and inactive segments of the same doublet assembly can coexist, even though all parts are exposed to ATP. The motor response is seen to be a localized event that is transmitted metachronally. The propagation of activation is an intrinsic property of structures in the interdoublet gap and does not require constituents of the cytosol other than ATP and Mg2+. Since it occurs in helical ribbons (3 + 0, 4 + 0, etc.), the propagation of activity must be independent of central axonemal structures; furthermore, it cannot be dependent on the integrity of the 9 + 2 cylinder nor on any feedback from large-scale features of the waveform.

Direct evidence for tension development between flagellar doublet microtubules.

In this study of isolated ribbons of flagellar doublet microtubules, we demonstrate that a resistance to sliding exists in the interdoublet gap. By photolytically releasing ATP from caged ATP, it has been possible to follow closely the responses of individual specimens. Distortion of the helical superstructure of the doublets, most often by a reduction in helical pitch, is interpreted as revealing the development of tension between doublets. Tension does not develop in the presence of vanadate.