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1.
J Anat ; 213(3): 336-41, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18537850

ABSTRACT

Electron microscopy has been used to investigate whether the transversely striated columns of the connecting piece in the neck region of guinea pig spermatozoa, undergo lengthening and shortening as a result of the forces generated during motility. Motile spermatozoa were subjected to near-instantaneous rapid freezing, followed by freeze-substitution fixation and epoxy embedment. Thin sections passing longitudinally through the striated columns revealed that the periodicity was indeed variable. The repeat period, taken to have an unstressed width of 60 nm, could be found extended to 75 nm in some specimens, and reduced to 54 nm in others. The estimates of the coefficients of variation were 6.6% for the width of the 'dense' band and 33.5% for the 'pale' band. The 'pale' band in the extended state showed longitudinal striae. Such variations in length, which - it is suggested - are physiological, and passively induced, would have functional implications for the flagellum - for both bend initiation and bend growth. Also, hypothetically, any mechanism that could increase the degree of compliance in these columns, such as perhaps phosphorylation of the constituent proteins, could permit the flagellum to develop the exaggerated bend angles and asymmetries of the 'hyperactivated' state.


Subject(s)
Sperm Midpiece/physiology , Animals , Compliance , Freeze Substitution , Guinea Pigs , Male , Microscopy, Electron , Sperm Midpiece/ultrastructure
2.
Reproduction ; 126(2): 259-70, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12887282

ABSTRACT

The hydrodynamic basis for the accumulation of spermatozoa at surfaces has been investigated. The general conclusion is that when spermatozoa arrive at a surface, they will remain there if the vector of the time-averaged thrust is directed towards that surface. This can arise in two basic ways. First, consider spermatozoa that maintain a three-dimensional waveform and roll (spin) as they progress: in this case, it is argued that the conical (rather than cylindrical) shape of the flagellar envelope will establish the direction-of-thrust necessary for capture by the surface. (Additional findings, for spermatozoa of this type, are that the swim-trajectory is curved and that the direction of its curvature reveals the roll-direction of the cell.) Second, consider spermatozoa that maintain a strictly two-dimensional waveform at the surface: in this case, spermatozoa can be captured because the plane-of-flattening of the sperm head is tilted slightly relative to the plane of the flagellar beat. The sperm head is acting as a hydrofoil and, in one orientation only, it comes to exert a pressure against the surface. (This pressure may possibly, in vivo, aid the penetration of the zona pellucida.) The hydrofoil action of sperm heads may explain any bias in the circling direction of spermatozoa that execute two-dimensional waves at surfaces. Finally, a more complex phenomenon is where interaction of the spermatozoa with the surface appears to induce a three-dimensional to two-dimensional conversion of the flagellar wave (thus permitting the hydrofoil effect described). This is characteristic of sperm with 'twisted planar' rather than helical waves. In mammalian spermatozoa, approximately half the beat cycle is planar and the other half generates a pattern of torque causing the head to roll clockwise (seen from ahead), producing a torsion of the neck region of the flagellum. It is the gradual suppression of this torsion, by either impedance at the solid boundary or by raised viscosity, that converts the 'twisted planar' shape into a planar wave.


Subject(s)
Sperm Motility/physiology , Spermatozoa/physiology , Animals , Cells, Cultured , Chinchilla , Male , Mice , Mice, Inbred Strains , Microscopy, Electron, Scanning , Rats , Rats, Wistar
3.
Biophys J ; 83(4): 2162-9, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12324433

ABSTRACT

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.


Subject(s)
Dyneins/metabolism , Movement , Sperm Motility , Sperm Tail/physiology , Spermatozoa/ultrastructure , Animals , Biophysical Phenomena , Biophysics , Male , Mice , Sperm Tail/metabolism , Sperm Tail/ultrastructure
4.
J Exp Biol ; 204(Pt 7): 1333-45, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11249842

ABSTRACT

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.


Subject(s)
Sea Urchins/physiology , Animals , Biomechanical Phenomena , Male , Methylcellulose , Solutions , Sperm Motility/physiology , Sperm Tail/physiology , Time Factors , Videotape Recording , Viscosity
5.
Cell Motil Cytoskeleton ; 42(2): 149-61, 1999.
Article in English | MEDLINE | ID: mdl-10215424

ABSTRACT

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.


Subject(s)
Birds/physiology , Flagella/physiology , Spermatozoa/physiology , Aging/physiology , Animals , Cell Movement/physiology , Columbidae/physiology , Coturnix/physiology , Male , Microscopy, Electron, Scanning , Microscopy, Video , Poultry/physiology , Saline Solution, Hypertonic/pharmacology , Songbirds/physiology , Spermatozoa/cytology , Spermatozoa/ultrastructure , Temperature , Viscosity
6.
Pflugers Arch ; 437(5): 771-5, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10087156

ABSTRACT

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.


Subject(s)
Chickens/physiology , Dyneins/metabolism , Phosphates/metabolism , Sperm Tail/enzymology , Animals , Calibration , Image Processing, Computer-Assisted , In Vitro Techniques , Kinetics , Male , Photolysis , Sperm Tail/ultrastructure , Ultraviolet Rays
7.
Cell Motil Cytoskeleton ; 39(3): 233-45, 1998.
Article in English | MEDLINE | ID: mdl-9519904

ABSTRACT

The sperm flagellum of Anguilla anguilla lacks outer dynein arms, radial spokes and central structures. Its characteristic motion has been obtained by studying cells swimming perpendicularly against, but not adhering to, the coverslip. The flagellum generates a sinistrally helical wave of rising, then falling, amplitude. The frequency of the wave, which can exceed 70 Hz, is inversely related to its maximum amplitude. As a reaction to the torque, the entire cell rolls (spins) in the opposite direction to that taken by points on the flagellum in the generation of the sinistral wave. However, because the head (which contributes on opposing torque) is asymmetrical, the axis of this counter-rotation is displaced laterally from the axis of the flagellar rotation. As a result, the flagellum precesses around the progression axis, with each point on the flagellum travelling along a special flagelloid curve, specified by the ratios of the two frequencies and the two radii for the circular motions. The instantaneous flagellar waveform (the flagelloid wave) is thus derived as a succession of phase-shifted points on the series of flagelloid curves along the axis of the cell's rotation. This adds complexity to the underlying, rather simple, helical geometry. Calculations suggest that the forward swimming speed of the sperm is greatly aided by the orientation and shape of the sperm head. The movement of latex beads was observed around sperm swimming against the coverslip and around sperm swimming freely. Bulk, vortical flows of fluid were seen in the former case and net lateral displacements in the latter; this is in accordance with hydrodynamic theory for low Reynolds number systems.


Subject(s)
Sperm Motility/physiology , Sperm Tail/physiology , Anguilla , Animals , Computer Simulation , Male , Microscopy, Video
8.
Cell Motil Cytoskeleton ; 39(3): 246-55, 1998.
Article in English | MEDLINE | ID: mdl-9519905

ABSTRACT

A further account is given of motility in this 9 + 0 flagellum, where the axoneme is of special interest because it is powered by only inner dynein arms. Under some circumstances, normal motility is inactivated and yet the flagellum swims (or appears to glide) forward, albeit much more slowly. The propulsive thrust in these cases is due to a vibratile motion of the flagellum. Vibratile motion has a very small amplitude and is very rapid, but a frequency could not be determined stroboscopically. Provided that the sperm head is in place, a vibratile sperm can be stimulated mechanically such that it instantly resumes and continues normal motility. This indicates that a suprathreshold deformation of the axoneme triggers normal motility and that the threshold is normally continuously exceeded by a self-generated fluid-mechanical interaction in which the sperm head plays a necessary part. Without a sperm head, the flagellum propels itself by vibratile motion. Some vibratile sperm, found to be stuck by their heads, perform also a slow rotatory (clockwise) bending at the base of the flagellum. When this happens, there is no rotation of the axonemal substance. Therefore, this is interpreted as sequential, clockwise, self-perpetuating, circumferential activity around the arrays of inner dynein arms. The phenomenon is considered to be a restricted representation of the rapid clockwise (i.e., sinistral) helical wave of normal motility.


Subject(s)
Sperm Motility/physiology , Sperm Tail/physiology , Adenosine Triphosphate/pharmacology , Animals , Eels , Male , Microscopy, Video , Sperm Tail/ultrastructure
9.
J Cell Sci ; 110 ( Pt 1): 85-94, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9010787

ABSTRACT

The highly motile, 9 + 0 sperm axoneme of Anguilla has inner dynein arms (IDAs) but not outer dynein arms. The in situ morphology of these IDAs is shown here to be essentially identical to the IDA morphology already seen in the axonemes of Chlamydomonas, Tetrahymena and Beroë, and in the sperm tails of echinoderms and several vertebrate species. In addition, this study demonstrates: (1) that the nexin (circumferential) links are present in Anguilla and are typical; (2) that IDA1 incorporates an archway, supported by a pillar-structure; (3) that images from thin sections and whole mounts are consistent with those from replicas of rapidly-frozen specimens; and (4) that the IDA and nexin link morphology is apparently unaffected by whether the axoneme is depleted of ATP, relaxed with ATP and vanadate, or inhibited by high ATP. An attempt has been made to reconcile the emergent morphology of the IDA complex with all earlier descriptions in the literature. From a detailed comparison of the results with published information on Chlamydomanas mutants, it is concluded that the nexin (circumferential) link is a major part of the 'dynein regulatory complex'.


Subject(s)
Dyneins/ultrastructure , Sperm Tail/ultrastructure , Anguilla , Animals , Freeze Etching , Male , Microtomy , Models, Structural , Species Specificity , Spermatozoa/ultrastructure , Subcellular Fractions/ultrastructure
10.
Exp Cell Res ; 220(2): 482-94, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7556458

ABSTRACT

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.


Subject(s)
Microtubules/physiology , Microtubules/ultrastructure , Sperm Tail/physiology , Sperm Tail/ultrastructure , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Coturnix , Male , Microscopy, Electron , Microtubules/drug effects , Models, Structural , Sperm Tail/drug effects , Video Recording
11.
J Submicrosc Cytol Pathol ; 27(3): 281-90, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7671210

ABSTRACT

Extremely fine threads have been seen, under dark-field illumination, extending from the tips of the sperm flagella of four avian and two mammalian species. They occur after a variable period of incubation in balanced salt solutions. The threads, never more than one per flagellum, can be as long as the flagellum itself. Often they are attached to the substrate by their remote end, tethering the cell and showing elastic deformation. When not stressed, they remain threadlike and undergo the Brownian motion characteristic of fine filaments. Shedding of the threads has been seen, as has their apparent retraction. When cockerel sperm are broken into proximal and distal segments which are respectively motile and immotile, the threads develop at the tips of only the proximal segments. This proves that the formation of the thread is not dependent on structures normally present at the natural tip of the flagellum. Each thread is a regular tubule of membrane, with a standard minimum diameter of 28 nm, and having blebs of membrane, containing vesicles, at irregular intervals. The tubular membrane is assumed to be plasmalemma and is equivalent, maximally, to less than 10% of the cell's calculated surface area. It is most likely that the thread is formed by extrusion, as a stalk bearing fluid-filled pinocytotic vesicles. Thread formation is prevented by adding bovine serum albumin to the diluent. The phenomenon may be relevant to understanding how spermatozoa are impaired by the 'dilution effect'.


Subject(s)
Sperm Tail/ultrastructure , Animals , Birds , Cell Membrane/ultrastructure , In Vitro Techniques , Male , Mammals , Mice , Microscopy/methods , Microscopy, Electron , Pinocytosis , Rats , Sperm Tail/physiology
12.
J Exp Biol ; 198(Pt 7): 1469-81, 1995 Jul.
Article in English | MEDLINE | ID: mdl-9319369

ABSTRACT

The distal region of the sperm flagellum of Gallus domesticus has been separated and purified. It consists of a 9+2 axoneme, without basal or accessory structures. Such distal segments have been demembranated and then reactivated, either by adding ATP or by releasing ATP photolytically from caged ATP: we find that they are capable of a period of independent motility. Bends form repetitively and travel towards the tip, though it is an abnormal, irregular pattern of beating. It is argued that this motility is not dependent on damage to the flagellum at the fracture site. Evidence is presented that the potential for such motility depends upon the existence of bends on the axoneme before the reactivation. The reactivated motility is short-lived: 50 % of the distal flagellar segments, placed in the reactivating solution, become quiescent and straight within 60 s. However, vigorous beating can be induced in such quiescent segments of axoneme by compressing one end with a glass microneedle. We record, provisionally, that the site of compression does not determine the direction in which bends move along the flagellar segment. The effect of compression in re-initiating motility suggests that a mechanical resistance is necessary, somewhere along the axoneme, for normal, sustained motility; it is proposed that the specialized basal structures, collectively, provide such a resistance in the intact flagellum.

13.
Exp Cell Res ; 215(2): 390-4, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7982478

ABSTRACT

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.


Subject(s)
Flagella/physiology , Flagella/ultrastructure , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/radiation effects , Animals , Biomechanical Phenomena , Chickens , Flagella/drug effects , In Vitro Techniques , Male , Microscopy, Electron , Microtubules/drug effects , Microtubules/physiology , Microtubules/ultrastructure , Photolysis , Sperm Tail/drug effects , Sperm Tail/physiology , Sperm Tail/ultrastructure , Vanadates/pharmacology
14.
Cell Motil Cytoskeleton ; 24(2): 109-18, 1993.
Article in English | MEDLINE | ID: mdl-8440024

ABSTRACT

In this work, we examine whether the "nexin" linkages of the flagellum can extend in length to accommodate interdoublet sliding. Flagellar bends of large angle were induced in bull spermatozoa by hypotonic treatment. It is argued that this produces large interdoublet displacements that are, nevertheless, still within physiological limits. Such flagella were examined by the rapid-freeze, deep-etch technique and the nexin linkages identified by their position in relation to the inner dynein arms and by their straplike, bipartite, morphology. They were found to bridge perpendicularly (or occasionally at an angle) between the A- and B-tubules of adjacent doublets. The nexin linkages were no more than approximately nm in length, even in regions in which approximately 200 nm of sliding could be inferred. Variable registration between adjacent nexin rows gave some further support to the assumption that sliding had indeed taken place. From this, it is concluded that elastic deformation of the links, such as would accommodate interdoublet sliding, does not occur; some form of displacement must occur between nexin and the adjacent B-tubule.


Subject(s)
Carrier Proteins/ultrastructure , Sperm Tail/ultrastructure , Animals , Cattle , Dyneins/ultrastructure , Freeze Etching , Hypotonic Solutions/pharmacology , Male , Microscopy, Electron , Microtubule-Associated Proteins , Sperm Motility , Sperm Tail/drug effects
15.
J Struct Biol ; 108(1): 6-13, 1992.
Article in English | MEDLINE | ID: mdl-1562433

ABSTRACT

The anterior lens capsule provides a thick, easily handled model system for the study of the organization of type IV collagen, the main component of basement membranes. We have used the technique of rapid freezing, deep-etch, and rotary replication to study the three-dimensional organization of the collagen skeleton in mammalian lens capsule after a variety of extraction procedures. In all cases the collagen appeared as a densely packed three-dimensional branching network of fine microfibrils. The organization of the microfibrils appears to show some regularity, with branch points approximately 40 nm apart. Most junctions are three-way and the network forms predominantly five-sided figures. This closely resembles the collagenous network described by Yurchenco and Ruben (1987, 1988) in human amniotic basement membrane and EHS tumor matrix, but extends their findings to another system for which X-ray diffraction data are available. The three-dimensional network is discussed in terms of molecular packing of type IV collagen in light of the information available from the diffraction data.


Subject(s)
Collagen/ultrastructure , Lens Capsule, Crystalline/ultrastructure , Animals , Cattle , Microscopy, Electron
16.
J Cell Sci ; 100 ( Pt 2): 319-28, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1836791

ABSTRACT

A study has been made of the inner dynein arm complex in the sperm flagellum of Gallus domesticus. It has been found that images of the complex made from highly contrasted, approx. 20 nm, sections are very largely compatible with images made from replicas of rapidly cryofixed material. This suggests that neither technique seriously distorts the native state. From both types of image, we conclude that the most proximal group of inner dynein arm heads (IDA 1) is related to spoke S1 and consists of 3 heads with fine connections to the B-tubule. IDA 2 consists of 2 such heads and is related to spoke S2. IDA 3 is apparently single headed and lies close to the A-attachment of spoke S3. This arrangement of IDAs repeats every 96 nm. Between the IDAs and outer dynein arms (ODAs), lying close to IDAs 2 and 3, is a strap-like linkage to the next B-tubule; it is argued that this represents the circumferential link, nexin. In sections, but not consistently in replicas, IDA2 lies closer to the plane of the nexin link than does IDA 1. In the presence of ATP and vanadate, little change is seen in the IDA complex except for an ill-defined alteration to IDA 1. It is speculated that the apparently smaller size of the inner arm heads (compared with the ODA major subunit) may have functional implications in relation to maximal sliding velocity.


Subject(s)
Chickens/anatomy & histology , Dyneins/ultrastructure , Adenosine Triphosphate/pharmacology , Animals , Dyneins/drug effects , Freeze Etching , Image Processing, Computer-Assisted , Male , Microscopy, Electron , Sperm Tail/metabolism , Sperm Tail/ultrastructure , Vanadates/pharmacology
17.
J Cell Sci ; 98 ( Pt 1): 17-26, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1829084

ABSTRACT

Demembranated sperm flagella from Gallus domesticus have been prepared by the rapid-freeze, deep-etch, rotary replica technique in order to study the three-dimensional morphology of the outer dynein arm (ODA)-ATPase complex. In general, the ODAs resemble most closely those from the sea urchin Strongylocentrotus described by W. S. Sale et al. In the 'rigor' condition, the ODA consists of a major subunit (the head), from which a slender link extends to the adjacent B-tubule (the B-link). A smaller, intermediate subunit lies adjacent to the head, distally, and a further extension of the complex, the minor subunit, continues distally beneath the head domain of the next arm complex, where it attaches to the A-tubule. In the presence of ATP and vanadate ('relaxed' condition), the attachment point of the B-link to the head is shifted to a more proximal position, and the minor subunit is no longer visible. This is interpreted as resulting from a rotation of the head. These features are demonstrated stereoscopically, and from several viewpoints. Image enhancement has been used to clarify and define the repetitive features of the dynein arrays. In addition, some of the axonemes have been imaged from highly contrasted 20 nm thin sections; the detection of B-links in such sections means that these slender structures cannot be considered artefacts of the rapid-freeze, deep-etch protocol.


Subject(s)
Dyneins/ultrastructure , Sperm Tail/enzymology , Adenosine Triphosphate/pharmacology , Animals , Chickens , Dyneins/antagonists & inhibitors , Fourier Analysis , Freeze Etching , Image Enhancement/methods , Intracellular Membranes/physiology , Male , Microscopy, Electron , Protein Conformation , Vanadates/pharmacology
18.
J Cell Sci ; 88 ( Pt 3): 361-71, 1987 Oct.
Article in English | MEDLINE | ID: mdl-3448100

ABSTRACT

Trypsin digestion of demembranated fowl spermatozoa caused a longitudinal splitting of the distal part of the axoneme. The resulting strands, consisting of groups of doublet microtubules, formed left-handed helices. On the evidence of electron micrographs, the digestion had caused the loss of dynein arms from the outer row; it is assumed that the doublets remained linked together by dynein arms of the inner row. When such helices were mechanically detached from the proximal flagellum and reactivated with adenosine triphosphate, they lengthened in an orderly way by inter-doublet sliding. All the doublets of the axoneme could be reactivated and in all instances the direction of sliding was the same as that reported for the cilia of Tetrahymena. Within the groups of doublets the measured inter-doublet displacements were generally similar, suggesting that the rates of sliding had been equivalent. These findings are contrasted with the differential pattern of activation that is assumed to occur in vivo.


Subject(s)
Flagella/physiology , Spermatozoa/physiology , Animals , Chickens , Flagella/drug effects , Flagella/ultrastructure , Male , Microscopy, Electron , Spermatozoa/drug effects , Spermatozoa/ultrastructure , Trypsin
19.
J Ultrastruct Res ; 90(3): 221-34, 1985 Mar.
Article in English | MEDLINE | ID: mdl-4078965

ABSTRACT

Detailed reconstructions of the flagellar tip (end-piece) in rodent spermatozoa have shown patterns of displacement between the termination points of the axonemal doublets (judging the terminations by the loss of electron density from the A-tubule). The patterns are in good agreement with those derived from sliding microtubule theory. In the hamster at least, the axis of major displacement passes approximately through doublet 1 and between doublets 5 and 6, though there may be some skewness in the clockwise direction. Microtubules derived from the plane at right angles to this (the central pair and presumably one or both of doublets 3 and 8) continue beyond the rest to the extreme tip, where they appear to be linked together at the cell membrane. This arrangement suggests that the tapering form of the end-piece, and of flagellar terminal filaments and ciliary tips in general, may be an adaptation to contain the sliding microtubules and prevent them impinging on the membrane overlying the tip.


Subject(s)
Flagella/ultrastructure , Microtubules/ultrastructure , Spermatozoa/ultrastructure , Animals , Cricetinae , Male , Mice , Microscopy, Electron , Models, Biological , Rats , Species Specificity
20.
J Cell Sci ; 67: 159-70, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6746770

ABSTRACT

Micrographs were made of free-swimming hamster spermatozoa using a high intensity xenon flash and two-colour darkground illumination - conditions that permit three-dimensional reconstruction of the instantaneous shape of the flagellum. From the waveforms observed, we constructed an account of the three-dimensional kinematics of this flagellum. We found that near-planar bends grew on the proximal 25% of the flagellum to reach a mean angle of 1 X 7 rad. Principal bends achieved a greater angle than reverse bends. As bends propagated they maintained their near-planarity and their angle, but decreased in radius of curvature. However, the plane of the more distal bends tended to become displaced, as though a predominantly clockwise torsion of the axoneme was developing in the interbend segment. This gave rise to a complex shape resembling a sinistral helix of reducing pitch and eccentricity. There are clear indications that not all cycles of bending lead to the same degree of three-dimensionality.


Subject(s)
Sperm Motility , Sperm Tail/physiology , Spermatozoa/physiology , Animals , Cricetinae , Male , Mesocricetus , Photomicrography
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