Department of Anatomy and the Laboratory for Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts, U.S.A.

Transcription

1 J. Cell Sci. 3, (1968) Printed in Great Britain THE TOPOGRAPHICAL RELATIONSHIP BETWEEN THE PLANE OF THE CENTRAL PAIR OF FLAGELLAR FIBRILS AND THE TRANSVERSE AXIS OF THE HEAD IN GUINEA-PIG SPERMATOZOA D. W. FAWCETT Department of Anatomy and the Laboratory for Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts, U.S.A. SUMMARY Earlier studies on the arrangement of sperm-tail components have suggested that a line through the central pair of fibrils is always perpendicular to the broad face of the flattened sperm head. The principal plane of bending of proximal regions of the tail was therefore assumed to coincide with the transverse axis of the head. The heads of guinea-pig epididymal sperm are flexed from the long axis of the tails and are stacked one upon the other in coherent packets. The parallel arrangement of the heads ensures that all of the associated tails will be similarly oriented, and certain planes of section transect heads and tails of the same sperm packet. Taking advantage of this circumstance, it was possible to demonstrate that in quiescent sperm of this species the plane of the central pair is not at 90 to the transverse axis of the head, but is inclined off the perpendicular and always in the same direction. Attention is drawn to examples from the literature on cilia and flagella, where there appears to be a similar inclination of the axis of the central pair with respect to external landmarks. INTRODUCTION In the locomotion of mammalian spermatozoa the heads appear to rotate around their axis of progression and it is the consensus among those who have analysed their movements cinematographically that the tail waves are more complex than those of cilia and simpleflagella (Gray, 1958; Rikmenspoel, 1962; Bishop, 1962; Brokaw, 1965, 1966). Theflexionsof sperm tails are believed to be mainly two-dimensional near their base but they become distinctly helical farther along the tail, and the three-dimensional character of the waves is presumed to be responsible for the observed rotation of the heads. Bradfield (1955) was among the first to propose a mechanism of flagellar movement based upon electron micrographs. He argued that three-dimensional movements could only be produced if the nine longitudinal contractile fibrils were helically disposed, or alternatively, if nine straight fibrils were activated sequentially at their base and the contractions were propagated along their length, out of phase, so that the contracted segments of successive fibres, at any given moment, described a helix. The numerous investigations of sperm in the ensuing years have failed to identify any

2 188 D. W. Fawcett structural component at the base of the flagellum which could act as a timer or commutator device to bring about the sequential activation of parallel fibrils. They have likewise failed to produce convincing evidence for a helical disposition of the fibrils. Thus, although a wealth of structural detail has been revealed in electron micrographs, the mechanism of sperm-tail movement remains as puzzling as ever. Any consistent correlation of an asymmetry of the head with the ultrastructure of the axial fibrils of the sperm tail or any new information bearing upon the relation of the arrangement of internal fibrils to the direction of beat is worth recording for what it may ultimately contribute to our understanding of sperm locomotion. For epithelial cilia it has not been difficult to relate the direction of beat to the asymmetry that results from the presence of two fibrils instead of one in the interior of the axial filament complex. In studies of cells on the gills of molluscs that have only two rows of cilia, the direction of beat was observed to be along the rows (Fawcett & Porter, 1954; Gibbons, 1961a). When these cilia were examined in transverse section with the electron microscope, the orientation of the plane of the central pair of fibrils was the same in all of the cilia along the row, and was approximately at right angles to the plane of the row. It was suggested, therefore, that the direction of ciliary beat is perpendicular to the plane passing through the central pair of fibrils. Corresponding information for sperm tails is less easily acquired. Being freeswimming cells, they have no fixed orientation or constant relation to external landmarks. However, in those mammalian species in which the head is markedly flattened, this asymmetry can be recognized in living, motile sperm and provides a plane of reference to which the disposition of tail components can be related. For spermatozoa of the bat (Fawcett, 1961; Fawcett & Ito, 1965) and of the bull (Gibbons, 1963) it has been reported that the plane of the central pair of fibrils is perpendicular to the transverse axis of the head. Reasoning from analogy with cilia, it has been concluded that the principal plane of bending of the proximal part of the sperm flagellum in those species, and possibly in other mammals, is parallel to the flat surfaces of the head. The studies on guinea-pig sperm reported here demonstrate that the plane of the central pair of fibrils in this species is not actually at a right angle to the transverse axis of the head but is rotated away from the perpendicular in a direction counterclockwise from the point of view of an observer looking along the flagellum from the base toward the tip. This observation suggests the need for investigation of the relations of the corresponding axes in other species and a re-examination of the widely accepted generalization that the movements of cilia are planar and perpendicular to the plane of the central pair of fibrils. MATERIALS AND METHODS All observations were made upon thin sections of the distal portions of the ductus epididymidis where it may be assumed that the stored spermatozoa in the lumen of the duct are non-motile. The material was fixed in 6-5 % glutaraldehyde buffered with s-collidine, washed in the buffer alone and subsequently immersed in 1 % osmium

3 Guinea-pig spermatozoa 189 tetroxide in the same buffer. Embedding was in Epon and thin sections exhibiting silver to pale yellow interference colours were stained with uranyl acetate and lead citrate and examined with an RCA EMU 3 G electron microscope. OBSERVATIONS The guinea-pig offers unique advantages for studies of the disposition of tail components in relation to the transverse axis of the head. The sperm head is large and the nucleus is inclined at an angle of about 45 to the axis of the tail. The exceptionally large acrosome in turn, makes an angle of about 135 with the flattened nucleus and has a convex upper surface and a deep concavity in its underside (Fawcett, 1965). As they pass through the epididymis the sperm heads become associated in packets or rouleaux of 4-14, which are stacked like nested spoons with the convex upper surface of one fitting into the concave under surface of the next (Figs. 2, 3). As a consequence of this stacking of the heads there is a corresponding grouping of the tails, and the individuals within the group all have a similar orientation of their cross-sections. If the plane of section is perpendicular to the long axis of one tail it will, of necessity, be perpendicular to all in the same group (Fig. 5 C). Thus, in studying guinea-pig epididymal sperm tails, one enjoys the same advantage of multiplicity of similar crosssections that one has in studies on epithelial cilia. A clear explanation of the criteria for recognizing the orientation of sperm from cross-sections will require the use of a rather complex descriptive terminology developed in previous publications. To avoid a tedious repetition of definitions available elsewhere, the pertinent components of the sperm tail are illustrated in transverse section and their descriptive terms are presented in Fig. 4. For observations of the kind attempted in the present study it is essential to select true cross-sections, for only in these are the images sufficiently sharp to resolve the central pair of fibrils as disinct circular profiles, and thus to permit drawing a line through their centres with some precision. One must also be able to distinguish subfibrils B and A of the doublets and the direction of the arms on subfibrils A, for these details constitute the structural basis for determining whether the cross-sections are being examined from the point of view of an observer looking along the flagellum from base to tip or from tip to base (Gibbons & Grimstone, i960). To ensure consistency and comparability, the illustrations are presented, wherever possible, with outer fibre number 1 at the left and numbers 5 and 6 on the right of the cross-section and with the arms on subfibrils A of the doublets directed clockwise. This is believed to be the pattern presented to a viewer looking from the base of the flagellum toward its tip (Gibbons & Grimstone, i960; Gibbons, 1961 b). Although the groups of associated sperm are randomly oriented in the lumen of the duct and sometimes intermingle, the criteria for recognition of the profiles belonging to the same group are quite clear. The cross-sections of tails belonging to sperm whose heads are associated in the same stack will necessarily have the same dorso-ventral orientation and the arms of the doublets of their axial filament complex will point in the same direction. For example, in Fig. 5, the tails in the sector marked C, obviously

4 190 D. W. Fawcett belong to the same group because (i) the arms on the doublets all point counterclockwise, (ii) the orientation of the central pair of fibrils is the same in all, and (iii) they are all in the same dorso-ventral relation because in all of the cross-sections the minor compartment containing fibre number 1 is on the left. The two tails in sector A, on the other hand, have a consistent orientation of the central pair, but clearly belong to a different group from those of sector C because fibre number 1 is on the right. Their dorso-ventral relations are therefore the reverse of those in sector C. The crosssections in sector B at the upper right represent an intermingling of 2 or more groups whose antero-posterior orientation can be assumed to be the same, since the arms on the doublets all point the same way, but whose dorso-ventral relations differ since fibre 1 is on the left in some, and on the right in others. Owing to the stacking of the guinea-pig sperm heads and their marked deflexion from the long axis of the flagellum there are planes of section that pass through heads and tails of the same sperm packet and still yield true cross-sections of the tails. Figure 6 illustrates such a section. The vertical stacking of the heads has resulted in some degree of alignment of the sections with each successive cross-section from the top to bottom of the figure representing a more caudal level. Cross-sections 1-4 are through the caudal portion of the heads of four successive sperm in the same packet. The line ruled across the micrograph is intended to represent the average orientation of the transverse axis of the heads. Section 5 passes through the connecting piece in the neck region and sections numbered 6-11 represent successive levels in the middle piece of six other spermatozoa in the same packet. Lines have been drawn on the electron micrographs of the cross-sections to indicate for each the plane passing through the centres of the central pair of fibrils. Contrary to the expectation raised by earlier published work, these lines are not found to be perpendicular to the line representing the transverse axis of the heads but instead are rotated counterclockwise so as to subtend an angle of uo with the transverse axis of the heads. A second group, illustrated in Fig. 7, shows a similar deviation of the plane of the central pair from the expected right angle. A dozen or more groups of spermatozoa were studied in this way, either selecting ones in which the direction of the arms on the doublets were clockwise in the original micrograph, or when this condition was not met, turning the negative over in printing so that the tail sections were always examined as though being viewed from base to tip. In all instances the plane of the central pair deviated from the perpendicular in the same direction. If it be accepted that waves at the base of the flagellum are mainly planar and that the direction of beat is perpendicular to the plane of the central pair, then one must conclude that the principal plane of bending in the proximal part of the tail does not exactly coincide with the transverse plane of the head but is rotated about 20 0 counterclockwise. The fibrous elements in the core of cilia and flagella are generally assumed to run straight and parallel throughout their length. Similarly in the sperm flagellum no change is detected in sections at successive points along its length in the position of the central pair of fibrils relative to the peripheral doublets, the outer fibres, or the columns of the fibrous sheath. However, this does not exclude the possibility that the

5 Guinea-pig spermatozoa 191 fibre complex and its sheath as a whole might exhibit a very gradual torsion along its length without any significant change in the positions of the fibres relative to each other. If such were the case the plane of the central pair would be expected to change its orientation with respect to the transverse axis of the head while the topographic relations of fibrils within the cross-sections would remain essentially constant. It is not feasible in the present material to explore this possibility over more than a small fraction of the total length of the sperm tail, but in the larger packets containing a dozen or more sperm one can extend the observations beyond the midpiece and into the first portion of the principal piece without losing the orientation of the heads. Figure 7 illustrates a packet of at least 13 sperm sectioned through the caudal portion of the lowermost head in the stack. The profile of the base of the head is at the top of the figure and the line immediately below is drawn parallel to its transverse axis. The next three cross-sections in order are through the distal part of the middle piece and its cytoplasmic droplet. The 9 cross-sections that follow are at successive levels along the principal piece. One can establish from a study of this group that there is no significant change in the inclination of the plane of the central pair throughout the length of the middle piece and that portion of the principal piece represented by the sections in this figure. It would be desirable to extend this type of analysis to more caudal levels in the tail, but where the sections of groups of tails do not include at least one head profile such observations are of doubtful validity. In the absence of this sure means of establishing the orientation of the transverse plane of the heads, one must make the assumption that where groups of tail profiles are disposed in a row, their direction of alignment is likely to be perpendicular to the plane of flattening of the heads. Such an assumption has been made in drawing the line AB on Fig. 8 to represent the probable transverse axis of the heads. In this group the orientation of the axis of the central pair shows slightly more variability than in some others, and its greater inclination in the more distal cross-sections at the bottom of the figure tends to keep open the possibility of further gradual counterclockwise torsion of the distal half of the tail. The farther caudal the plane of section, however, the more likely it becomes that the rows of longflexibletails will be passively skewed to one side or the other from the expected perpendicular relation to the transverse axis of the heads. The resulting obliquity of the row would then lead to erroneous interpretations. To cite an extreme example, if one assumes that the row of cross-sectional profiles in Fig. 9 is perpendicular to heads whose transverse plane is parallel to the top of the page, then the inclination of the lines through the central pairs would indicate that the tail elements had undergone a counterclockwise rotation of about 90 from their original position. Actually this does not seem to apply. The tails of this group seem to have fanned out laterally to form a row parallel instead of perpendicular to the transverse plane of the heads. If this interpretation is correct, the line AB representing the transverse axis of the heads would be parallel to the side of the page, and the orientation of the central pair would be exactly the same as it has been shown to be in the more proximal segments of the tail. From the present material, therefore, it can safely be stated that, in the guinea-pig

6 192 D. W. Fawcett spermatozoon, the line through the central pair of tail fibrils in the midpiece is not perpendicular to the transverse axis of the head, but inclined 20 or more. This same relationship prevails as far distally as the first part of the principal piece, and possibly farther. The possibility that there is a gradual torsion of the axis of the central pair of fibrils in more distal regions of the tail cannot be excluded. The relative inconstancy of the direction of alignment of the tail profiles at levels beyond the first portion of the principal piece does not permit valid inferences as to the orientation in the caudal region where the plane of reference provided by sections of heads is not available. DISCUSSION There have been two previous efforts to determine the relationship of the axis of the sperm tail to the plane of flattening of the sperm head. One of these was based upon study of serial transverse sections of the head and middle piece of a single carefully oriented bull sperm (Gibbons, 1963). The other depended upon a unique organization of the mitochondrial sheath of the bat spermatozoon that enabled one to determine the relation of these axes from oblique sections passing through the head and tangential to the middle piece (Fawcett, 1962). Both investigators concluded that the plane of the central pair of fibrils was perpendicular to the longer transverse axis of the head, and that bending movements of the proximal parts of the tail were probably in a plane parallel to the flat surface of the head. In neither study was the method capable of detecting minor deviations from perpendicular. In both, the sample was very small and no indication could be given as to the constancy of the relations described. In the present study, by taking advantage of the unique mode of aggregation of epididymal sperm, it has been possible to ascertain that the plane of the central pair of flagellar fibrils is not perpendicular to the transverse axis of the flattened head, but is at an angle of This relationship was found consistently in a dozen or more packets of sperm and may be presumed to be a constant feature. In marked contrast to the constancy of this precise angular relation of the axis of tail to that of the head in the guinea-pig spermatozoon is the considerable variability in the site of implantation of the tail along the base of the nucleus. Retzius (1909) reported, and I can confirm, that the tail is sometimes attached in the midline but may be eccentric in either direction. It is tempting to infer from the variability of this character that it is less critical for sperm locomotion than is the more constant relation of the axis of the central pair of flagellar fibrils to the transverse axis of the head. In the first electron-microscopic studies of epithelial cilia (Fawcett & Porter, 1954) the orientation of the central pair of fibrils was found to be the same in all of the cilia and it was concluded that the direction of their beat was perpendicular to the line drawn through the centres of these fibrils. Slight departures from this perpendicular relationship were noted from time to time in these early studies but in the period when embedding for electron microscopy was in methacrylate, such minor deviations could reasonably be attributed to deformation by the knife during sectioning. This general relationship was subsequently reaffirmed in studies employing more advanced technical methods (Gibbons, 1961a) and has been widely accepted.

7 Guinea-pig spermatozoa 193 When an oblique orientation of the axis of the fibril bundle was observed in the present study, it was tempting to try to relate the difference in the character of beat of cilia and sperm tails to this asymmetry. However, upon re-examining the literature with respect to the orientation of cross-sectional axes of cilia andflagellain relation to external planes of reference, one finds in many of the published micrographs a consistent slight departure from the perpendicular, like that described here for sperm B Fig. 1. Tracings from published electron micrographs by other investigators illustrating flagella, other than those of sperm, which show a similar obliquity of the axis of the central pair of fibrils with respect to external planes of reference. A, Flagella of Pseudotrichonympha in a fiagellar groove. After fig. 47 of Gibbons & Grimstone (i960). B, Two free flagella of Trichonympha lying parallel to the cell surface (below). After fig. 21 of Gibbons & Grimstone (i960). C, Cross-sections of cilia on the lateral cells of the gills oielliptio. The line at the right is parallel to the lateral surface of the cell. After fig. 30 of Satir (19656). tails. Gibbons & Grimstone (i960) reported that the line joining the centralfibrilsof the flagella of Trichonympha almost invariably makes an angle of with the plane of the groove from which the flagella emerge (Fig. 1 A). They illustrate some cross-sections in which the angle is considerably greater. In the gill cilia oielliptio, Satir (1963) could

8 194 D- W- Fawcett define the axis of the cross-sections quite precisely and reported that in resting cilia this angle was about 70 0 to the surface of the cells (Fig. 1C). This angle, representing a departure of 20 0 from the perpendicular, is close to that reported here for the deflexion of the axis of the sperm tail from the transverse axis of the head. Thus what appeared, at first, to be an unexpected and novel asymmetry of the sperm tail, seems instead to be simply one of many features shared by cilia, flagella and sperm tails. The common occurrence of this deviation from perpendicular in material not prone to distortion in preparation, and the consistency of its direction, indicate that it is real and point to the need for systematic re-examination of the generalization relating direction of beat to the axis of the central pair of fibrils. It is now generally accepted that the mammalian sperm tail executes helical movements but there is still no agreement as to how three-dimensional waves are generated by the two sets of longitudinal fibres observed in electron micrographs. Several observations suggest that the tail movements are the resultant of two distinct motor components which can become dissociated under certain conditions. Bull sperm which have been subjected to cold shock or which are in poor condition for other reasons, swim in tight circles and fail to show rotation of the head (Rikmenspoel, 1962). Under these conditions the tail is said to exhibit two-dimensional undulations. Here evidently the torsional component of the movement has been lost while the ability to produce flat waves has persisted. In ageing squid sperm, there is a marked decrease in activity of the tail which facilitates observation of the nature of its wave motion (Bishop, 1958). In such preparations two components of the movements can be distinguished: a twodimensional bending wave largely limited to the proximal segment of the tail and a rotation on the longitudinal axis that appears to be generated by the distal portion. In glycerin-extracted sperm tails the planar bending component can be reactivated by ATP but such preparations never show rotational movements. Dissociation of the components of the movements may not be confined to shocked, exhausted, or extracted sperm. There is some indication of their sequential development in sperm maturation. Blandau & Rummery (1964) have found that rat sperm from the caput epididymidis swim in circles, whereas those from the cauda show normal progressive movements. One can speculate that in rat sperm, as in those of the bull, the circling sperm may be producing only two-dimensional undulations. If this is so, it is suggestive that in the continuing physiological differentiation of sperm in their passage through the epididymis, the capacity for forming two-dimensional waves may develop first and the mechanism responsible for the torsional movements of the tails may be acquired later. The existence of two components to the wave motion which can be dissociated suggests the possibility that these may reside in different structural components, and leads naturally to the speculation that the axial filament complex (9 + 2) may produce mainly two-dimensional undulations and that the torsional movement may be superimposed upon the flat waves by the activity of the nine large outer fibres. Consistent with this view is the observation of Gray (1958) that the rotational movements of distal portions of mammalian sperm tails (with the complement of fibres) are more marked than are those in sperm of invertebrates (with the simple 9 + 2

9 Guinea-pig spermatozoa 195 complex) (Gray, 1955). The outer coarse fibres, however, are best developed in the anterior portion of the tail and it is evident that if the rotation of sperm depends upon a torsion in the contractile or supporting elements of the tail, this twist must be located in its distal portion. In the present study of quiescent sperm no clear evidence was obtained of a proximo-distal change in the relation of the structural elements of the tail to each other or in their orientation with respect to the plane offlatteningof the head. Most investigators agree that the internal fibres probably run straight along the entire length of quiescent cilia, flagella and sperm tails, but because longitudinal sections seldom contain lengths that stay in the plane of section for more than 5 /i, it has not been possible to exclude the possibility that the fibre complex as a whole might have a long-pitched spiral course (Gibbons & Grimstone, i960). The geometry of the stacked sperm of guinea-pigs, studied here, has made it possible to establish that the orientation of the axis of the central fibres with reference to an external landmark remains constant throughout the entire length of the middle piece and for a substantial portion of the principal piece. This represents a segment several times longer than has previously been studied. It is true, however, that the portion accessible to analysis was limited to the segment of the tail where planar bending movements predominate. It has yet to be determined whether or not there is a twist in the structural elements of still more distal portions of the tail where the rotational movements are most obvious. The relations described here apply to the tails of the inactive sperm in the epididymal duct. One cannot conclude from the constancy of orientation of tail components observed in quiescent sperm that the same would also apply to actively swimming sperm. Satir (1963, 1965 a) has reported that, in quiescent epithelial cilia, the axes of cross-sections are all parallel as would be expected if the fibrils ran straight from base to tip. He insists, however, that this is not representative of the situation during ciliary beat. In his experience, activated epithelial cilia that have been instantly immobilized by the fixative present a more complex picture, with the axes of crosssections much more variable in their orientation. This he interprets to mean either that the fibrils do not run straight in beating cilia, or their beat is not strictly twodimensional as is commonly believed. Were it possible to study the relation of the fibre pattern to the plane of the head in active sperm, more variability might well be observed. In investigating ejaculated bull sperm Lindahl & Drevius (1964) were able to examine cross-sections of proximal and distal regions of the same spermatozoon by taking advantage of the fact that when subjected to hypotonic medium a permanent exaggerated bending of the tail occurs (Drevius, 1963). In electron micrographs of the pairs of cross-sections obtained by sectioning these strongly recurved tail loops, there was evidence of torsion of the more distal segment of the tail with respect to the orientation of components in the more proximal segment. The torsion occurred in either direction. If these authors are correct in assuming that the hairpin bends and loops produced by treatment with hypotonic solution represent merely an exaggeration of the movements of normal swimming, then their results clearly indicate a degree of torsion of the longitudinal fibres in the tail of active sperm which we have not found in the quiescent epididymal sperm.

10 196 D. W. Fawcett Since the earliest description of the pattern of fibrils, investigators of cilia and flagella have been puzzled as to the function of the central pair. Some consider them to be stiffening skeletal elements against which the others contract. It has also been suggested that the central pair are the contractile members, and that the outer nine doublets conduct information (Inoue, 1959). Others have favoured the converse interpretation, namely that the central pair transmit instructions for co-ordination of the contractile activities of the outer doublets (Bradfield, 1955; Cleland & Rothschild, 1959). Another interpretation considers both to be contractile and proposes that the central pair and fibrils number 3 and 8 undergo a slight shift of position upon activation to produce, on the side of shortening, an hexagonal array of 6 fibrils around a single central fibre a pattern reminiscent of that found in the arrangement of the actin and myosin filaments of muscle (Harris, 1961). The present paper contributes little to a solution of this problem of the function of the central pair, but it establishes topographical relations of these elements to extraflagellar landmarks. These findings may be helpful in the elaboration of future theories of the mechanism of flagellar motion. This research was supported by grants GM and HD from the Institute of General Medical Sciences, National Institutes of Health, U.S. Public Health Service. REFERENCES BISHOP, D. (1958). Motility of the sperm flagellum. Nature, Lond. 182, BISHOP, D. (1962). Sperm motility. Physiol. Rev. 42, BLANDAU, R. J. & RUMMERY, R. E. (1964). The relationship of swimming movements of spermatozoa to their fertilizing capacity. Fert. Steril. 15, BRADFIELD, J. R. G. (1955). Fibre patterns in animal flagella and cilia. Symp. Soc. exp. Biol. 9, BROKAW, C. J. (1965). Non-sinusoidal bending waves of sperm flagella. J. exp. Biol. 43, BROKAW, C. J. (1966). Bend propagation alongflagella.nature, Lond. 209, 161. CLELAND, K. W. & LORD ROTHSCHILD (1959). The bandicoot spermatozoon: an electron microscope study of the tail. Proc. R. Soc. B 150, DREVIUS, L. O. (1963). Spirilization of mammalian spermatozoa in hypotonic media. Nature, Lond. 197, FAWCETT, D. W. (1961). Cilia and flagella. In The Cell, vol. 2 (ed. J. Brachet & A. E. Mirsky), pp New York: Academic Press. FAWCETT, D. W. (1962). Sperm tail structure in relation to the mechanism of movement. In Spermatozoon Motility (ed. D. W. Bishop), pp Washington: American Association for the Advancement of Science. FAWCETT, D. W. (1965). The anatomy of the mammalian spermatozoon with particular reference to the guinea pig. Z. Zellforsch. mikrosk. Anat. 67, FAWCETT, D. W. & ITO, S. (1965). The fine structure of bat spermatozoa. Am. J. Anat. 116, FAWCETT, D. W. & PORTER, K. R. (1954). A study of the fine structure of ciliated epithelia. J. Morphol. 92, GIBBONS, I. R. (1961a). The relationship between the fine structure and direction of beat in gill cilia of a lamellibranch mollusc. J. biophys. biochem. Cytol. n, GIBBONS, I. R. (19616). Structural asymmetry in cilia andflagella."nature, Lond. 190, GIBBONS, I. R. (1963). A method for obtaining serial sections of known orientation from single spermatozoa. J. Cell Biol. 16,

11 Guinea-pig spermatozoa 197 GIBBONS, I. R. & GRIMSTONE, A. V. (i960). Onflagellar structure in certainflagellates.j. biophys. biochem. Cytol. 7, GRAY, J. (1955)- The movement of sea urchin spermatozoa. J.exp.Biol 32, GRAY, J. (1958). The movement of the spermatozoa of the bull. J. exp. Biol. 35, HARRIS, J. E. (1961). The mechanics of ciliary movement. In The Cell and the Organism (ed. J. A. Ramsey & V. B. Wigglesworth), pp Cambridge University Press. INOUE, S. (1959). Motility of cilia and the mechanism of mitosis. In Biophysical Science (ed. J. L. Oncley), pp New York: Wiley. LINDAHL, P. E. & DREVIUS L. O. (1964). Observations on bull spermatozoa in a hypotonic medium related to sperm mobility mechanisms Expl Cell Res. 36, RIKMENSPOEL, R. (1962). Biophysical approaches to the measurement of sperm motility. In Spermatozoan Motility (ed. D. W. Bishop), pp Washington: American Association for the Advancement of Science. SATIR, P. (1963). Studies on cilia. The fixation of the metachronal wave. J. Cell Biol. 18, SATIR, P. (1965 a). Studies on cilia. II. Examination of the distal region of the ciliary shaft and the role of the filaments in motility. J. Cell Biol. 26, SATIR, P. (19656). Structure and function in cilia andflagella.protoplasmatologia 3, {Received 25 August 1967)

12 198 D. W. Fawcett Fig. 2. A low-power electron micrograph of a parasagittal section through one of the rouleaux or stacks of guinea-pig sperm as they occur in the distal segments of the epididymis. Fig. 3. A transverse section through a similar packet of sperm heads showing how they stack with the convexity of one fitting into the concavity of the next like nested spoons. Fig. 4. A cross-section through the principal piece of a guinea-pig sperm tail presented here for orientation of the reader unfamiliar with the terminology used in the paper.

13 Journal of Cell Science, Vol. 3, No. 2 Longitudinal Column Outer Fibers Doublet of Axial Filament Complex Minor Compartment Major Compartment Flagellar Membrane Central Fibrils Rib of the Fibrous Sheath Plane of the Central Pair D. W. FAWCETT (Facing p. 198)

14 Fig. s. An electron micrograph of a section of the epididymal duct illustrating the criteria by which one can recognize groups of cross-sectional profiles of tails belonging to the same packet. The field has been divided into sectors A, B and C for convenience of description. It is evident that the cross-sections of tails in sector C are associated with the same packet of stacked heads, because the orientation of the central pair is consistent throughout the group; the minor compartment is on the upper left in all (indicated by L adjacent to the cross-section); and the arms on subfibre A all point counterclockwise. The two profiles in sector A, although at different levels, were probably also associated with adherent sperm heads, for the same criteria apply except that the minor compartment is to the right. These sperm are headed in the same direction as those in sector C, but their dorso-ventral relations are the reverse, hence they could not be part of the same group. In sector B all sperm are headed in the same direction as indicated by consistent counterclockwise direction of the arms on the doublets but they represent an intermingling of two different groups because the minor compartment is at the upper right in some and lower left in others. In many instances, the groups are sufficiently separated to make one less dependent upon these criteria.

15 Journal of Cell Science, Vol. 3, No. 2 D. W. FAWCETT

16 Fig. 6. A section transecting a packet of spermatozoa in such a way as to include sections of the lowermost heads and cross-sections of the neck and middle piece of sperm, whose heads are at higher levels in the stack. For orientation see the inset where lines corresponding to the plane of section have been superimposed on drawings of a stack of sperm seen from behind (right) and in parasagittal section (left). Sections numbered 1-4 pass through sperm heads at successively more caudal levels. Section 5 is through the connecting piece; 6 and 7 transect the anterior portion of the middle piece; 8-10 are farther caudal in the midpiece at the level of the fusiform protoplasmic droplet. Notice that the lines indicating the axis of the central pair of flagellar fibres are not perpendicular to AB, which represents the transverse axis of the heads, but are at an angle of about 70 0 to it.

17 Journal of Cell Science, Vol. 3, No. 2 D. W. FAWCETT

18 Fig. 7. Section through another sperm packet at a more caudal level than Fig. 6. Only one section of a head is included but this suffices to establish the orientation of the transverse axis of the heads (AB). From above downward are shown: a section through the upper part of the middle piece; 2 through the middle piece at the level of the cytoplasmic droplet; and 9 sections through the anterior portion of the principal piece. Here again it is evident that the axes of the central flagellar fibrils depart from the expected orientation perpendicular to the transverse plane of the head, and this orientation persists without significant change throughout the length of the middle piece and for a considerable distance into the principal piece. Thus over this anterior region of the guinea-pig sperm there is no evidence of a twist in the outer coarse fibres, or the axial bundle.

19 Journal of Cell Science, Vol. 3, No. 2 D. W. FAWCETT

20 Fig. 8. In an effort to extend observations to more caudal levels of the tails, one can identify aligned groups of profiles of consistent orientation. In the absence of a section through one of the heads one must assume that the tails associated with a stack of heads would tend to be aligned perpendicular to their transverse axis. This assumption seems justified in the case of the group shown here. Inspection of the axes of the crosssections suggests that their orientation remains about the same through the middle of the principal piece. The hazards of attempting to extend the analysis this far along the tails are illustrated in Fig. 9. Fig. 9. Although a rare and extreme example, examination of the group of aligned cross-sections shown here makes it clear that one cannot safely assume that the transverse axis of the heads is perpendicular to the direction of alignment of the tail profiles. To account for the orientation of the axes of the central pair in this group one must conclude that the tails have fanned out laterally in a row parallel to the transverse axis of the heads. Thus the mode of analysis used here to relate the orientation of the contractile elements to the position of the head is only applicable to those anterior regions of the flagellum where at least one head profile is included in the section.

21 Journal of Cell Science, Vol. 3, No. 2 A B B D. W. FAWCETT

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