© J968 by Academic Press Inc.
j. tJLTRASTRtJC~URERESEARCrt23, 81-97 (1968)
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Unusual Features of the Neck and Middle-Piece of Snake Spermatozoa 1 DAVID W . HAMILTON AND DON W . FAWCETT
Department of Anatomy and the Laboratories for Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115 Received February 16, 1968 In the course of an investigation on reptilian spermiogenesis, we have observed in the sperm of the king snake, Lampropeltis getulus, and the boa, Constrictor constrictor, a nonmitochondrial component of the middle-piece sheath that has not been reported heretofore. This component consists of conspicuous plaques of dense material interspersed among the mitochondria and spaced at irregular intervals along the length of the middle-piece. The plaques, along with the neck cylinder described earlier by Austin (1), are unique to snake sperm and it will be shown that both have a common origin from the coalescence of material first deposited in dense spherical granules in close topographical relationship to the ends of mitochondria that gather around the distal centriole and base of the tail at a late stage of spermiogenesis. The function of both the plaques and the neck cylinder in mature sperm is not clear, but possible functional implications will be discussed in light of homologies between snake sperm and mammalian sperm. Austin (1) and Boisson and Mattei (2) describe several aspects of the structure of the neck and middle-piece of snake spermatozoa that clearly distinguish them from mammalian spermatozoa. A m o n g these is the absence of cross-banding in the longitudinal columns of the connecting piece; the presence of two typical centrioles instead of one; a unique concentric arrangement of the mitochondrial and fibrous sheaths in the middle-piece; and the occurrence of an additional structure, the neck cylinder, encircling the base of the flagellum in the neck region. In the course of investigations on reptilian spermiogenesis we have observed in sperm of the king snake, Lampropeltis getulus, and the boa, Constrictor constrictor, an additional component of the mitochondrial sheath that has not been reported heretofore. This consists of conspicuous plaques of dense material interspersed among the mitochondria and spaced at irregular intervals along the length of the middle1 Supported by grant H D 02344 from the Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service. 6-- 681837 J . Ultrastructure Research
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piece. These bodies a p p e a r to be c o m p o s e d of the same substance as the neck cylinder. The m o d e of f o r m a t i o n of b o t h the bodies a n d the neck cylinder during spermiogenesis is described here a n d certain m i n o r differences in our i n t e r p r e t a t i o n of the structure of the neck cylinder, connecting piece, a n d l o n g i t u d i n a l fibers of the tail are discussed. MATERIALS AND METHODS Epididymal ducts and testes of Lampropeltis and Constrictor were fixed either with Karnovsky's (9) formaldehyde-glutaraldehyde mixture or with 3.5% glutaraldehyde buffered with 0.2 M s-collidine. After postfixation in osmium tetroxide the tissues were quickly dehydrated through a graded series of ethanols and embedded in Epon 812. Sections with silver to silver-gold interference colors were collected on uncoated copper grids and viewed on an R C A 3-G electron microscope. OBSERVATIONS The m a j o r features of the s p e r m tail in Lampropeltis have been described by A u s t i n (1) a n d only those features will be r e c o u n t e d here t h a t are essential to the interpret a t i o n of the new findings. T h e c u s t o m a r y subdivision of the tail of the s p e r m a t o z o o n into neck, middle-piece, p r i n c i p a l piece, a n d endpiece is difficult to a p p l y to snake s p e r m a t o z o a , i n a s m u c h as the structural c o m p o n e n t s that are n o r m a l l y used to define the various segments in the m a m m a l i a n s p e r m have quite different t o p o g r a p h i c a l relationships in the snake. This is p a r t i c u l a r l y true of the o r g a n i z a t i o n of the neck a n d middle-piece. I n m a m m a l i a n s p e r m the neck is defined as the region between the c a u d a l pole of the nucleus a n d the first circumferential elements of the m i t o c h o n d r i a l sheath. It contains p r i m a r i l y the segmented columns c o m p r i s i n g the connecting piece, a single centriole, a n d two or m o r e longitudinally oriented m i t o c h o n d r i a . In snake s p e r m a t o zoa, b o t h p r o x i m a l a n d distal centrioles are present in the neck of the m a t u r e s p e r m (Figs. 1, 2, a n d 6). The distal centriole is oriented in the axis of the flagellum a n d constitutes its b a s a l b o d y . The p r o x i m a l centriole is i n t e r p o s e d between the nucleus a n d All figures shown here are taken from specimens of Lampropeltis getulus. FIas. 1 and 2. The major features of the neck region in the mature sperm of Lampropeltis can be seen in these micrographs. The neck cylinder (Nk C) immediately surrounds the columns of the connecting piece. Anteriorly the cylinder abuts against the rim of the articular fossa, and caudally it is in relationship both to the fibrous sheath (Fb S) and to the mitochondrial sheath (Mt S). The proximal centriole, which is shown in cross section in Fig. i, is embedded within dense material that is continuous with the columns of the connecting piece. The central density in the proximal centriole is regularly found in mature sperm: its significance is unknown. In Fig, 2 the plane of section is at right angles to that in Fig. 1 and the contiguity of the proximal centriole with the columns of the connecting piece is more clearly shown. Multiple layers of membranes (Mb S)can be seen extending from the base of the sperm head toward the annulus. Examples of dense plaques (Dn P) interpersed with the mitochondria can be seen in both figures. Fig. 1, × 31,300; Fig. 2, × 29,000.
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the distal centriole, making an angle of about 80 degrees with the latter (Fig. 2). Both are enclosed within a connecting piece composed of homogeneous dense material that appears to be identical and continuous with that comprising the nine outer coarse fibers of the tail. Encircling the connecting piece and centrioles is a dense structure which extends from the nucleus where it abuts against the rim of the articular fossa, to the anterior margin of the mitochondrial sheath. This component, termed the neck cylinder by Austin (1), is not represented in mammalian sperm. It was interpreted by Austin (1) as forming a thin continous layer between the articular fossa of the nucleus and the base of the flagellum and was therefore described as forming a chamber closed proximally but open distally. In our experience the homogeneous dense substance of the columns of the connecting piece formed by anterior continuations of the outer coarse fibers does form a thin plate over the proximal centriole. This is complementary in contour to the articular fossa of the nucleus and closely coapted to it. We are unable, however, to confirm the presence of a second layer continuous with the neck cylinder and interposed between these surfaces as depicted by Austin (1). Our confidence that the dense layer between the proximal centriole and the concavity in the nucleus is indeed a part of the connecting piece and not the neck cylinder, stems from the observation that the layer is present before the neck cylinder has developed (Fig. 12). The caudal rim of the neck cylinder marks the beginning of the mitochondrial sheath in snake sperm and, thus, technically the lower margin of the neck. The sheath terminates, as in mammals, at the annulus, which is at the junction of the middlepiece and principal piece. Unlike the closely packed, regularly shaped mitochondria in the middle-piece of mammalian spermatozoa, those of the snake are highly contorted and variable in their orientation (Fig. 4). Their predominant direction is in a loose spiral around the flagellum, but they may run longitudinally for short distances. In mammals, the extent of the mitochondrial sheath is taken as defining the length of the middle-piece, and the circumferential elements of the fibrous sheath are found exclusively in the principal piece. In the snake, on the other hand, the fibrous sheath is found not only in the principal piece, but also in the middle piece, concentric with the mitochondrial sheath and interposed between it and the longitudinal fibers of the tail (Figs. 1, 2, and 5). The structure of the snake sperm is therefore very much
FIGS. 3-5. The disposition of the dense plaques (Dn P) along the length of the middle-piece, within the mitochondrial sheath (Mt), is shown in Fig. 3. Note that the plaques are not membrane bounded. When viewed enface as in Fig. 4, the plaques are irregular in outline and can be seen to be distributed more or less randomly among the contorted mitochondria (Mt). In Fig. 5, a plaque has been cut in such a way that one can see the dense profiles of a plaque edge-on at either side of the groove occupied by the flagellum, and the less dense outline (at arrows) of the plaque in seen on the flat as it extends around behind the flagellum. Fig. 3, x 29.003; Fig. 4, x 30,009; Fig. 5, × 30,009.
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as though there has been an intussusception, or telescoping, of the anterior portion of the fibrous sheath into the midpiece. Interspersed among the mitochondria are pleomorphic dense bodies which in three-dimensional configuration, are curved plaques of variable size and outline, with their concave surface apposed to the underlying fibrous sheath of the middlepiece (Figs. 3, 4, and 5). The thickness of these plaques is approximately the same as that of the mitochondrial sheath, and in earlier studies they were evidently mistaken for unusually dense mitochondria. They are not limited by a membrane, however, and are composed of a granular- textured dense material identical in electron micrographs to that comprising the neck cylinder (Figs. 1-9). They are distributed at irregular intervals along the length of the middle-piece as elongate dark profiles that stand out in marked contrast to the lighter and predominantly round profiles of the mitochondria (Fig. 3). The dense plaques vary in their circumferential, as well as in their longitudinal, dimensions. The majority extend around only a fraction of the circumference (Fig. 8), but the largest may almost completely encircle the fibrous sheath. In Fig. 9, for example, only a single mitochondrial profile is interposed between the ends of a plaque that forms a nearly complete ring around the fibrous sheath. Another unusual apsect of sperm cell tail structure in the snake is the occurrence of multiple layers of membranes that extend around the circumference of the sperm from the head to the end of the middle-piece (Figs. 1-9). These were noted by Austin (1), but he considered that they were possibly an artifact of fixation. In our material these are always present, although the fixation appears, by other criteria, to be free of gross artifact. It has not been possible to determine the source of these membranes during spermatogenesis. Their functional significance is obscure, but it is possible that they provide a source of endogenous phospholipid that can be utilized as a source of energy for motility as is reported by Rothschild and Cleland (11) to be the case for Echinus sp. In cross sections, such as in Figs. 6-9, one invariably finds tubules (approximately 500/k in diameter) in close relationship to the sperm; these can be seen in longitudinal sections as well in Figs. 1-3. The tubules are extracellular and form a loosely organized sheath over the whole longitudinal extent of the sperm during its sojourn in the epididymal ducts. To our knowledge extracellular tubules of this sort have not been described in relation to other vertebrate spermatozoa, although in invertebrates Reger (10) has reported that tubules, approximately one-half the size of those shown here, surround the nuclear region of spermotozoa of the isopod Asellus sp. The origin and function of these tubules are unknown.
Formation of the mitoehondrial sheath The major features of spermatid development in the king snake resemble those described for mammalian sperm (3, 6). Associated with the later phases of growth
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FIGS. 6-8. These three cross sections of the sperm tail are representative of images that would be obtained at the levels indicated by corresponding numbers in Fig. 2. Fig. 6 is a cross section through the neck region. Note that the columns of the connecting piece (Cn P) are partially surrounded by the neck cylinder (Nk C). Within the distal centriole one can see dense material central to the triplets and material of similar density associated with one member of the central pair of tubules. At a level presumably at the junction of the distal centriole and axial filaments complex (Fig. 7), the outer coarse fibers are present and in continuity with the slender wedge-shaped columns that extend between the triplets of the centriole. Fibers 3 and 8, which are largest in Fig. 7, are fused farther caudad with the fibrous sheath (Fb S) (Fig. 8, at arrows). The other seven outer dense fibers are attached to the doublets of the axial filament complex. The concentric layers of membranes are quite apparent in these cross sections, and in addition one can see extracellular tubular structures-of unknown provenance that are closely associated with the surface of the mature sperm. These tubules, which are about twice the diameter of intracellular microtubules, can also be seen in longitudinal section in Fig. 1 and 2. Dn P, dense plaque; Mb S, membrane sheath; Mt, mitochondrial sheath. Fig. 6, x 29,000; Fig. 7, x 30,000; Fig. 8, x 26,800. FIG. 9. In this figure a dense plaque (Dn P) extends around the whole circumference of the sperm tail, except for the interposition of one small mitochondrial (Mt) profile. Fb S, fibrous sheath; MbS, membrane sheath, x 29,000.
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FIG. 12. Here the connecting piece (Cn P) is fully formed and the manchette, which extended around the neck at the stage depicted in Fig. 10, has regressed in its caudal portion. The structure marked X is representative of membrane-associated dense bodies that are first seen at this stage and appear to undergo maturation along with other organelles in the spermatid. These bodies are closely associated with the inner aspect of the plasmalemma throughout most of their existence (Fig. 13), but eventually disintegrate within the cytoplasm of the spermatic just prior to its release of the cell into the seminiferous tubule (Fig. 18, arrows). An, annulus; Ce, centriole; Ncl, nucleus. × 40,500.
FIG. 10. This stage of development of the spermatid is just precedent to formation of the neck cylinder. The columns of the connecting piece (Cn P) are forming, and the annulus (An) is present. The whole neck complex is surrounded by an amorphous material, but there is no indication of precursors to the neck cylinder or dense plaques of the mitochondrial sheath. Ce, centriole; Mn, manchette, x 27,000. F~G. 11. Transverse section through the distal centriole (Ce) at a stage of development comparable to that of Fig. 10. Note the extensions from the columns of the connecting piece (Cn P) passing centrally between the triplets of the centriole. Mn, manchette, x 27,000.
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of the acrosome there is a progressive condensation of the spermatid nucleus, and a differentiation of structures in the neck enclosing the centrioles and providing for attachment of the flagellum to the nucleus. In relatively late spermatids (Fig. 10) one observes an ill-defined accumulation of granular material around the proximal part of the axial filament complex (Fig. 11). Spermatids at this stage are considered to be just precedent to the stage of formation of the neck cylinder and of the plaques of the mitochondrial sheath. The nucleus at this stage is fully condensed, and the microtubules of the manchette are aligned longitudinally in a cylinder encircling the nucleus and extending caudally into the cytoplasm of the elongated spermatid. Mitochondria are scattered throughout the spermatid cytoplasm at this stage. At a later stage they gather around the centriolar apparatus of the neck (Fig. 13), before entering into the formation of the mitochondrial sheath of the middle-piece. During the development of the neck region, nine dense columns arise in close relationship to the nine triplets of the distal centriole (Fig. 10). These enlarge and extend anteriorly to envelope the proximal centriole and form a structure analogous to the connecting piece of the mammalian sperm, but lacking the cross-striated substructure characteristic of the latter. This connecting piece assumes its adult form (Fig. 12) before the precursors of the neck cylinder and middle-piece plaques become apparent. The manchette than regresses and mitochondria migrate toward the neck region and become oriented radially, with their ends aligned along the margins of a clear zone around the connecting piece (Fig. 13). Concurrently with the establishment of this relationship of the mitochondria to the neck region, a number of spherical dense bodies appear along the margin of the clear zone and among the ends of the associated mitochondria. In the further differentiation of the spermatid these dense bodies progressively enlarge and become less ordered in their arrangement than at the time of their first appearance (Figs. 14 and 15). Many of them subsequently coalesce and become associated with the base of the nucleus to form the neck cylinder (Figs. 16 and 17). With the caudal displacement of the annulus, the mitochondria become disposed around the fibrous sheath to form the mitochondrial sheath of the middle-piece. Some of the dense bodies that do not take part in formation of the neck cylinder are carried caudad along with the mitochondria and give rise to the dense plaques described above as distributed at irregular intervals along the length of the middle-piece (Fig. 18). A prominent feature of spermiogenesis in snakes is the accumulation of lipid droplets around the nucleus. In early stages they are scattered throughout the cytoplasm, but after regression of the manchette, they congregate around the nucleus (Fig. 13) and persist in this relationship throughout the remainder of spermiogenesis. They are cast off in the residual body shortly before release of the sperm from the germinal epithelium. Contrary to the reports by Sud and Meek (12) and Boisson and Mattei
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FIG. 13. After regression of the manchette, mitochondria aggregate and become aligned around the periphery of the neck region. At this stage, dense spherical bodies (Dn B) arise in relationship to the ends of the mitochondria (Mr). At this time, too, lipid droplets (Lp) accumulate around the nucleus. Fb S, fibrous sheath, x 27,000.
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(2), we have not seen any evidence of the formation of these lipid droplets from mitochondria. At a stage represented by Fig. 12, one first begins to see dense bodies (X in Fig. 12) closely apposed to the plasmalemma. As maturation of the spermatid progresses these bodies change shape somewhat (Fig. 13) but nonetheless remain closely apposed to the membranes and are found distributed over most of the inner surface of the spermatid plasmalemma. These bodies eventually disintegrate within the spermatid cytoplasm, and remnants of them can be seen in residual bodies of Regaud. DISCUSSION In describing the neck cylinder, Austin (1) recognized that it was apparently not represented in spermatozoa of other animals and commented upon the desirability of tracing its origin in spermiogenesis. The present study has now established that it is formed by coalescence of material first deposited in dense spherical granules in close topographical relationship to the ends of the mitochondria that gather around the distal centriole and base of the tail at a late stage of spermiogenesis. It is not possible from our observations definitely to attribute the formation of the granules either to the mitochondria or to the centrioles, or to establish their homology with any of the components of the mammalian sperm. Granules similar to those described here were seen by Boisson and Mattei (2) in late spermatids of Python sebae, but these authors considered them to be satellites to the centrioles and did not follow their development through to the mature sperm. Austin's suggestion that the basal plate lying between the nucleus and the proximal centriole of the mammalian spermatozoon might represent a much reduced mammalian homologue of the neck cylinder, is not borne out by our observations. The edge of the neck cylinder abuts upon the nuclear envelope in a ring around the implantation fossa. The cylinder is not closed at its anterior end by a layer applied to the base of the nucleus. Moreover, it has recently been shown that the basal plate is a product of the proximal centriole and begins to form at a very early stage of spermiogenesis before the centrioles and base of the flagellum become associated with the caudal pole of the nucleus (8). The dense plaques among the mitochondria of the middle-piece, which are described here for the first time, were found to arise by coalescence of those dense granules of the spermatid which do not enter into formation of the neck cylinder. They therefore share a common origin with the neck cylinder and are presumably of similar composition. The function of both remains obscure. The interposition of the fibrous sheath between the mitochondria and the longitudinal fibers of the tail is apparently unique to snake sperm. In other species in which
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FIGS. 14 and 15. The precise alignment of mitochondria and the formative stages of the dense bodies seen in Fig. 13 is transient. In later stages of spermiogenesis, the dense bodies (Db B) enlarge and become dispersed among the mitochondria (Mt). In the longitudinal section of a spermatid in Fig. 14, the distribution seems to be random, but in a cross section, Fig. 15, the bodies seem to be clustered around the distal centriole. Cn P, connecting piece; Lp, lipid droplets; Ncl, nucleus. Fig. 14, x 54,000; Fig. 15, x 54,000.
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FIGS. 16 and 17. At the stage depicted here, the dense granules in the neck region have begun to coalesce to form the neck cylinder (Nk C) and have become attached to the rim of the articular fossa anteriorly. Fig. 16, x 27,000; Fig. 17, × 29,200.
outer coarse fibers are found, there is an intimate relationship between the mitochondria and the outer fibers. Fawcett (4) and Fawcett and Ito (7) have put forward the suggestion that the outer coarse fibers are contractile and that the close structural relationship usually found between the mitochondria and the fibers may serve to provide an energy source necessary for their contraction. This view must be modified in the light of the findings reported here, for in the snake the mitochondria seem to be completely isolated. The fibrous sheath is interposed between them and the longitudinal fibers of the tail; caudally they are limited by the annulus, and anteriorly the fibrous sheath ends in direct relationship to a medially directed flange of the neck cylinder (Figs. 1 and 2). Only rarely does one see instances such as are illustrated in Fig. 2 in which a mitochondrion extends outside this compartment. It is difficult to imagine how an energy source, such as adenosine triphosphate (ATP) could diffuse to the outer coarse fibers through the relatively thick fibrous sheath. It is not known when during its lifetime a snake spermatozoon is motile, but it seems reasonable to assume that during its passage through the excurrent ducts it is immotile, as in mammals, and that motility (and thus the need for large amounts of ATP) occupies
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Fro. 18. Granules that do not participate in formation of the neck cylinder are carried along with the mitochondria into the tail, where they coalesce and alter their shape to form the plaques seen in the mitochondrial sheath of the mature sperm. In this oblique view of three spermatids, the granules can be seen among the mitochondria forming the mitochondria sheath around the flagellum. The arrows indicate the remnants of granules of another kind that are associated with the plasmalemma. These granules are beginning to break up and become dispersed in the cytoplasm, which will be cast off as the residual body of Regaud. × 27,000.
only a short part of its life span. Possibly a buildup of reserve A T P is part of the maturation process of the sperm during its passage t h r o u g h the epididymis, both in species with a well developed mitochondrial sheath and in those without it, and the intimate structural relationship f o u n d in m a n y species between the outer coarse fibers and the mitochondria m a y well be fortuitous. A n o t h e r exceptional feature of snake sperm, commented u p o n by Austin (1) is the fact that the outer coarse fibers 3 and 8 are largest, whereas in the anterior part of the m a m m a l i a n sperm tail these are smallest. In mammals, they also terminate first, and their place is occupied t h r o u g h o u t most of the length of the principal piece by inward prolongations of the dorsal and ventral columns of the fibrous sheath. In the snake the large fibers 3 and 8 do not terminate but apparently become fused to the dorsal and ventral aspects of the fibrous sheath whereas the other seven coarse
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fibers remain attached to the corresponding doublets (Fig. 8 and 9). The predominance in size of the lateral coarse fibers 1, 5, and 6 or 1, 9, 5, and 6 in mammals has been interpreted as evidence that these fibers probably contribute importantly to the bending movements of the flagellum. The size predominance of 3 and 8 in the snake, seemed to Austin (1) to invalidate the argument that the large size of the lateral fibers in the mammal was related to their effectiveness as motor elements. However, since fibers 3 and 8 are incorporated into the structure of the fibrous sheath in the snake, they probably serve in longitudinal stabilization of the sheath rather than as agents of motility. If this is true, the disposition of the remaining seven fibers would still seem to be consistent with the idea that they contribute to the bending movements of the tail. It is, nevertheless, surprising that the coarse fibers in the snake are not better developed. A consideration of a cross section of the tail (Fig. 7) with its sheath of membranes, its mitochondrial sheath, and its fibrous sheath all arranged concentrically, would suggest the need for an unusually powerful motor apparatus to overcome their combined resistance to bending. Among the atypical features of the distal centriole of snake sperm, Austin described an inner row of nine slender dense columns central to the triplets and of an additional dense longitudinal column associated with one member of the central pair of tubules. These findings are confirmed in the present study. It was found that the anterior continuations of the outer dense fibers of the tail which form the connecting piece of the snake sperm are in continuity with these inner columns via thin radially oriented laminae that pass between the triplets (Figs. 8 and 11). Although discrete dense columns central to the triplets are not usually found in centrioles of mammalian sperm, the dense material that gives rise to the basal plate and to the striated columns of the connecting piece arises within the proximal and distal centrioles respectively and extends radially between the triplets to form the peripheral structures that ultimately coalesce to make up the surrounding connecting piece (8). The outer coarse fibers of the tail do not arise at the neck and grow distally by additions to their ends as appears to be the case in the outgrowth of the doublets of the axial filaments complex. Instead they arise by deposition of dense material over the outer aspect of the doublets along much of their length. The slender dense fibers initially formed are thickened by progressive addition of material to their surface. The outer fibers formed in this manner later become separated from the doublets on which they were initially deposited, except at their caudal ends where they remain continuous. In mammals the peripheral structures arising from centrioles display a distinct cross-striation with a periodicity relating them to rootlets of cilia. The dense homogeneous peripheral fibers of the tail display an abrupt discontinuity in texture where they are joined end-to-end with the nine cross-striated columns of the connecting piece. This has been interpreted as indicating a significant difference in the chemical
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nature of these two components (5). Studies of snake sperm show that a connecting piece can be formed which is similar in form to that of mammals but lacks crossstriations and evidently is composed of the same homogeneous dense material as the outer tail fibers. The similarity in form and mode of origin of the mammalian and reptilian connecting piece raises the questidn whether the striated material of the one, and nonstriated material of the other, represent different centriolar products or simply alternative modes of polymerization of the same molecular subunits. REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12.
AUSTIN, C. R., J. Ultrastruct. Res. 12, 452 (1965). BOISSON,C. and MAITEL X., Ann. Sci. Nat. 8, 363 (1966). BURGOS, M. H. and FAWCETT,D. W., J. Biophys. Biochem. Cytol. 1, 287 (1955). FAWCETT,D. W., "Spermatozoan Motility," p. 147. American Association for the Advancement of Science, Washington, 1962. Z. Zellforsch. 67, 279 (1965). FAWCETr, D. W. and HOLLENBERH,R. D., Z. Zellforseh. 60, 276 (1963). FAWCETT,D. W. and ITO, S., Am. J. Anat. 116, 567 (1965). FAWCETT,D. W. and PHILLIPS,D. M., in preparation. KARNOVSKY,M., J. Cell Biol. 27, 137A (1965). REGER, J. F., J. Ultrastruct. Res. 11, 181 (1964). ROTHSCHILD,Lord and CLELAND, K. W., J. Exptl. Biol. 29, 66 (1952). SUD, B. N. and MEEK, G. A., Anat. Record 157, 329 (1967).
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