Cell Tiss. Res. 164, 121-132 (1975) - 9 by Springer-Verlag 1975
Ultrastructural Changes in Spermatozoa of the Brush-Tailed Possum, Trichosurus vulpecula (Marsupialia), during Epididymal Transit Part I: The Flagellum H.R. Harding, F.N. Carrick and C.D. Shorey* Department of General Studies and School of Zoology, University of New South Wales, Kensington, Australia and Department of Histology and Embryology, University of Sydney, Australia
Summary. During epididymal transit, a fibre network and an array of vesicles develop in the posterior two-thirds of the midpiece in sperm of the Brush-tailed possum, Trichosurus vulpecula. The fibre network is developed by the time the sperm reach the corpus epididymidis, and is composed of evenly spaced, helically arranged fibres lying immediately beneath the plasma membrane. The angle of these fibrous helices is always counter to that of the underlying mitochondrial helix. Separating the fibre network f r o m the mitochondria is a layer of granular material which develops at the same time, and over the same length of the midpiece as the fibre network. A somewhat tenuous fibre network is found between the fibrous sheath and plasma m e m b r a n e in the anterior principal piece of sperm from all regions of the epididymis. The array of vesicles is developed by the time sperm reach the cauda epididymidis. The vesicles resemble pinocytotic vesicles; some appear as invaginations of the plasma membrane, and are open to the medium surrounding the spermatozoon by a narrow neck, while others are entirely enclosed within the spermatozoon, and lie at varying distances between the plasma m e m b r a n e and the layer of granular material. Key words: Sperm flagellum - Trichosurus vulpecula (Marsupial) - Epididymal maturation - Transmission and Scanning electron microscopy.
Introduction Bedford and Calvin (1974) pointed out that in m a m m a l s no obvious alteration has yet been found to occur in the ultrastructure of the sperm tail during epididySend offprint requests to." H.R. Harding, Department of General Studies, University of New South Wales, P.O. Box l, Kensington, 2033, N.S.W., Australia.
* The authors would like to acknowledge the assistance of Miss Dianne Higginbotham of the Electron Microscope Unit, University of Sydney for expert assistance with the scanning electron microscopy. Also to Dr. D.J.H. Cockayne, Director of the Electron Microscope Unit, for use of the transmission electron microscope facilities.
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mal transit, other than that associated with the cytoplasmic droplet. However, changes at the molecular level, involving an increase in disulphide bonding, have been shown to occur in the sperm tails of a number of eutherians, and the metatherians, Didelphys marsupialis virginiana and Trichosurus vulpecula, as sperm pass down the epididymis (Calvin and Bedford, 1971 ; Bedford and Calvin, 1974). This paper reports the occurrence of two significant changes in the ultrastructure of the sperm tail of the Australian marsupial, Trichosurus vulpeeula (Kerr), during epididymal transit. At the same time, unusual modfications also occur in the acrosomal region of the sperm head in this species (Fig. 6) and these will be described in a subsequent paper. Although there have been numerous studies into maturation of the eutherian spermatozoon as it passes down the epididymis (Orgebin-Crist, 1969; Bedford, 1974), only one prior study, that of Phillips (1970) on the South American woolly opossum, Caluromysphilander, has investigated the ultrastructure of metatherian spermatozoa during epididymal transit. The changes described by Phillips (1970) however, are mainly concerned with loss of the cytoplasmic droplet, and with pairing of spermatozoa. The latter, involving a coupling of spermatozoa in the acrosomal region, is an occurrence characteristic of American marsupials during epididymal passage (Biggers, 1966). In addition, there have been relatively few published reports of sperm tail ultrastructure in marsupials. Epididymal spermatozoa have been examined in the North American opossum, Didelphys marsupialis virginiana (Holstein, 1965), the South American woolly opossum, Caluromys philander (Phillips, 1970) and the bandicoot, Perameles nasuta (Cleland and Rothschild, 1959; Sapsford et al., 1970). However, for the latter species, only the principal piece is described. Observations on mature spermatids are included in the extensive reports on spermiogenesis in Perameles nasuta (Sapsford and Rae, 1969; Sapsford et al., 1969, 1970). Brief descriptions of spermatozoa of Didelphys marsupialis virginiana (Fawcett, 1970; Phillips, 1974), and the Australian marsupial mouse, Antechinus (Cleland, 1965), do not indicate the region from which these sperm were obtained. At the commencement of this study, no observations on the ultrastructure of spermatozoa of Trichosurus vulpecula had been published. However, Olson (1975) recently described a fibre network beneath the plasma membrane in both the midpiece and principal piece of the spermatozoon from the cauda epididymidis of this species. The present study confirms the presence of this fibre network, but shows also that in the midpiece it develops during epididymal transit, as does an unusual collection of vesicles, which were not reported by Olson (1975).
Fig. 1. Longitudinal section through the head (H), midpiece (MP) and anterior principal piece (PP) of a spermatozoon from the cauda epididymidis. The anterior extent of the fibre network and underlying granular layer is shown arrowed, two-thirds along the midpiece from the annulus (AN). x 7 700 Fig. 2. Scanning electron micrograph of a spermatozoon from the cauda epididymidis. Note the apparent folding in the plasma membrane of the anterior one-third of the midpiece (arrowed). x 6400
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Materials and Methods Spermatozoa from the caput, corpus and cauda epididymidis, and from the vas deferens were examined. Sperm were obtained from three adult male Brush-tailed possums (Trichosurus vulpecula) captured in the Sydney metropolitan area. The epididymal samples were taken from the proximal part of the caput region, the central part of the corpus and the distal part of the cauda, prior to its tapering and reflection towards the vas deferens. Spermatozoa were extracted from the epididymis by excision of the appropriate region which was then extensively lacerated with a sharp razor blade under fixative and shaken vigorously with fixative in a stoppered centrifuge tube; after which the remaining epididymal tissue was removed from the suspension and discarded. The contents of the vas deferens were either flushed with fixative from a syringe or squeezed between the fingers into a tube of fixative. After fixation for 30 to 60 minutes at room temperature in modified Karnovsky's fixative with picric acid added, sperm were spun down at about 1900 RCF for 10 minutes, and washed overnight in cacodylate/sucrose buffer. Postfixation was carried out in the cold for 60 minutes using osmium tetroxide/cacodylate/glucose and stained for 30 minutes in uranyl acetate prior to dehydration. Material for examination under the scanning electron microscope was obtained from each of the above sperm suspensions prior to centrifugation. The appropriate suspension was drawn up into a clean Pasteur pipette, a drop deposited on a glass coverslip, and immediately withdrawn leaving a deposit of cells to dry on the coverslip. Fragments of coverslip were mounted on aluminium stubs using double sided adhesive tape. When dried, samples were rinsed in distilled water to remove sediment of picric acid which produces a granular background in the scanning electron micrographs. Specimens were then rotary shadowed with gold. Specimens were examined using a Philips EM 201 or EM 300 for transmission electron microscopy, and JEOL JSM-U3 scanning electron microscope.
Results
Midpiece Fibre Network Transverse and longitudinal sections of the midpiece region of sperm from the corpus (Fig. 5) and cauda (Fig. 8) epididymidis and from the vas deferens (Fig. 9) show a single layer of evenly spaced (about 110 nm between centres) rounded aggregates of electron dense material, of about 60 nm diameter, lying immediately beneath the plasma membrane. Superficial longitudinal sections (Fig. 7) show that these form regular helices which occur in the posterior twothirds of the midpiece (Fig. 1). The direction of the fibrous helices is always counter to that of the underlying mitochondrial helix (Fig. 8). Scanning electron micrographs (Fig. 2) suggest that these fibrous helices may have a supportive function, since the anterior region of the midpiece which lacks these helices, has a rather folded appearance in contrast to the firm appearance of the more distal regions. Beneath these fibrous helices, and separating them from the mitochondria, is a layer of unevenly dispersed granular material (Figs. 5, 9). This layer starts Fig. 3. Longitudinal section of the midpiece in a spermatozoon from the caput epididymidis. A F C axial filament complex; M T mitochondrion; P M plasma membrane. Note absence of the fibre network and granular layer. • 62 000 Fig. 4. Longitudinal section of the principal piece in a caput sperm. FS fibrous sheath; P N principal piece fibre network, x 72 000
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Fig. 5. Transverse section of the midpiece of a spermatozoon from the corpus epididymidis. A midpiece fibre network (MN) and layer of granular material (GM) are developed, however vesicles are not found between the plasma membrane (PM) and granular layer. Note the absence of the principal piece fibre network in arrowed section & t h e posterior principal piece, x 66000
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abruptly and coincidentally with the overlying fibrous helices, about one-third the distance along the midpiece from its anterior margin, and similarly they terminate together at the annulus (Figs. 1, 6). Neither the fibrous helices, nor this granular layer are present in sperm from the caput epididymidis (Fig. 3). Instead, an irregular cytoplasmic matrix surrounds the mitochondria, but is not formed into a definite layer. Principal Piece Fibre Network. In transverse and longitudinal sections of the principal piece of sperm from the caput (Fig. 4), corpus (Fig. 6) and cauda (Fig. 8) epididymidis, and from the vas deferens a somewhat irregular array of rounded aggregates of electron dense material is seen to be interspersed with less organized electron dense material. Superficial longitudinal sections (Fig. 6) show these rounded aggregates to form rather tenuous fibres wound around the fibrous sheath. These fibres are thinner (about 35 nm), less regular in both spacing and angle of orientation, and generally more widely separated from the plasma membrane, than those of the midpiece (Figs. 4, 6). The principal piece fibre network terminates some distance from the end piece of the flagellum (Fig. 5). Sections of the testis reveal that the principal piece fibre network is already present in the flagellum of advanced spermatids. Midpiece Vesicles. In the midpiece region of sperm from the cauda epididymidis and vas deferens a number of vesicles of maximum diameter about 90 nm are observed lying between the plasma membrane and the layer of granular material described above (Figs. 7, 9, 10). Transverse and longitudinal sections show that these vesicles are bound by a single trilaminar membrane (Fig. 10), and that they pass through a number of stages. Some of the vesicles appear as invaginations of the plasma membrane and are open to the medium surrounding the spermatozoon by a narrow neck (Fig. 10). Others are entirely enclosed within the spermatozoon and vary in their distance of separation from the plasma membrane (Fig. 9). The more superficially located vesicles are seen in transverse (Fig. 9) and longitudinal sections (Fig. 8) to lie between the fibres of the midpiece helices, and superficial longitudinal sections (Fig. 7) show that with this exception, the vesicles have no regularity in their arrangement. The anterior termination of the midpiece fibres and underlying granular layer also marks the anterior limit of the location of the vesicles, and likewise, distally these structures terminate simultaneously at the annulus (Fig. 8). It is striking that these vesicles are not found in the midpiece of sperm from the caput and corpus epididymidis (Figs. 3, 5), nor are they found at any stage in the principal piece of the sperm (Figs.4, 8). Discussion
Midpiece Fibre Network. An unusual fibre network is found in the midpiece of distal epididymal spermatozoa of Trichosurus vulpecula. This structure was Fig. 6. Longitudinalsectionsof spermatozoafrom the corpus epididymidis.Coincidentterminations of the granular layer and the midpiece fibre network occur anteriorly (arrowed) and posteriorly at the annulus (AN). Note the unusual structure in the acrosomal region (AR). FS fibrous sheath, N nucleus, PN principal piece fibre network, x 20000
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recently described by Olson (1975), and his observations are confirmed here. However, this study extends his observations by showing that the midpiece network develops during epididymal transit, being found in sperm from the corpus epididymidis and more distal regions of the tract, but not in sperm from the caput epididymidis. The present study also notes that the direction of winding of the fibrous helices is always counter to that of the mitochondrial helix. The functional significance of this fibre network is not clear, and while no suggestions were given by Olson (1975), certain of the observations made in the present study deserve comment. The anterior region of the midpiece which lacks fibrous helices, has a rather folded appearance in contrast to the firm appearance of the more distal regions where the helices are present. On this evidence it seems reasonable to suggest that the midpiece fibre network may have a supportive function. In addition, the opposite direction of winding of the fibrous helices to the mitochondrial helix, is of interest when considering the spermatozoon structures which may have potential to modify flagellar movement. No information is available on spermatozoon motility in Trichosurus, but it would be interesting to determine whether any temporal correlation exists between the development of the midpiece fibre network and changes in spermatozoon motility patterns during epididymal transit. Observations on the cytoplasmic droplet will be reported in a subsequent paper, but it is worth noting here, that the midpiece fibre network and vesicles develop after the shedding of the droplet. A midpiece fibre network of the type described here, has not been reported in the sperm of any eutherian species. However, in the midpiece of guinea pig sperm, freeze-fracture techniques reveal circumferential arrays of 6 to 8 nm particles associated with the plasma membrane overlying the mitochondria (Friend and Fawcett, 1974; K oehler and Gaddum-Rosse, 1975). Although these particle strands have a somewhat similar position and orientation to the possum fibrous helices, there are significant differences. The guinea pig particle strands are intramembranous and only about one-tenth the diameter of the possum fibrous helices. Among non-mammalian vertebrates, the spermatozoon of the spiny dogfish, Squalus suckleyi, has a helical sheath of filaments, the fibrous midpiece sheath, closely surrounding the mitochondria (Stanley, 1971). However, neither this fibrous sheath, nor the particle strands in the guinea pig closely resemble the possum midpiece fibre network. Midpiece fibrous helices have not been reported in the sperm of other marsupial species. There are indications in the literature, however, of their presence in at least some species. In published micrographs of Caluromysphilander (Phillips, 1970) and Didelphys marsupialis virginiana (Phillips, 1974) sperm, longitudinal sections suggest that this might be the case, although further investigation would be necessary to determine this with certainty. For Australian marsupials, Cleland (1965) in a brief abstract, reports the presence in the midpiece of an accessory
Fig. 7. Superficial longitudinal section of the midpiece of a spermatozoon from the cauda epididymidis. Note the orientation of the midpiece vesicles (V) with respect to the midpiece fibre network (MN). x 36 000
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Fig. 8. Longitudinal section through the midpiece and anterior principal piece of a spermatozoon from the cauda epididymidis. Midpiece vesicles (V) are now present. Attention is drawn to the contra-rotation of the mitochondrial helix (MT) and helices of the midpiece fibre network (MAr) by the extended arrows. P N principal piece fibre network, x 44000
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Fig. 9. Transverse section of the midpiece of a spermatozoon from the vas deferens. GM layer of granular material; MN midpiece fibre network; PM plasma membrane; Ve midpiece vesicle enclosed within the cytoplasm of the spermatozoon; Vs superficial midpiece vesicle open to the extracellular space, x 81000 Fig. 10. High power view of a superficial midpiece vesicle (Vs) of a spermatozoon from the vas deferens. B Vboundary trilaminar membrane of the midpiece vesicle; GM layer of granular material ; MN fibre of midpiece fibre network; MTmitochondrion; PM plasma membrane, x 210000
p e r i m i t o c h o n d r i a l sheath which is a m o r p h o u s in Perameles nasuta, b u t spiral in the dasyurids, Antechinus a n d Sminthopsis. We are currently investigating the sperm o f a range of A u s t r a l i a n m a r s u p i a l s for the presence of these midpiece fibrous helices. Principal Piece Fibre N e t w o r k . The o b s e r v a t i o n s in this study agree with the recent report of O l s o n (1975) o n the principal piece fibre n e t w o r k in sperm
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of Trichosurus. However, the present study shows also that the network is present in sperm from all regions of the epididymis and mature spermatids in the testis. This is in contrast to the later development of the midpiece fibrous helices, and suggests that the two networks may have separate origins. A similar rather irregular array of fibres has been described in the principal piece of sperm from the American marsupials Caluromys philander (Phillips, 1970) and Didelphys marsupialis virginiana (Holstein, 1965). Sapsford et al., (1970) found a narrow granular accessory sheath to surround the principal piece sheath in the bandicoot spermatozoon. However, this differs from the network in Trichosurus in its regular granular nature and close attachment to the principal piece sheath. At present there is no evidence of the function of the principal piece fibre network. Midpiece Vesicles. The presence of vesicles in the midpiece of the spermatozoon has not been reported for other mammals. A notable finding of this study is that an irregular array of vesicles develops in the midpiece of sperm of T. vulpecula during epididymal transit. The vesicles develop slightly later than the midpiece fibrous helices, appearing in sperm from the cauda but not the corpus epididymidis. It is unusual that Olson (1975) did not report these vesicles in sperm from the cauda epididymidis of T. vulpecula. A possible explanation may lie in the different fixation techniques used. Whilst the function of these vesicles is not apparent, it seems likely that they may be pinocytotic vesicles absorbing some substance from the epididymal fluid. There is no certain evidence of this in the present study, but use of appropriate marking techniques in a later study should enable this to be determined. The possibility of such an absorptive function for these vesicles is interesting in view of the increasing knowledge of the secretory functions of the eutherian epididymis, and of changes in the eutherian spermatozoon during epididymal transit (Orgebin-Crist, 1969; White, 1973; Bedford, 1974). The possibility of a relationship between these vesicles and the midpiece fibre network and granular layer is suggested by the coincidence of their extent in the midpiece. Of course it is equally possible that their simultaneous anterior termination may be caused by another factor which is not as yet apparent. If a relationship does exist, then it is difficult to suggest what this might be. Perhaps the fibrous helices make some structural compensation for alteration in the sperm surface caused by the vesicles.
References Bedford, J.M. : Report of a workshop : Maturation of the fertilizing ability of mammalian spermatozoa in the male and female reproductive tract. Biol. Reprod. II, 346 362 (1974) Bedford, J.M., Calvin, H.I. : Changes in -S-S- linked structures of the sperm tail during epididymal maturation, with comparative observations in sub-mammalian species. J. exp. Zool. 187, 181-204 (1974) Biggers, J.D. : Reproduction in male marsupials, Symp. zool. Soc. Lond. 15, 251-280 (1966) Calvin, H.I., Bedford, J.M. : Formation of disulphide bonds in the nucleus and accessory structures
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of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Fertil. Suppl. 13, 65 75 (1971) Cleland, K.W.: Electron microscopic structure of the Dasyuroid sperm. J. Anat. (Lond.) 99, 953 (Abstract) (1965) Cleland, K.W., Lord Rothschild: The bandicoot spermatozoon: and electron microscope study of the tail. Proc. roy. Soc. B 150, 2 4 4 2 (1959) Fawcett, D.W. : A comparative view of sperm ultrastructure. Biol. Reprod., Suppl. 2, 90-127 (1970) Friend, D.S., Fawcett, D.W.: Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol. 63, 641-664 (1974) Holstein, A.F. : Elektronenmikroskopische Untersuchungen am Spermatozoon des Opossum (Didelphys virginiana Kerr). Z. Zellforsch. 65, 904--914 (1965) Koehler, J.K., Gaddum-Rosse, P. : Media induced alterations of the membrane associated particles of the guinea pig sperm tail. J. Ultrastruct. Res. 51,106-118 (1975) Olson, G.: Observations on the ultrastructure of a fiber network in the flagellum of sperm of the Brush tailed Phalanger, Trichosurus vulpecula. J. Ultrastruct. Res. 50, 193-198 (1975) Orgebin-Crist, M.C.: Studies on the function of the epididymis. Biol. Reprod. Suppl. I, 155-175 (1969) Phillips, D.M. : Ultrastructure of spermatozoa of the Woolly Opossum Caluromys philander. J. Ultrastruct. Res. 33, 381 397 (1970) Phillips, D.M. : Spermiogenesis. Academic Press: New York 1974 Sapsford, C.S., Rae, C.A. : Ultrastructural studies on Sertoli cells and spermatids in the bandicoot and ram during the movement of mature spennatids into the lumen of the seminiferous tubule. Aust. J. Zool. 17, 415445 (1969) Sapsford, C.S., Rae, C.A., Cleland, K.W.: Ultrastructural studies on maturing spermatids and on Sertoli cells in the bandicoot Perameles nasuta Geoffroy (Marsupialia). Aust. J. Zool. 17, 195- 292 (1969) Sapsford, C.S., Rae, C.A., Cleland, K.W.: Ultrastructural studies on the development and form of the principal piece sheath of the bandicoot spermatozoon. Aust. J. Zool. 18, 2 1 4 8 (1970) Stanley, H.P.: Fine structure of spermiogenesis in the Elasmobranch fish Squalus suckleyi. II. Late stages of differentiation and structure of the mature spermatozoon. J. Ultrastruct. Res. 36, 103-118 (1971) White, I.G.: Biochemical aspects of spermatozoa and their environment in the male reproductive tract. J. Reprod. Fertil., Suppl. 18, 225 235 (1973)
Receired August 18, 1975
Ultrastructural Changes in Spermatozoa of the Brush-Tailed Possum, Trichosurus vulpecula (Marsupialia), during Epididymal Transit Part I: The Flagellum H.R. Harding, F.N. Carrick and C.D. Shorey* Department of General Studies and School of Zoology, University of New South Wales, Kensington, Australia and Department of Histology and Embryology, University of Sydney, Australia
Summary. During epididymal transit, a fibre network and an array of vesicles develop in the posterior two-thirds of the midpiece in sperm of the Brush-tailed possum, Trichosurus vulpecula. The fibre network is developed by the time the sperm reach the corpus epididymidis, and is composed of evenly spaced, helically arranged fibres lying immediately beneath the plasma membrane. The angle of these fibrous helices is always counter to that of the underlying mitochondrial helix. Separating the fibre network f r o m the mitochondria is a layer of granular material which develops at the same time, and over the same length of the midpiece as the fibre network. A somewhat tenuous fibre network is found between the fibrous sheath and plasma m e m b r a n e in the anterior principal piece of sperm from all regions of the epididymis. The array of vesicles is developed by the time sperm reach the cauda epididymidis. The vesicles resemble pinocytotic vesicles; some appear as invaginations of the plasma membrane, and are open to the medium surrounding the spermatozoon by a narrow neck, while others are entirely enclosed within the spermatozoon, and lie at varying distances between the plasma m e m b r a n e and the layer of granular material. Key words: Sperm flagellum - Trichosurus vulpecula (Marsupial) - Epididymal maturation - Transmission and Scanning electron microscopy.
Introduction Bedford and Calvin (1974) pointed out that in m a m m a l s no obvious alteration has yet been found to occur in the ultrastructure of the sperm tail during epididySend offprint requests to." H.R. Harding, Department of General Studies, University of New South Wales, P.O. Box l, Kensington, 2033, N.S.W., Australia.
* The authors would like to acknowledge the assistance of Miss Dianne Higginbotham of the Electron Microscope Unit, University of Sydney for expert assistance with the scanning electron microscopy. Also to Dr. D.J.H. Cockayne, Director of the Electron Microscope Unit, for use of the transmission electron microscope facilities.
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mal transit, other than that associated with the cytoplasmic droplet. However, changes at the molecular level, involving an increase in disulphide bonding, have been shown to occur in the sperm tails of a number of eutherians, and the metatherians, Didelphys marsupialis virginiana and Trichosurus vulpecula, as sperm pass down the epididymis (Calvin and Bedford, 1971 ; Bedford and Calvin, 1974). This paper reports the occurrence of two significant changes in the ultrastructure of the sperm tail of the Australian marsupial, Trichosurus vulpeeula (Kerr), during epididymal transit. At the same time, unusual modfications also occur in the acrosomal region of the sperm head in this species (Fig. 6) and these will be described in a subsequent paper. Although there have been numerous studies into maturation of the eutherian spermatozoon as it passes down the epididymis (Orgebin-Crist, 1969; Bedford, 1974), only one prior study, that of Phillips (1970) on the South American woolly opossum, Caluromysphilander, has investigated the ultrastructure of metatherian spermatozoa during epididymal transit. The changes described by Phillips (1970) however, are mainly concerned with loss of the cytoplasmic droplet, and with pairing of spermatozoa. The latter, involving a coupling of spermatozoa in the acrosomal region, is an occurrence characteristic of American marsupials during epididymal passage (Biggers, 1966). In addition, there have been relatively few published reports of sperm tail ultrastructure in marsupials. Epididymal spermatozoa have been examined in the North American opossum, Didelphys marsupialis virginiana (Holstein, 1965), the South American woolly opossum, Caluromys philander (Phillips, 1970) and the bandicoot, Perameles nasuta (Cleland and Rothschild, 1959; Sapsford et al., 1970). However, for the latter species, only the principal piece is described. Observations on mature spermatids are included in the extensive reports on spermiogenesis in Perameles nasuta (Sapsford and Rae, 1969; Sapsford et al., 1969, 1970). Brief descriptions of spermatozoa of Didelphys marsupialis virginiana (Fawcett, 1970; Phillips, 1974), and the Australian marsupial mouse, Antechinus (Cleland, 1965), do not indicate the region from which these sperm were obtained. At the commencement of this study, no observations on the ultrastructure of spermatozoa of Trichosurus vulpecula had been published. However, Olson (1975) recently described a fibre network beneath the plasma membrane in both the midpiece and principal piece of the spermatozoon from the cauda epididymidis of this species. The present study confirms the presence of this fibre network, but shows also that in the midpiece it develops during epididymal transit, as does an unusual collection of vesicles, which were not reported by Olson (1975).
Fig. 1. Longitudinal section through the head (H), midpiece (MP) and anterior principal piece (PP) of a spermatozoon from the cauda epididymidis. The anterior extent of the fibre network and underlying granular layer is shown arrowed, two-thirds along the midpiece from the annulus (AN). x 7 700 Fig. 2. Scanning electron micrograph of a spermatozoon from the cauda epididymidis. Note the apparent folding in the plasma membrane of the anterior one-third of the midpiece (arrowed). x 6400
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Materials and Methods Spermatozoa from the caput, corpus and cauda epididymidis, and from the vas deferens were examined. Sperm were obtained from three adult male Brush-tailed possums (Trichosurus vulpecula) captured in the Sydney metropolitan area. The epididymal samples were taken from the proximal part of the caput region, the central part of the corpus and the distal part of the cauda, prior to its tapering and reflection towards the vas deferens. Spermatozoa were extracted from the epididymis by excision of the appropriate region which was then extensively lacerated with a sharp razor blade under fixative and shaken vigorously with fixative in a stoppered centrifuge tube; after which the remaining epididymal tissue was removed from the suspension and discarded. The contents of the vas deferens were either flushed with fixative from a syringe or squeezed between the fingers into a tube of fixative. After fixation for 30 to 60 minutes at room temperature in modified Karnovsky's fixative with picric acid added, sperm were spun down at about 1900 RCF for 10 minutes, and washed overnight in cacodylate/sucrose buffer. Postfixation was carried out in the cold for 60 minutes using osmium tetroxide/cacodylate/glucose and stained for 30 minutes in uranyl acetate prior to dehydration. Material for examination under the scanning electron microscope was obtained from each of the above sperm suspensions prior to centrifugation. The appropriate suspension was drawn up into a clean Pasteur pipette, a drop deposited on a glass coverslip, and immediately withdrawn leaving a deposit of cells to dry on the coverslip. Fragments of coverslip were mounted on aluminium stubs using double sided adhesive tape. When dried, samples were rinsed in distilled water to remove sediment of picric acid which produces a granular background in the scanning electron micrographs. Specimens were then rotary shadowed with gold. Specimens were examined using a Philips EM 201 or EM 300 for transmission electron microscopy, and JEOL JSM-U3 scanning electron microscope.
Results
Midpiece Fibre Network Transverse and longitudinal sections of the midpiece region of sperm from the corpus (Fig. 5) and cauda (Fig. 8) epididymidis and from the vas deferens (Fig. 9) show a single layer of evenly spaced (about 110 nm between centres) rounded aggregates of electron dense material, of about 60 nm diameter, lying immediately beneath the plasma membrane. Superficial longitudinal sections (Fig. 7) show that these form regular helices which occur in the posterior twothirds of the midpiece (Fig. 1). The direction of the fibrous helices is always counter to that of the underlying mitochondrial helix (Fig. 8). Scanning electron micrographs (Fig. 2) suggest that these fibrous helices may have a supportive function, since the anterior region of the midpiece which lacks these helices, has a rather folded appearance in contrast to the firm appearance of the more distal regions. Beneath these fibrous helices, and separating them from the mitochondria, is a layer of unevenly dispersed granular material (Figs. 5, 9). This layer starts Fig. 3. Longitudinal section of the midpiece in a spermatozoon from the caput epididymidis. A F C axial filament complex; M T mitochondrion; P M plasma membrane. Note absence of the fibre network and granular layer. • 62 000 Fig. 4. Longitudinal section of the principal piece in a caput sperm. FS fibrous sheath; P N principal piece fibre network, x 72 000
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Fig. 5. Transverse section of the midpiece of a spermatozoon from the corpus epididymidis. A midpiece fibre network (MN) and layer of granular material (GM) are developed, however vesicles are not found between the plasma membrane (PM) and granular layer. Note the absence of the principal piece fibre network in arrowed section & t h e posterior principal piece, x 66000
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abruptly and coincidentally with the overlying fibrous helices, about one-third the distance along the midpiece from its anterior margin, and similarly they terminate together at the annulus (Figs. 1, 6). Neither the fibrous helices, nor this granular layer are present in sperm from the caput epididymidis (Fig. 3). Instead, an irregular cytoplasmic matrix surrounds the mitochondria, but is not formed into a definite layer. Principal Piece Fibre Network. In transverse and longitudinal sections of the principal piece of sperm from the caput (Fig. 4), corpus (Fig. 6) and cauda (Fig. 8) epididymidis, and from the vas deferens a somewhat irregular array of rounded aggregates of electron dense material is seen to be interspersed with less organized electron dense material. Superficial longitudinal sections (Fig. 6) show these rounded aggregates to form rather tenuous fibres wound around the fibrous sheath. These fibres are thinner (about 35 nm), less regular in both spacing and angle of orientation, and generally more widely separated from the plasma membrane, than those of the midpiece (Figs. 4, 6). The principal piece fibre network terminates some distance from the end piece of the flagellum (Fig. 5). Sections of the testis reveal that the principal piece fibre network is already present in the flagellum of advanced spermatids. Midpiece Vesicles. In the midpiece region of sperm from the cauda epididymidis and vas deferens a number of vesicles of maximum diameter about 90 nm are observed lying between the plasma membrane and the layer of granular material described above (Figs. 7, 9, 10). Transverse and longitudinal sections show that these vesicles are bound by a single trilaminar membrane (Fig. 10), and that they pass through a number of stages. Some of the vesicles appear as invaginations of the plasma membrane and are open to the medium surrounding the spermatozoon by a narrow neck (Fig. 10). Others are entirely enclosed within the spermatozoon and vary in their distance of separation from the plasma membrane (Fig. 9). The more superficially located vesicles are seen in transverse (Fig. 9) and longitudinal sections (Fig. 8) to lie between the fibres of the midpiece helices, and superficial longitudinal sections (Fig. 7) show that with this exception, the vesicles have no regularity in their arrangement. The anterior termination of the midpiece fibres and underlying granular layer also marks the anterior limit of the location of the vesicles, and likewise, distally these structures terminate simultaneously at the annulus (Fig. 8). It is striking that these vesicles are not found in the midpiece of sperm from the caput and corpus epididymidis (Figs. 3, 5), nor are they found at any stage in the principal piece of the sperm (Figs.4, 8). Discussion
Midpiece Fibre Network. An unusual fibre network is found in the midpiece of distal epididymal spermatozoa of Trichosurus vulpecula. This structure was Fig. 6. Longitudinalsectionsof spermatozoafrom the corpus epididymidis.Coincidentterminations of the granular layer and the midpiece fibre network occur anteriorly (arrowed) and posteriorly at the annulus (AN). Note the unusual structure in the acrosomal region (AR). FS fibrous sheath, N nucleus, PN principal piece fibre network, x 20000
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recently described by Olson (1975), and his observations are confirmed here. However, this study extends his observations by showing that the midpiece network develops during epididymal transit, being found in sperm from the corpus epididymidis and more distal regions of the tract, but not in sperm from the caput epididymidis. The present study also notes that the direction of winding of the fibrous helices is always counter to that of the mitochondrial helix. The functional significance of this fibre network is not clear, and while no suggestions were given by Olson (1975), certain of the observations made in the present study deserve comment. The anterior region of the midpiece which lacks fibrous helices, has a rather folded appearance in contrast to the firm appearance of the more distal regions where the helices are present. On this evidence it seems reasonable to suggest that the midpiece fibre network may have a supportive function. In addition, the opposite direction of winding of the fibrous helices to the mitochondrial helix, is of interest when considering the spermatozoon structures which may have potential to modify flagellar movement. No information is available on spermatozoon motility in Trichosurus, but it would be interesting to determine whether any temporal correlation exists between the development of the midpiece fibre network and changes in spermatozoon motility patterns during epididymal transit. Observations on the cytoplasmic droplet will be reported in a subsequent paper, but it is worth noting here, that the midpiece fibre network and vesicles develop after the shedding of the droplet. A midpiece fibre network of the type described here, has not been reported in the sperm of any eutherian species. However, in the midpiece of guinea pig sperm, freeze-fracture techniques reveal circumferential arrays of 6 to 8 nm particles associated with the plasma membrane overlying the mitochondria (Friend and Fawcett, 1974; K oehler and Gaddum-Rosse, 1975). Although these particle strands have a somewhat similar position and orientation to the possum fibrous helices, there are significant differences. The guinea pig particle strands are intramembranous and only about one-tenth the diameter of the possum fibrous helices. Among non-mammalian vertebrates, the spermatozoon of the spiny dogfish, Squalus suckleyi, has a helical sheath of filaments, the fibrous midpiece sheath, closely surrounding the mitochondria (Stanley, 1971). However, neither this fibrous sheath, nor the particle strands in the guinea pig closely resemble the possum midpiece fibre network. Midpiece fibrous helices have not been reported in the sperm of other marsupial species. There are indications in the literature, however, of their presence in at least some species. In published micrographs of Caluromysphilander (Phillips, 1970) and Didelphys marsupialis virginiana (Phillips, 1974) sperm, longitudinal sections suggest that this might be the case, although further investigation would be necessary to determine this with certainty. For Australian marsupials, Cleland (1965) in a brief abstract, reports the presence in the midpiece of an accessory
Fig. 7. Superficial longitudinal section of the midpiece of a spermatozoon from the cauda epididymidis. Note the orientation of the midpiece vesicles (V) with respect to the midpiece fibre network (MN). x 36 000
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Fig. 8. Longitudinal section through the midpiece and anterior principal piece of a spermatozoon from the cauda epididymidis. Midpiece vesicles (V) are now present. Attention is drawn to the contra-rotation of the mitochondrial helix (MT) and helices of the midpiece fibre network (MAr) by the extended arrows. P N principal piece fibre network, x 44000
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Fig. 9. Transverse section of the midpiece of a spermatozoon from the vas deferens. GM layer of granular material; MN midpiece fibre network; PM plasma membrane; Ve midpiece vesicle enclosed within the cytoplasm of the spermatozoon; Vs superficial midpiece vesicle open to the extracellular space, x 81000 Fig. 10. High power view of a superficial midpiece vesicle (Vs) of a spermatozoon from the vas deferens. B Vboundary trilaminar membrane of the midpiece vesicle; GM layer of granular material ; MN fibre of midpiece fibre network; MTmitochondrion; PM plasma membrane, x 210000
p e r i m i t o c h o n d r i a l sheath which is a m o r p h o u s in Perameles nasuta, b u t spiral in the dasyurids, Antechinus a n d Sminthopsis. We are currently investigating the sperm o f a range of A u s t r a l i a n m a r s u p i a l s for the presence of these midpiece fibrous helices. Principal Piece Fibre N e t w o r k . The o b s e r v a t i o n s in this study agree with the recent report of O l s o n (1975) o n the principal piece fibre n e t w o r k in sperm
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of Trichosurus. However, the present study shows also that the network is present in sperm from all regions of the epididymis and mature spermatids in the testis. This is in contrast to the later development of the midpiece fibrous helices, and suggests that the two networks may have separate origins. A similar rather irregular array of fibres has been described in the principal piece of sperm from the American marsupials Caluromys philander (Phillips, 1970) and Didelphys marsupialis virginiana (Holstein, 1965). Sapsford et al., (1970) found a narrow granular accessory sheath to surround the principal piece sheath in the bandicoot spermatozoon. However, this differs from the network in Trichosurus in its regular granular nature and close attachment to the principal piece sheath. At present there is no evidence of the function of the principal piece fibre network. Midpiece Vesicles. The presence of vesicles in the midpiece of the spermatozoon has not been reported for other mammals. A notable finding of this study is that an irregular array of vesicles develops in the midpiece of sperm of T. vulpecula during epididymal transit. The vesicles develop slightly later than the midpiece fibrous helices, appearing in sperm from the cauda but not the corpus epididymidis. It is unusual that Olson (1975) did not report these vesicles in sperm from the cauda epididymidis of T. vulpecula. A possible explanation may lie in the different fixation techniques used. Whilst the function of these vesicles is not apparent, it seems likely that they may be pinocytotic vesicles absorbing some substance from the epididymal fluid. There is no certain evidence of this in the present study, but use of appropriate marking techniques in a later study should enable this to be determined. The possibility of such an absorptive function for these vesicles is interesting in view of the increasing knowledge of the secretory functions of the eutherian epididymis, and of changes in the eutherian spermatozoon during epididymal transit (Orgebin-Crist, 1969; White, 1973; Bedford, 1974). The possibility of a relationship between these vesicles and the midpiece fibre network and granular layer is suggested by the coincidence of their extent in the midpiece. Of course it is equally possible that their simultaneous anterior termination may be caused by another factor which is not as yet apparent. If a relationship does exist, then it is difficult to suggest what this might be. Perhaps the fibrous helices make some structural compensation for alteration in the sperm surface caused by the vesicles.
References Bedford, J.M. : Report of a workshop : Maturation of the fertilizing ability of mammalian spermatozoa in the male and female reproductive tract. Biol. Reprod. II, 346 362 (1974) Bedford, J.M., Calvin, H.I. : Changes in -S-S- linked structures of the sperm tail during epididymal maturation, with comparative observations in sub-mammalian species. J. exp. Zool. 187, 181-204 (1974) Biggers, J.D. : Reproduction in male marsupials, Symp. zool. Soc. Lond. 15, 251-280 (1966) Calvin, H.I., Bedford, J.M. : Formation of disulphide bonds in the nucleus and accessory structures
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of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Fertil. Suppl. 13, 65 75 (1971) Cleland, K.W.: Electron microscopic structure of the Dasyuroid sperm. J. Anat. (Lond.) 99, 953 (Abstract) (1965) Cleland, K.W., Lord Rothschild: The bandicoot spermatozoon: and electron microscope study of the tail. Proc. roy. Soc. B 150, 2 4 4 2 (1959) Fawcett, D.W. : A comparative view of sperm ultrastructure. Biol. Reprod., Suppl. 2, 90-127 (1970) Friend, D.S., Fawcett, D.W.: Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol. 63, 641-664 (1974) Holstein, A.F. : Elektronenmikroskopische Untersuchungen am Spermatozoon des Opossum (Didelphys virginiana Kerr). Z. Zellforsch. 65, 904--914 (1965) Koehler, J.K., Gaddum-Rosse, P. : Media induced alterations of the membrane associated particles of the guinea pig sperm tail. J. Ultrastruct. Res. 51,106-118 (1975) Olson, G.: Observations on the ultrastructure of a fiber network in the flagellum of sperm of the Brush tailed Phalanger, Trichosurus vulpecula. J. Ultrastruct. Res. 50, 193-198 (1975) Orgebin-Crist, M.C.: Studies on the function of the epididymis. Biol. Reprod. Suppl. I, 155-175 (1969) Phillips, D.M. : Ultrastructure of spermatozoa of the Woolly Opossum Caluromys philander. J. Ultrastruct. Res. 33, 381 397 (1970) Phillips, D.M. : Spermiogenesis. Academic Press: New York 1974 Sapsford, C.S., Rae, C.A. : Ultrastructural studies on Sertoli cells and spermatids in the bandicoot and ram during the movement of mature spennatids into the lumen of the seminiferous tubule. Aust. J. Zool. 17, 415445 (1969) Sapsford, C.S., Rae, C.A., Cleland, K.W.: Ultrastructural studies on maturing spermatids and on Sertoli cells in the bandicoot Perameles nasuta Geoffroy (Marsupialia). Aust. J. Zool. 17, 195- 292 (1969) Sapsford, C.S., Rae, C.A., Cleland, K.W.: Ultrastructural studies on the development and form of the principal piece sheath of the bandicoot spermatozoon. Aust. J. Zool. 18, 2 1 4 8 (1970) Stanley, H.P.: Fine structure of spermiogenesis in the Elasmobranch fish Squalus suckleyi. II. Late stages of differentiation and structure of the mature spermatozoon. J. Ultrastruct. Res. 36, 103-118 (1971) White, I.G.: Biochemical aspects of spermatozoa and their environment in the male reproductive tract. J. Reprod. Fertil., Suppl. 18, 225 235 (1973)
Receired August 18, 1975
