- Email: [email protected]
Structural specializations in the flagellar plasma membrane of opossum spermatozoa
JOURNAL OF ULTRASTRUCTURE RESEARCH 59, 207-221 (1977)
Structural Specializations in the Flagellar Plasma Membrane of Opossum Spermatozoa 1 GARY E. OLSON, 2 MIRIAM LIFSICS, DON W. FAWCETT, AND DAVID W. HAMILTON
Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115 Received December 14, 1976 A correlated thin section, freeze fracture, and scanning electron microscopic study has been made of membrane specializations in the flagellum of opossum spermatozoa. In flagellar cross sections, the plasma membrane over most of the midpiece has a regularly scalloped contour. Scanning microscopy shows that the scallops course parallel to the long axis of the flagellum as nearly parallel ridges. A mat of amorphous or finely filamentous material 25-30 nm thick is applied against the cytoplasmic surface of the plasma membrane wherever the membrane is in the scalloped configuration. In freeze fracture replicas the intramembranous particles in the .midpiece are found closely aggregated into rows several particles wide which correspond in spacing to the scallops seen in thin sections. Optical diffraction of freeze fracture replicas shows that the particles within the rows are packed in a highly ordered two dimensional lattice. The membrane between rows is relatively particle free. The linear aggregations of intramembranous particles extend distally to the annulus and terminate. Over specific portions of the annulus, where the plasma membrane is closely associated with the underlying dense matrix, the intramembranous particles are densely packed. The plasma membrane of the principal piece shows neither scalloping nor any undercoat on its cytoplasmic surface. Freeze fracture replicas show a longitudinal differentiation in the membrane of the principal piece which is generally composed of a double row of particles but which in some regions may be 5-6 particles in width.
tively charged colloidal iron (5, 29) to the sperm membrane reveals differences in charge and in the abundance of specific carbohydrate groups over different regions of the sperm surface. In the few species whose sperm have been studied by freeze-fracturing, distinctive patterns of the membrane intercalated particles have been demonstrated over the acrosome, the postacrosomal sheath and even in different regions of the flagellum (8-10, 16, 17, 25). In the midpiece of the guinea pig sperm flagellum the intramembranous particles are associated in linear arrays like strings of beads coursing circumferentially (10, 17). In the corresponding region of the mouse spermatozoon particles may be aggregated into local paracrystalline arrays (25). A spe' Research supported by USPHS Grants HD-04290, cialization of more general occurrence has HD-08258, and HD-02344. 2 NIH Postdoctoral Fellow. Present address: De- been described in the principal piece. It consists of two closely associated rows of partment of Anatomy, Vanderbilt University Medical School, Nashville, Tenn. 37232. particles running longitudinally in the
Regional specialization of the sperm surface for specific functions is reflected in local differences in behavior, in chemical composition and in internal structure of the plasma membrane. For example, in those species where epididymal sperm associate either in pairs or in extensive rouleaux, the membrane overlying the acrosome is invariably the site of cell-to-cell adherence (7, 8, 10, 23). Moreover, during the acrosome reaction only the membrane overlying the acrosome undergoes vesiculation and fusion with the acrosomal membrane (2, 7, 8, 28). On the other hand, the initial events in gamete fusion during fertilization appear to involve only the postacrosomal region of the head membrane (1, 7, 8). The binding of lectins (20) and posi-
207 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
OLSON ET AL.
208
membrane overlying dense fiber number one. T h i s s p e c i a l i z a t i o n , s o m e t i m e s r e f e r r e d to a s t h e z i p p e r , b e g i n s a t t h e a n n u l u s a n d e x t e n d s c a u d a l l y for t h e g r e a t e r p a r t o f t h e l e n g t h o f t h e p r i n c i p a l p i e c e (8, 10, 25). T h i s s p e c i a l i z a t i o n a p p e a r s to b e quite stable, whereas the circumferential beaded strands dissociate into a random pattern of i n t r a m e m b r a n o u s particles u p o n e x p o s u r e to h y p o t o n i c s o l u t i o n s o r environmental conditions favoring sperm c a p a c i t a t i o n (11, 18). T h e t r u e f u n c t i o n a l significance of these ordered particle arrays in the sperm flagellar membrane rem a i n s to b e d e t e r m i n e d . Previous transmission electron microscope s t u d i e s of m a r s u p i a l s p e r m a t o z o a h a v e d r a w n a t t e n t i o n to u n u s u a l s u r f a c e p a t t e r n s i n t h e f l a g e l l a r m e m b r a n e (7, 21 23) b u t t h e s e h a v e n o t b e e n s t u d i e d h e r e t o fore by the freeze-fracturing technique. T h e p r e s e n t i n v e s t i g a t i o n is c o n c e r n e d with specializations of the flagellar membrane of spermatozoa from the American o p o s s u m , Didelphis virginiana. D i s t i n c tive longitudinal ridges observed on the midpiece in thin sections and scanning electron micrographs are found in freezef r a c t u r e p r e p a r a t i o n s to b e r e l a t e d to a n unusual ordered arrangement of intramembranous particles. MATERIALS AND METHODS
Thin Sections Mature male opossums, Didelphis virginiana, were anesthetized with ether .and the epididymis and testis were removed. The epididymis was subdivided into four distinct regions; the caput, proximal corpus, distal corpus, and cauda. The tissue was fixed in 4% glutaraldehyde buffered with 0.2 M scollidine (pH 7.2) for 2-4 hr at 4°C. After 2 buffer washes the tissue was postfixed in 1% OsO4 buffered with 0.2 M s-collidine, dehydrated through an ethanol series and propylene oxide and embedded in Epon 812. Thin sections cut on an LKB-Huxley ultramicrotome were stained with uranyl acetate and lead citrate and examined in a JEOL 100S electron microscope at 80 kV.
Scanning Electron Microscopy Sperm were gently squeezed from minced epididymal tubules into a phosphate buffered saline solu-
tion (0.145 M NaC1, 0.01 M Na2HPOJNaH2PO4, pH 7.0). An equal volume of the 4% glutaraldehyde/ collidine fixative was immediately added to the sperm suspension. A few drops of the suspension were placed on a glass coverslip which had previously been coated with poly-L-lysine. The sperm adhering to the coverslip were washed in buffer, postfixed with 1% OSO4, dehydrated through ethanol and amyl acetate and either air dried or critical point dried from liquid CO2. Fragments of the coverslip were attached to metal stubs and coated with palladium-gold in a Technics Hummer II. The specimens were examined at 25 kV in a JEOL JSM 35S scanning electron microscope.
Freeze Fracture Sperm were fixed for 30 min in a mixture of 2% glutaraldehyde and 2.5% paraformaldehyde in 0.2 M cacodylate buffer for 0.5 hr. The sperm were washed in buffer and suspended in buffer containing 20% glycerol. Concentrated .sperm suspensions were mounted on gold discs and frozen in Freon 22. The specimens were then freeze fractured by standard techniques in a Balzers BAF 301 freeze fracture unit.
Optical Diffraction and Image Processing Image processing was performed with an optical diffractometer constructed on a 6 m double rail optical bench (Ealing Corp.) and equipped with a 3 mW helium-neon laser (Spectra Physics). The technique employed was basically that of Klug and De Rosier (15). Individual areas on electron microscope plates were masked with black tape along the boundaries of area to be diffracted; the specimens were scanned in an optical diffractometer and optical transforms were photographically recorded. For optical filtering a diffraction mask was made from recorded transforms; in some instances a 400 mesh copper screen with a light transmittance of 30% was placed over the origin of the mask. The mask was then placed at the diffraction plane on the optical bench and the resulting filtered image was photographically recorded. RESULTS
:"
The head of the opossum spermatozoon is V - s h a p e d w i t h o n e a r m of t h e V s o m e what larger than the other. In the case of s i n g l e s p e r m , t h e h e a d is a t a r i g h t a n g l e to t h e a x i s o f t h e t a i l . T h e a c r o s o m e is l i m i t e d to t h e s u r f a c e o f o n e w i n g o f t h e V and the tail inserts in a deep implantation fossa on the same wing a short distance f r o m t h e a p e x ( F i g s : 1 a n d 2). S p e r m a t o z o a f r o m t h e c a u d a e p i d i d y m i d i s of t h e opos-
OPOSSUM SPERM MEMBRANE SPECIALIZATION
209
FIG. 1. Longitudinal section of sperm head in which nucleus has an asymmetric V-shaped profile. The connecting piece (CP) is associated with an extensive membranous network, The posterior ring (PR) is located at the lateral margin of the nucleus. Distal to the posterior ring the plasma membrane covering the midpiece possesses a prominent fuzzy coat on its cytoplasmic surface (arrows). x 23 000. FIG. 2. High magnification view of spermatozoan neck region. The long connecting piece (CP) inserts into a hemisphere-shaped implantation fossa (I). A dense layer of amorphous material is applied against the outer nuclear membrane in the implantation fossa. At its anterior end the connecting piece consists of two distinct materials and more distally the proximal centriole is embedded in the connecting piece matrix. A crystall0id (C) is present in the matrix of the membranous network, x 42 000.
210
OLSON E T A L .
sum are typically conjoined in pairs with the two members adherent along their apposed acrosomal surfaces. To achieve this relationship the V-shaped heads of two sperm are rotated to bring the apex of the two V's anterior and their acrosomal surfaces into apposition. The complex formed by the conjoined heads then is shaped like a trident with the center tine composed of the two adherent acrosomal arms. The apex is forward and the diverging free arms directed posteriorly. The tails insert by slender curved connecting pieces into the implantation fossa which is located on the major wing of the sperm head. A thin coating of amorphous material lines the spherical implantation fossa and the small round capitulum of the connecting piece articulates within the implantation fossa in a ball and socket relationship (Fig. 2). The capitulum of the connecting piece is composed of two distinct components: an electron dense, amorphous layer forms the articular element which inserts into the implantation fossa (Fig. 2) and a homogeneous less electron dense material forms the bulk of the connecting piece and is continuous with the striated columns (Fig. 2). A mass of concentric membranes organized around a crystalloid is also found between the diverging wings or lobes of the nucleus and m a y be analogous to the membranous scrolls found in some other species (Figs. 1 and 2). Throughout most of the length of the midpiece the plasma membrane is remarkably specialized in structure. A 25-30 nm thick undercoat of fuzzy, dense material is found on the cytoplasmic surface of the membrane which has a regularly scalloped appearance (Fig. 3). The amorphous material coating the inner aspect of the plasma membrane also appears as a continuous layer of uniform thickness in longitudinal sections of the midpiece (Fig. 4). Scanning electron micrographs reveal numerous parallel ridges on the midpiece which run roughly parallel to the flagellar long axis (Fig. 5). These ridges terminate at the an-
nulus (Fig. 5). The ridges seen in scanning electron microscopy obviously correspond to the scallops of the plasma membrane observed in cross sections of the midpiece. Replicas of freeze fractured cauda epididymal spermatozoa show a striking organization of the intramembranous particles in the flagellar midpiece (Figs. 6, 7, 13). The particles cluster into roughly parallel rows 30-50 n m in width, which extend throughout much of the midpiece. Generally each major row is three particles wide but occasionally they m a y be up to five particles wide (Figs. 6, 7, 13). The individual particles average 6-8 n m in diameter. Not all rows are of equal length for a few abruptly terminate along the midpiece (Figs. 6, 7, 13). The membrane between rows is 30-50 n m wide and is usually devoid of particles. Toward the anterior end of the midpiece the particle rows abruptly terminate; the membrane area anterior to the termination of the rows of particles corresponds to the plasma membrane covering the nucleus and contains a sparse population of randomly distributed particles (Fig. 6). Distally the organized particle rows extend to the annulus and terminate (Fig. 13). Within the major rows the intramembranous particles appear to aggregate into a highly ordered two-dimensional lattice. Diffraction patterns of individual rows show a single prominent layer line indexing between 16-18 n m -1 (Figs. 8-10). Axially the particles are aligned into subrows like strings of beads and as determined from diffraction patterns and reconstructed images the particles exhibit a center to center spacing of 16-18 nm (Figs. 810). The three neighboring subrows are slightly staggered with respect to each other so that a line between adjacent particles in neighboring subrows is inclined at about 60° with respect to the long axis of the rows (Figs. 8-10). The plasma membrane retains its scalloped configuration over a portion of the annulus which is located at the midpiece-
211
OPOSSUM SPERM MEMBRANE SPECIALIZATION
¸%¸¸,¸¸/~
J
J
c
%.
/ \
J
/
/
/
® FIG. 3. Cross section, through distal region of midpiece of two paired spermatozoa. The plasma membrane has a regular scalloped appearance as well as a submembranous coating of amorphous material (arrows). × 43 000.
/N
FIG. 4. Longitudinal section through sperm midpiece. The m e m b r a n e scallops seen in cross section are not obvious but the m a t e r i a l coating the cytoplasmic face of the plasma m e m b r a n e appears as a continuous layer along the flagellar long axis (arrows). x 64 000. FIG. 5. S c a n n i n g electron micrograph showing a segment of the midpiece and principal piece. The midpiece is characterized by the presence of longitudinally oriented, parallel ridges which extend to the a n n u l u s (A). No ridges are seen on the principal piece, x 42 000. 212
OPOSSUM SPERM MEMBRANE SPECIALIZATION
213
FIG. 6. Freeze fracture replica showing anterior region of sperm midpiece. In the region of the midpiece characterized by the presence of m e m b r a n e scallops replicas reveal t h a t the i n t r a m e m b r a n o u s particles are aggregated into n u m e r o u s parallel rows, each 3-5 particles wide. The particle rows t e r m i n a t e at the anterior end of the midpiece; the plasma m e m b r a n e anterior to the t e r m i n a t i o n of the particle rows overlies the nucleus a n d is characterized by a r a n d o m particle distribution × 72 000.
FIG. 7. Freeze fracture replica showing the intramembranous particle a r r a n g e m e n t s in the sperm midpiece. Note t h a t some rows show an abrupt termination (arrows) and t h a t the membrane between adjacent rows is relatively particle free. x 78 000. 214
OPOSSUM SPERM MEMBRANE SPECIALIZATION
215
Fro. 8. High magnification view of one of the particle rows from sperm midpiece region. The 6-8 nm diameter particles are packed into a two-dimensional lattice. Fro. 9. Diffraction pattern arising from one particle row. The prominent layer line indexes at 16-18 nm -~ and shows two prominent off-meridional reflections and a single discrete reflection near the meridian. The size of the two large off-meridional reflections compared to the discrete meridional reflection suggests that long-range disruptions in the particle lattice arise predominantly from dislocations between adjacent particle subrows. FIa. 10. hnage obtained by layer line filtering diffraction pattern in Fig. 14. In the reconstruction zero order intensity was reduced with a 30% transmittance screen placed over the origin of the mask. The lattice of the particles is clearly shown. principal piece b o u n d a r y (Fig. 12). The ann u l u s consists of a n extensive circumferential sheet of fibrous m a t e r i a l positioned u n d e r t h e last g y r e of t h e midpiece mitochondrial s h e a t h (Figs. 11 and 12). As this sheet of fibrous m a t e r i a l approaches the p l a s m a m e m b r a n e it bifurcates into a Yshaped configuration (Fig. 11). T h e p l a s m a m e m b r a n e involutes rostrally at the bifurcation of the a n n u l u s and is closely associated w i t h the u n d e r l y i n g a n n u l a r m a t r i x (Fig. 12). Thus, in cross sections t h r o u g h the a n n u l u s t h r e e views of the p l a s m a m e m b r a n e are a p p a r e n t (Fig. 12). T h e oute r m o s t profile of the p l a s m a m e m b r a n e is scalloped like the m e m b r a n e o v e r l y i n g the midpiece and it is not i n t i m a t e l y associ-
ated with the u n d e r l y i n g dense m a t e r i a l of the annulus; however, the i n n e r two profiles of the p l a s m a m e m b r a n e , w h e r e the p l a s m a m e m b r a n e follows the bifurcation of the a n n u l u s , are not scalloped and the m e m b r a n e is closely associated with the u n d e r l y i n g dense m a t e r i a l composing the a n n u l u s (Fig. 12). Freeze f r a c t u r e replicas of the a n n u l u s show t h a t the rows of particles in the midpiece t e r m i n a t e w h e r e the p l a s m a mereb r a n e reflects rostrally to follow the underlying bifurcation of the a n n u l u s (Fig. 13). The p l a s m a m e m b r a n e is r e l a t i v e l y particle free at the point of its rostral reflection b u t t h e r e appears to be a n a b u n d a n c e of particles in the region w h e r e the p l a s m a
FIG. 11. Longitudinal section showing the plate-like a n n u l u s (An); note t h a t it bifurcates as it approaches the plasma membrane. The m e m b r a n e involutes rostrally to foliow the bifurcation, x 66 000. FIG. 12. Cross section through the a n n u l u s reveals three views of plasma membrane. The outermost m e m b r a n e profile is in the scalloped configuration(s) characteristic of the midpiece. The other 2 profiles of the plasma m e m b r a n e are closely associated with the underlying m a t r i x of the a n n u l u s (arrows). x 60 000. 216
OPOSSUM SPERM MEMBRANE SPECIALIZATION
membrane involutes and is most closely associated with the underlying dense matrix of the annulus (Fig. 13). The plasma membrane shows no specialized configuration in the principal piece region and there is no mat of fuzzy material associated with the cytoplasmic surface of the plasma membrane (Fig. 11). There is a network of fibrillar material surrounding the ribs of the fibrous sheath but it does not appear to preferentially associate with the plasma membrane (Fig. 11)o This fibrillar network is confined to the anteriormost portion of the principal piece. Most intramembranous particles in the principal piece region show a random arrangement (Figs. 14-16). However, the ~zipper," the one structural specialization of the principal piece plasma membrane seen in sperm of other species, is present and extends throughout much of the length of the principal piece (Fig. 14). The zipper has a rather striking heterogeneity with respect to particle arrangement. Most often it consists of only a double row of particles, but sometimes it is 5 to 6 particles wide (Figs. 14-16). Discontinuities in the zipper are frequently seen but they are generally short and the zipper continues after the short gap (Fig. 14). DISCUSSION
Freeze fracture studies have demonstrated considerable heterogeneity over the sperm surface in the size, arrangement, and relative density of intramembrane particles. In different species a variety of ordered particle arrays have been found in the membranes associated with the sperm head (8, 10, 16, 17, 25), but with the exception of mouse and guinea pig sperm (10, 17, 25) previous studies have found a random particle array in the midpiece plasma membrane. In the mouse sperm midpiece small patches of ordered particle arrays are occasionally found (25) but in the guinea pig midpiece the particles associate into linear arrays which wrap circumferentially about the flagel-
217
lum and precisely overlie the gyres of the mitochondrial sheath (10, 17). Although the organized intramembrane particle rows and mitochondria do not show this precise structural interrelationship in opossum sperm, the particle rows are only found in the plasma membrane which directly overlies the mitochondrial sheath suggesting a functional relationship between the plasma membrane and mitochondrial energy generating apparatus. Since organized particle arrays are not universally found in the midpiece of sperm of different species the functional significance of the relationship remains obscure. The rows of particles revealed in freeze fracture replicas of Didelphis sperm are only seen in regions of the midpiece that in thin sections or scanning electron microscopy display a scalloped plasma membrane configuration. Although there is a direct correspondence between the spacing of the particle rows and the scallops, the precise localization of the particle rows within or between adjacent scallops remains to be elucidated. Several different mechanisms could be responsible for aggregating the particles into the ordered rows: for example, factors in the luminal fluid of the epididymis acting of the cell surface could induce particle aggregation via interaction with surface receptors or by altering the configuration of the intrinsic membrane proteins so that aggregation is favored. Also, peripheral components acting at the cytoplasmic face of the membrane could influence particle aggregation. On the basis of the morphological evidence a correlation clearly exists between the presence of the submembranous coat on the cytoplasmic face of the membrane and the presence of membrane scallops. At the anterior limits of the midpiece there is no prominent fuzzy coating on the cytoplasmic face of the membrane and the membrane is not scalloped; freeze fracture replicas further show that this anterior portion of the midpiece is characterized by a random particle array. In addition, the
FIG. 13. Freeze fracture replica showing annulus. The midpiece particle arrays t e r m i n a t e at the a n n u l u s (A). A scarcity of particles is seen where the plasma m e m b r a n e folds back to follow the underlying m a t r i x of the a n n u l u s b u t there appears to be a n abundance of particles where the plasma m e m b r a n e is closely associated with the a n n u l u s (arrows). × 71 000. 218
OPOSSUM SPERM MEMBRANE SPECIALIZATION
219
FIas. 14-16. Freeze fracture replicas of principal piece region showing the "zipper" which consists of a linear a r r a y of particles. Frequently the zipper consists of only a double row of particles b u t occasionally it is up to 6-7 particles wide. Short discontinuities in the zipper are often seen (arrows). Fig. 14, x 42 000; Fig. 15, x 60 000; Fig. 16,.x 60 000.
220
OLSON ET AL.
midpiece plasma membrane of immature caput epididymal spermatozoa is not scalloped and the submembranous coating is lacking (unpublished data); whether or not the intramembrane particles of immature sperm are organized into regular rows remains to be determined. Similar maturational changes appear to take place in sperm of other marsupial species as neither the plasma membrane associated accessory fibers of phalanger sperm (13, 26) nor the membrane scallops of Wooley opossum sperm (22) are observed in sperm from the initial portion of the epididymis. Although a direct structural contact between the intramembranous particles revealed in freeze fracture replicas of Didelphis spermatozoa and the material coating the cytoplasmic face of the membrane seen in thin sections remains to be demonstrated, it is suggested that the material coating the inner aspect of the membrane may play a functional role in organizing and/or anchoring the intramembrane particles into the ordered arrays observed in freeze fracture replicas. Such transmembrane control has been demonstrated in the red cell membrane where the peripheral protein spectrin appears to be capable of influencing the mobility of intramembranous particles as well as the distribution of external cell surface glycoproteins (6, 19). In Didelphis sperm the organized rows of particles are only present in the midpiece and do not extend distally beyond the annulus. Nonetheless, distinctive particle accumulations are present within restricted areas of the plasma membrane overlying the annulus. The peculiar morphology of the annulus of opossum spermatozoa has been documented in previous thin section studies (5, 14, 22, 23). The particle rows present in the midpiece terminate as the plasma membrane folds back rostrally to follow the bifurcation of the underlying annulus; replicas further show that at the point of folding, the plasma membrane is relatively particle
free. However, within the bifurcation of the annulus the plasma membrane is closely associated with the underlying dense matrix which forms the annulus, and in replicas the overlying membrane clearly shows a high particle density suggesting a structural relationship between the annulus and overlying plasma membrane. Similar densely packed arrays of particles over the annulus have been seen in replicas of sperm of other species (8,10, 18, 25). The linear array of particles seen in replicas is the most striking membrane differentiation of the principal piece. A double row of particles termed the ~zipper" has been observed in replicas of the principal piece of guinea pig, rat, and mouse spermatozoa (5,10,18, 25) and a similar particle array has been shown in the flagellum of earthworm spermatozoa (3). In Didelphis spermatozoa the zipper consists primarily of a double row of particles, but it appears to be more irregular than the zipper of rat, guinea pig, or mouse sperm as it shows frequent discontinuities and short stretches where it is up to 5-6 particles wide. The striking and different particle arrangements in the midpiece and principal piece membranes presumably reflect underlying functional differences in these two membranes. Intramembranous particles have been implicated in a variety of transmembrane phenomena. For example, in the red cell there appears to be a structural relationship between the intramembrane particles and some surface glycoproteins (27) and since some membrane glycoproteins span the membrane (4) it has been suggested that the intramembrane particles revealed by freeze fracture represent hydrophobic segments of the glycoprotein. A correspondence between the presence of intramembrane particles and external glycoprotein coat is not obligatory though, as in some cells particle free membrane regions possess a prominent surface coat (24). A spatial relationship between
OPOSSUM SPERM MEMBRANE SPECIALIZATION
the disposition of externally disposed surface components and the intramembrane particles in the spermatozoan flagellar membrane remains to be demonstrated. Available data indicates that changes in both the organization of the intramembrane particles as well as the disposition and abundance of the plasma membrane glycoprotein coat may accompany certain physiological changes in spermatozoa. During capacitation rabbit spermatozoa show a reduction in concanavalin A binding sites on the flagellar membrane (12), and during incubation under capacitating conditions the midpiece plasma membrane of guinea pig sperm loses the organized strands of intramembrane particles (11, 18). Whether or not similar changes in membrane architecture occur in opossum sperm during capacitation is under investigation However, the elaborate structural specializations present in the midpiece plasma membrane should permit visual assessment of structural modifications occurring at the inner and outer membrane surface to be correlated with changes in the arrangement of intramembrane particles revealed by freeze fracture. REFERENCES 1. BARROS, C., AND FRANKLIN, L. E., J. Cell Biol. 37, C13 (1968). 2. BARROS, C., BEDFORD, J. M., FRANKLIN, L. E., AND AUSTIN, C. R., J. Cell Biol. 34, C1 (1967). 3. BERGSTROM, B. B., AND HENLEY, C., J. Ultrastruct. Res. 42, 551 (1973). 4. BRETSHER,M. S., J. Mol. Biol. 59, 351 (1971). 5. COOPER,G. W., AND BEDFORD, J. M., Anat. Rec. 169, 300 (1971).
221
6. ELGSAETER,A., AND BRANTON, D., J. Cell Biol. 63, 1018 (1974). 7. FAWCETT, D. W., Biol. Reprod. Suppl. 2, 90 (1970). 8. FAWCETT,D. W., Develop. Biol. 44,394 (1975). 9. FLECHON, J. E., J. Microsc. 19, 59 (1975). 10. FRIEND, D. S., AND FAWCETT, D. W., J. Cell Biol. 63, 641 (1974). 11. FRIEND, D. S., AND RUDOLF,I., J. Cell Biol. 63, 466 (1974). 12. GORDON,M., DANDEKAR,P. V., AND RARTOSZEwIcz, W., J. Reprod. Fert. 36, 211 (1974). 13. HARDING,H. R., CARRICK,F. N., AND SHOREY, C. D., Cell Tissue Res. 164, 121 (1975). 14. HOLSTEIN,A. F., Z. ZeUforsch, 65,904 (1965). 15. KLUG, A. AND DE ROSIER, D. J., Nature (London) 212, 29 (1966). 16. KOEHLER, J. K., J. Ultrastruct. Res. 33, 598 (1970). 17. KOEHLER, J. K., J. Microsc. 18, 263 (1973). 18. KOEHLER, J. K., AND GADDUM-ROSSE,P., J. Ultrastruct. Res. 51, 106 (1975). 19. NICOLSON, G. L., AND PAINTER, R. G., J. Cell Biol. 59, 395 (1973). 20. NICOLSON, G. L., AND YANAGAMACHI,R., Science 184, 1294 (1974). 21. OLSON, G., J. Ultrastruct. Res. 50, 193 (1975). 22. OLSON, G. E., AND HAMILTON,D. W . , A n a t . Rec. 186, 387 (1976). 23. PHILLIPS, D. M., J. Ultrastruct. Res. 33, 381 (1970). 24. PINTODA SILVA,P., MARTINEZ-PALOMO,A., AND GONZALEZ-ROBLES, A., J. Cell Biol. 64, 538 (1975). 25. STACKPOLE,C. W., AND DEVORKIN,D., J. Ultrastruct. Res. 49, 167 (1974). 26. TEMPLE-SMITH, P. D., AND BEDFORD, J. M., Anat. Rec. 184, 545 (Abstr.) (1976). 26. TILLACK,T. W., SCOTT,R. E., AND MARCHESI,V. T., J. Exp. Med. 135, 1209 (1972). 27. YANAGIMACHI,R., AND NODA, Y. D., J. Ultrastruct. Res. 31," 465 (1970). 28. YANAGIMACHI,R., NODA, Y. D., FUJIMOTO, M., AND NICOLSON, G. L., Amer. J. Anat. 135, 497 (1972).
Structural Specializations in the Flagellar Plasma Membrane of Opossum Spermatozoa 1 GARY E. OLSON, 2 MIRIAM LIFSICS, DON W. FAWCETT, AND DAVID W. HAMILTON
Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115 Received December 14, 1976 A correlated thin section, freeze fracture, and scanning electron microscopic study has been made of membrane specializations in the flagellum of opossum spermatozoa. In flagellar cross sections, the plasma membrane over most of the midpiece has a regularly scalloped contour. Scanning microscopy shows that the scallops course parallel to the long axis of the flagellum as nearly parallel ridges. A mat of amorphous or finely filamentous material 25-30 nm thick is applied against the cytoplasmic surface of the plasma membrane wherever the membrane is in the scalloped configuration. In freeze fracture replicas the intramembranous particles in the .midpiece are found closely aggregated into rows several particles wide which correspond in spacing to the scallops seen in thin sections. Optical diffraction of freeze fracture replicas shows that the particles within the rows are packed in a highly ordered two dimensional lattice. The membrane between rows is relatively particle free. The linear aggregations of intramembranous particles extend distally to the annulus and terminate. Over specific portions of the annulus, where the plasma membrane is closely associated with the underlying dense matrix, the intramembranous particles are densely packed. The plasma membrane of the principal piece shows neither scalloping nor any undercoat on its cytoplasmic surface. Freeze fracture replicas show a longitudinal differentiation in the membrane of the principal piece which is generally composed of a double row of particles but which in some regions may be 5-6 particles in width.
tively charged colloidal iron (5, 29) to the sperm membrane reveals differences in charge and in the abundance of specific carbohydrate groups over different regions of the sperm surface. In the few species whose sperm have been studied by freeze-fracturing, distinctive patterns of the membrane intercalated particles have been demonstrated over the acrosome, the postacrosomal sheath and even in different regions of the flagellum (8-10, 16, 17, 25). In the midpiece of the guinea pig sperm flagellum the intramembranous particles are associated in linear arrays like strings of beads coursing circumferentially (10, 17). In the corresponding region of the mouse spermatozoon particles may be aggregated into local paracrystalline arrays (25). A spe' Research supported by USPHS Grants HD-04290, cialization of more general occurrence has HD-08258, and HD-02344. 2 NIH Postdoctoral Fellow. Present address: De- been described in the principal piece. It consists of two closely associated rows of partment of Anatomy, Vanderbilt University Medical School, Nashville, Tenn. 37232. particles running longitudinally in the
Regional specialization of the sperm surface for specific functions is reflected in local differences in behavior, in chemical composition and in internal structure of the plasma membrane. For example, in those species where epididymal sperm associate either in pairs or in extensive rouleaux, the membrane overlying the acrosome is invariably the site of cell-to-cell adherence (7, 8, 10, 23). Moreover, during the acrosome reaction only the membrane overlying the acrosome undergoes vesiculation and fusion with the acrosomal membrane (2, 7, 8, 28). On the other hand, the initial events in gamete fusion during fertilization appear to involve only the postacrosomal region of the head membrane (1, 7, 8). The binding of lectins (20) and posi-
207 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
OLSON ET AL.
208
membrane overlying dense fiber number one. T h i s s p e c i a l i z a t i o n , s o m e t i m e s r e f e r r e d to a s t h e z i p p e r , b e g i n s a t t h e a n n u l u s a n d e x t e n d s c a u d a l l y for t h e g r e a t e r p a r t o f t h e l e n g t h o f t h e p r i n c i p a l p i e c e (8, 10, 25). T h i s s p e c i a l i z a t i o n a p p e a r s to b e quite stable, whereas the circumferential beaded strands dissociate into a random pattern of i n t r a m e m b r a n o u s particles u p o n e x p o s u r e to h y p o t o n i c s o l u t i o n s o r environmental conditions favoring sperm c a p a c i t a t i o n (11, 18). T h e t r u e f u n c t i o n a l significance of these ordered particle arrays in the sperm flagellar membrane rem a i n s to b e d e t e r m i n e d . Previous transmission electron microscope s t u d i e s of m a r s u p i a l s p e r m a t o z o a h a v e d r a w n a t t e n t i o n to u n u s u a l s u r f a c e p a t t e r n s i n t h e f l a g e l l a r m e m b r a n e (7, 21 23) b u t t h e s e h a v e n o t b e e n s t u d i e d h e r e t o fore by the freeze-fracturing technique. T h e p r e s e n t i n v e s t i g a t i o n is c o n c e r n e d with specializations of the flagellar membrane of spermatozoa from the American o p o s s u m , Didelphis virginiana. D i s t i n c tive longitudinal ridges observed on the midpiece in thin sections and scanning electron micrographs are found in freezef r a c t u r e p r e p a r a t i o n s to b e r e l a t e d to a n unusual ordered arrangement of intramembranous particles. MATERIALS AND METHODS
Thin Sections Mature male opossums, Didelphis virginiana, were anesthetized with ether .and the epididymis and testis were removed. The epididymis was subdivided into four distinct regions; the caput, proximal corpus, distal corpus, and cauda. The tissue was fixed in 4% glutaraldehyde buffered with 0.2 M scollidine (pH 7.2) for 2-4 hr at 4°C. After 2 buffer washes the tissue was postfixed in 1% OsO4 buffered with 0.2 M s-collidine, dehydrated through an ethanol series and propylene oxide and embedded in Epon 812. Thin sections cut on an LKB-Huxley ultramicrotome were stained with uranyl acetate and lead citrate and examined in a JEOL 100S electron microscope at 80 kV.
Scanning Electron Microscopy Sperm were gently squeezed from minced epididymal tubules into a phosphate buffered saline solu-
tion (0.145 M NaC1, 0.01 M Na2HPOJNaH2PO4, pH 7.0). An equal volume of the 4% glutaraldehyde/ collidine fixative was immediately added to the sperm suspension. A few drops of the suspension were placed on a glass coverslip which had previously been coated with poly-L-lysine. The sperm adhering to the coverslip were washed in buffer, postfixed with 1% OSO4, dehydrated through ethanol and amyl acetate and either air dried or critical point dried from liquid CO2. Fragments of the coverslip were attached to metal stubs and coated with palladium-gold in a Technics Hummer II. The specimens were examined at 25 kV in a JEOL JSM 35S scanning electron microscope.
Freeze Fracture Sperm were fixed for 30 min in a mixture of 2% glutaraldehyde and 2.5% paraformaldehyde in 0.2 M cacodylate buffer for 0.5 hr. The sperm were washed in buffer and suspended in buffer containing 20% glycerol. Concentrated .sperm suspensions were mounted on gold discs and frozen in Freon 22. The specimens were then freeze fractured by standard techniques in a Balzers BAF 301 freeze fracture unit.
Optical Diffraction and Image Processing Image processing was performed with an optical diffractometer constructed on a 6 m double rail optical bench (Ealing Corp.) and equipped with a 3 mW helium-neon laser (Spectra Physics). The technique employed was basically that of Klug and De Rosier (15). Individual areas on electron microscope plates were masked with black tape along the boundaries of area to be diffracted; the specimens were scanned in an optical diffractometer and optical transforms were photographically recorded. For optical filtering a diffraction mask was made from recorded transforms; in some instances a 400 mesh copper screen with a light transmittance of 30% was placed over the origin of the mask. The mask was then placed at the diffraction plane on the optical bench and the resulting filtered image was photographically recorded. RESULTS
:"
The head of the opossum spermatozoon is V - s h a p e d w i t h o n e a r m of t h e V s o m e what larger than the other. In the case of s i n g l e s p e r m , t h e h e a d is a t a r i g h t a n g l e to t h e a x i s o f t h e t a i l . T h e a c r o s o m e is l i m i t e d to t h e s u r f a c e o f o n e w i n g o f t h e V and the tail inserts in a deep implantation fossa on the same wing a short distance f r o m t h e a p e x ( F i g s : 1 a n d 2). S p e r m a t o z o a f r o m t h e c a u d a e p i d i d y m i d i s of t h e opos-
OPOSSUM SPERM MEMBRANE SPECIALIZATION
209
FIG. 1. Longitudinal section of sperm head in which nucleus has an asymmetric V-shaped profile. The connecting piece (CP) is associated with an extensive membranous network, The posterior ring (PR) is located at the lateral margin of the nucleus. Distal to the posterior ring the plasma membrane covering the midpiece possesses a prominent fuzzy coat on its cytoplasmic surface (arrows). x 23 000. FIG. 2. High magnification view of spermatozoan neck region. The long connecting piece (CP) inserts into a hemisphere-shaped implantation fossa (I). A dense layer of amorphous material is applied against the outer nuclear membrane in the implantation fossa. At its anterior end the connecting piece consists of two distinct materials and more distally the proximal centriole is embedded in the connecting piece matrix. A crystall0id (C) is present in the matrix of the membranous network, x 42 000.
210
OLSON E T A L .
sum are typically conjoined in pairs with the two members adherent along their apposed acrosomal surfaces. To achieve this relationship the V-shaped heads of two sperm are rotated to bring the apex of the two V's anterior and their acrosomal surfaces into apposition. The complex formed by the conjoined heads then is shaped like a trident with the center tine composed of the two adherent acrosomal arms. The apex is forward and the diverging free arms directed posteriorly. The tails insert by slender curved connecting pieces into the implantation fossa which is located on the major wing of the sperm head. A thin coating of amorphous material lines the spherical implantation fossa and the small round capitulum of the connecting piece articulates within the implantation fossa in a ball and socket relationship (Fig. 2). The capitulum of the connecting piece is composed of two distinct components: an electron dense, amorphous layer forms the articular element which inserts into the implantation fossa (Fig. 2) and a homogeneous less electron dense material forms the bulk of the connecting piece and is continuous with the striated columns (Fig. 2). A mass of concentric membranes organized around a crystalloid is also found between the diverging wings or lobes of the nucleus and m a y be analogous to the membranous scrolls found in some other species (Figs. 1 and 2). Throughout most of the length of the midpiece the plasma membrane is remarkably specialized in structure. A 25-30 nm thick undercoat of fuzzy, dense material is found on the cytoplasmic surface of the membrane which has a regularly scalloped appearance (Fig. 3). The amorphous material coating the inner aspect of the plasma membrane also appears as a continuous layer of uniform thickness in longitudinal sections of the midpiece (Fig. 4). Scanning electron micrographs reveal numerous parallel ridges on the midpiece which run roughly parallel to the flagellar long axis (Fig. 5). These ridges terminate at the an-
nulus (Fig. 5). The ridges seen in scanning electron microscopy obviously correspond to the scallops of the plasma membrane observed in cross sections of the midpiece. Replicas of freeze fractured cauda epididymal spermatozoa show a striking organization of the intramembranous particles in the flagellar midpiece (Figs. 6, 7, 13). The particles cluster into roughly parallel rows 30-50 n m in width, which extend throughout much of the midpiece. Generally each major row is three particles wide but occasionally they m a y be up to five particles wide (Figs. 6, 7, 13). The individual particles average 6-8 n m in diameter. Not all rows are of equal length for a few abruptly terminate along the midpiece (Figs. 6, 7, 13). The membrane between rows is 30-50 n m wide and is usually devoid of particles. Toward the anterior end of the midpiece the particle rows abruptly terminate; the membrane area anterior to the termination of the rows of particles corresponds to the plasma membrane covering the nucleus and contains a sparse population of randomly distributed particles (Fig. 6). Distally the organized particle rows extend to the annulus and terminate (Fig. 13). Within the major rows the intramembranous particles appear to aggregate into a highly ordered two-dimensional lattice. Diffraction patterns of individual rows show a single prominent layer line indexing between 16-18 n m -1 (Figs. 8-10). Axially the particles are aligned into subrows like strings of beads and as determined from diffraction patterns and reconstructed images the particles exhibit a center to center spacing of 16-18 nm (Figs. 810). The three neighboring subrows are slightly staggered with respect to each other so that a line between adjacent particles in neighboring subrows is inclined at about 60° with respect to the long axis of the rows (Figs. 8-10). The plasma membrane retains its scalloped configuration over a portion of the annulus which is located at the midpiece-
211
OPOSSUM SPERM MEMBRANE SPECIALIZATION
¸%¸¸,¸¸/~
J
J
c
%.
/ \
J
/
/
/
® FIG. 3. Cross section, through distal region of midpiece of two paired spermatozoa. The plasma membrane has a regular scalloped appearance as well as a submembranous coating of amorphous material (arrows). × 43 000.
/N
FIG. 4. Longitudinal section through sperm midpiece. The m e m b r a n e scallops seen in cross section are not obvious but the m a t e r i a l coating the cytoplasmic face of the plasma m e m b r a n e appears as a continuous layer along the flagellar long axis (arrows). x 64 000. FIG. 5. S c a n n i n g electron micrograph showing a segment of the midpiece and principal piece. The midpiece is characterized by the presence of longitudinally oriented, parallel ridges which extend to the a n n u l u s (A). No ridges are seen on the principal piece, x 42 000. 212
OPOSSUM SPERM MEMBRANE SPECIALIZATION
213
FIG. 6. Freeze fracture replica showing anterior region of sperm midpiece. In the region of the midpiece characterized by the presence of m e m b r a n e scallops replicas reveal t h a t the i n t r a m e m b r a n o u s particles are aggregated into n u m e r o u s parallel rows, each 3-5 particles wide. The particle rows t e r m i n a t e at the anterior end of the midpiece; the plasma m e m b r a n e anterior to the t e r m i n a t i o n of the particle rows overlies the nucleus a n d is characterized by a r a n d o m particle distribution × 72 000.
FIG. 7. Freeze fracture replica showing the intramembranous particle a r r a n g e m e n t s in the sperm midpiece. Note t h a t some rows show an abrupt termination (arrows) and t h a t the membrane between adjacent rows is relatively particle free. x 78 000. 214
OPOSSUM SPERM MEMBRANE SPECIALIZATION
215
Fro. 8. High magnification view of one of the particle rows from sperm midpiece region. The 6-8 nm diameter particles are packed into a two-dimensional lattice. Fro. 9. Diffraction pattern arising from one particle row. The prominent layer line indexes at 16-18 nm -~ and shows two prominent off-meridional reflections and a single discrete reflection near the meridian. The size of the two large off-meridional reflections compared to the discrete meridional reflection suggests that long-range disruptions in the particle lattice arise predominantly from dislocations between adjacent particle subrows. FIa. 10. hnage obtained by layer line filtering diffraction pattern in Fig. 14. In the reconstruction zero order intensity was reduced with a 30% transmittance screen placed over the origin of the mask. The lattice of the particles is clearly shown. principal piece b o u n d a r y (Fig. 12). The ann u l u s consists of a n extensive circumferential sheet of fibrous m a t e r i a l positioned u n d e r t h e last g y r e of t h e midpiece mitochondrial s h e a t h (Figs. 11 and 12). As this sheet of fibrous m a t e r i a l approaches the p l a s m a m e m b r a n e it bifurcates into a Yshaped configuration (Fig. 11). T h e p l a s m a m e m b r a n e involutes rostrally at the bifurcation of the a n n u l u s and is closely associated w i t h the u n d e r l y i n g a n n u l a r m a t r i x (Fig. 12). Thus, in cross sections t h r o u g h the a n n u l u s t h r e e views of the p l a s m a m e m b r a n e are a p p a r e n t (Fig. 12). T h e oute r m o s t profile of the p l a s m a m e m b r a n e is scalloped like the m e m b r a n e o v e r l y i n g the midpiece and it is not i n t i m a t e l y associ-
ated with the u n d e r l y i n g dense m a t e r i a l of the annulus; however, the i n n e r two profiles of the p l a s m a m e m b r a n e , w h e r e the p l a s m a m e m b r a n e follows the bifurcation of the a n n u l u s , are not scalloped and the m e m b r a n e is closely associated with the u n d e r l y i n g dense m a t e r i a l composing the a n n u l u s (Fig. 12). Freeze f r a c t u r e replicas of the a n n u l u s show t h a t the rows of particles in the midpiece t e r m i n a t e w h e r e the p l a s m a mereb r a n e reflects rostrally to follow the underlying bifurcation of the a n n u l u s (Fig. 13). The p l a s m a m e m b r a n e is r e l a t i v e l y particle free at the point of its rostral reflection b u t t h e r e appears to be a n a b u n d a n c e of particles in the region w h e r e the p l a s m a
FIG. 11. Longitudinal section showing the plate-like a n n u l u s (An); note t h a t it bifurcates as it approaches the plasma membrane. The m e m b r a n e involutes rostrally to foliow the bifurcation, x 66 000. FIG. 12. Cross section through the a n n u l u s reveals three views of plasma membrane. The outermost m e m b r a n e profile is in the scalloped configuration(s) characteristic of the midpiece. The other 2 profiles of the plasma m e m b r a n e are closely associated with the underlying m a t r i x of the a n n u l u s (arrows). x 60 000. 216
OPOSSUM SPERM MEMBRANE SPECIALIZATION
membrane involutes and is most closely associated with the underlying dense matrix of the annulus (Fig. 13). The plasma membrane shows no specialized configuration in the principal piece region and there is no mat of fuzzy material associated with the cytoplasmic surface of the plasma membrane (Fig. 11). There is a network of fibrillar material surrounding the ribs of the fibrous sheath but it does not appear to preferentially associate with the plasma membrane (Fig. 11)o This fibrillar network is confined to the anteriormost portion of the principal piece. Most intramembranous particles in the principal piece region show a random arrangement (Figs. 14-16). However, the ~zipper," the one structural specialization of the principal piece plasma membrane seen in sperm of other species, is present and extends throughout much of the length of the principal piece (Fig. 14). The zipper has a rather striking heterogeneity with respect to particle arrangement. Most often it consists of only a double row of particles, but sometimes it is 5 to 6 particles wide (Figs. 14-16). Discontinuities in the zipper are frequently seen but they are generally short and the zipper continues after the short gap (Fig. 14). DISCUSSION
Freeze fracture studies have demonstrated considerable heterogeneity over the sperm surface in the size, arrangement, and relative density of intramembrane particles. In different species a variety of ordered particle arrays have been found in the membranes associated with the sperm head (8, 10, 16, 17, 25), but with the exception of mouse and guinea pig sperm (10, 17, 25) previous studies have found a random particle array in the midpiece plasma membrane. In the mouse sperm midpiece small patches of ordered particle arrays are occasionally found (25) but in the guinea pig midpiece the particles associate into linear arrays which wrap circumferentially about the flagel-
217
lum and precisely overlie the gyres of the mitochondrial sheath (10, 17). Although the organized intramembrane particle rows and mitochondria do not show this precise structural interrelationship in opossum sperm, the particle rows are only found in the plasma membrane which directly overlies the mitochondrial sheath suggesting a functional relationship between the plasma membrane and mitochondrial energy generating apparatus. Since organized particle arrays are not universally found in the midpiece of sperm of different species the functional significance of the relationship remains obscure. The rows of particles revealed in freeze fracture replicas of Didelphis sperm are only seen in regions of the midpiece that in thin sections or scanning electron microscopy display a scalloped plasma membrane configuration. Although there is a direct correspondence between the spacing of the particle rows and the scallops, the precise localization of the particle rows within or between adjacent scallops remains to be elucidated. Several different mechanisms could be responsible for aggregating the particles into the ordered rows: for example, factors in the luminal fluid of the epididymis acting of the cell surface could induce particle aggregation via interaction with surface receptors or by altering the configuration of the intrinsic membrane proteins so that aggregation is favored. Also, peripheral components acting at the cytoplasmic face of the membrane could influence particle aggregation. On the basis of the morphological evidence a correlation clearly exists between the presence of the submembranous coat on the cytoplasmic face of the membrane and the presence of membrane scallops. At the anterior limits of the midpiece there is no prominent fuzzy coating on the cytoplasmic face of the membrane and the membrane is not scalloped; freeze fracture replicas further show that this anterior portion of the midpiece is characterized by a random particle array. In addition, the
FIG. 13. Freeze fracture replica showing annulus. The midpiece particle arrays t e r m i n a t e at the a n n u l u s (A). A scarcity of particles is seen where the plasma m e m b r a n e folds back to follow the underlying m a t r i x of the a n n u l u s b u t there appears to be a n abundance of particles where the plasma m e m b r a n e is closely associated with the a n n u l u s (arrows). × 71 000. 218
OPOSSUM SPERM MEMBRANE SPECIALIZATION
219
FIas. 14-16. Freeze fracture replicas of principal piece region showing the "zipper" which consists of a linear a r r a y of particles. Frequently the zipper consists of only a double row of particles b u t occasionally it is up to 6-7 particles wide. Short discontinuities in the zipper are often seen (arrows). Fig. 14, x 42 000; Fig. 15, x 60 000; Fig. 16,.x 60 000.
220
OLSON ET AL.
midpiece plasma membrane of immature caput epididymal spermatozoa is not scalloped and the submembranous coating is lacking (unpublished data); whether or not the intramembrane particles of immature sperm are organized into regular rows remains to be determined. Similar maturational changes appear to take place in sperm of other marsupial species as neither the plasma membrane associated accessory fibers of phalanger sperm (13, 26) nor the membrane scallops of Wooley opossum sperm (22) are observed in sperm from the initial portion of the epididymis. Although a direct structural contact between the intramembranous particles revealed in freeze fracture replicas of Didelphis spermatozoa and the material coating the cytoplasmic face of the membrane seen in thin sections remains to be demonstrated, it is suggested that the material coating the inner aspect of the membrane may play a functional role in organizing and/or anchoring the intramembrane particles into the ordered arrays observed in freeze fracture replicas. Such transmembrane control has been demonstrated in the red cell membrane where the peripheral protein spectrin appears to be capable of influencing the mobility of intramembranous particles as well as the distribution of external cell surface glycoproteins (6, 19). In Didelphis sperm the organized rows of particles are only present in the midpiece and do not extend distally beyond the annulus. Nonetheless, distinctive particle accumulations are present within restricted areas of the plasma membrane overlying the annulus. The peculiar morphology of the annulus of opossum spermatozoa has been documented in previous thin section studies (5, 14, 22, 23). The particle rows present in the midpiece terminate as the plasma membrane folds back rostrally to follow the bifurcation of the underlying annulus; replicas further show that at the point of folding, the plasma membrane is relatively particle
free. However, within the bifurcation of the annulus the plasma membrane is closely associated with the underlying dense matrix which forms the annulus, and in replicas the overlying membrane clearly shows a high particle density suggesting a structural relationship between the annulus and overlying plasma membrane. Similar densely packed arrays of particles over the annulus have been seen in replicas of sperm of other species (8,10, 18, 25). The linear array of particles seen in replicas is the most striking membrane differentiation of the principal piece. A double row of particles termed the ~zipper" has been observed in replicas of the principal piece of guinea pig, rat, and mouse spermatozoa (5,10,18, 25) and a similar particle array has been shown in the flagellum of earthworm spermatozoa (3). In Didelphis spermatozoa the zipper consists primarily of a double row of particles, but it appears to be more irregular than the zipper of rat, guinea pig, or mouse sperm as it shows frequent discontinuities and short stretches where it is up to 5-6 particles wide. The striking and different particle arrangements in the midpiece and principal piece membranes presumably reflect underlying functional differences in these two membranes. Intramembranous particles have been implicated in a variety of transmembrane phenomena. For example, in the red cell there appears to be a structural relationship between the intramembrane particles and some surface glycoproteins (27) and since some membrane glycoproteins span the membrane (4) it has been suggested that the intramembrane particles revealed by freeze fracture represent hydrophobic segments of the glycoprotein. A correspondence between the presence of intramembrane particles and external glycoprotein coat is not obligatory though, as in some cells particle free membrane regions possess a prominent surface coat (24). A spatial relationship between
OPOSSUM SPERM MEMBRANE SPECIALIZATION
the disposition of externally disposed surface components and the intramembrane particles in the spermatozoan flagellar membrane remains to be demonstrated. Available data indicates that changes in both the organization of the intramembrane particles as well as the disposition and abundance of the plasma membrane glycoprotein coat may accompany certain physiological changes in spermatozoa. During capacitation rabbit spermatozoa show a reduction in concanavalin A binding sites on the flagellar membrane (12), and during incubation under capacitating conditions the midpiece plasma membrane of guinea pig sperm loses the organized strands of intramembrane particles (11, 18). Whether or not similar changes in membrane architecture occur in opossum sperm during capacitation is under investigation However, the elaborate structural specializations present in the midpiece plasma membrane should permit visual assessment of structural modifications occurring at the inner and outer membrane surface to be correlated with changes in the arrangement of intramembrane particles revealed by freeze fracture. REFERENCES 1. BARROS, C., AND FRANKLIN, L. E., J. Cell Biol. 37, C13 (1968). 2. BARROS, C., BEDFORD, J. M., FRANKLIN, L. E., AND AUSTIN, C. R., J. Cell Biol. 34, C1 (1967). 3. BERGSTROM, B. B., AND HENLEY, C., J. Ultrastruct. Res. 42, 551 (1973). 4. BRETSHER,M. S., J. Mol. Biol. 59, 351 (1971). 5. COOPER,G. W., AND BEDFORD, J. M., Anat. Rec. 169, 300 (1971).
221
6. ELGSAETER,A., AND BRANTON, D., J. Cell Biol. 63, 1018 (1974). 7. FAWCETT, D. W., Biol. Reprod. Suppl. 2, 90 (1970). 8. FAWCETT,D. W., Develop. Biol. 44,394 (1975). 9. FLECHON, J. E., J. Microsc. 19, 59 (1975). 10. FRIEND, D. S., AND FAWCETT, D. W., J. Cell Biol. 63, 641 (1974). 11. FRIEND, D. S., AND RUDOLF,I., J. Cell Biol. 63, 466 (1974). 12. GORDON,M., DANDEKAR,P. V., AND RARTOSZEwIcz, W., J. Reprod. Fert. 36, 211 (1974). 13. HARDING,H. R., CARRICK,F. N., AND SHOREY, C. D., Cell Tissue Res. 164, 121 (1975). 14. HOLSTEIN,A. F., Z. ZeUforsch, 65,904 (1965). 15. KLUG, A. AND DE ROSIER, D. J., Nature (London) 212, 29 (1966). 16. KOEHLER, J. K., J. Ultrastruct. Res. 33, 598 (1970). 17. KOEHLER, J. K., J. Microsc. 18, 263 (1973). 18. KOEHLER, J. K., AND GADDUM-ROSSE,P., J. Ultrastruct. Res. 51, 106 (1975). 19. NICOLSON, G. L., AND PAINTER, R. G., J. Cell Biol. 59, 395 (1973). 20. NICOLSON, G. L., AND YANAGAMACHI,R., Science 184, 1294 (1974). 21. OLSON, G., J. Ultrastruct. Res. 50, 193 (1975). 22. OLSON, G. E., AND HAMILTON,D. W . , A n a t . Rec. 186, 387 (1976). 23. PHILLIPS, D. M., J. Ultrastruct. Res. 33, 381 (1970). 24. PINTODA SILVA,P., MARTINEZ-PALOMO,A., AND GONZALEZ-ROBLES, A., J. Cell Biol. 64, 538 (1975). 25. STACKPOLE,C. W., AND DEVORKIN,D., J. Ultrastruct. Res. 49, 167 (1974). 26. TEMPLE-SMITH, P. D., AND BEDFORD, J. M., Anat. Rec. 184, 545 (Abstr.) (1976). 26. TILLACK,T. W., SCOTT,R. E., AND MARCHESI,V. T., J. Exp. Med. 135, 1209 (1972). 27. YANAGIMACHI,R., AND NODA, Y. D., J. Ultrastruct. Res. 31," 465 (1970). 28. YANAGIMACHI,R., NODA, Y. D., FUJIMOTO, M., AND NICOLSON, G. L., Amer. J. Anat. 135, 497 (1972).