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The spermatozoon of arthropoda XIII. The cell surface
© 1971 by Academic Press~ Inc.
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J. ULTRASTRtlCrURERESEARCH35, 582-605 (1971)
The Spermatozoon of Arthropoda X I I I . The Cell Surface BACCIO BACCETTI. ELISA BIGLIARDI AND FLORIANA ROSATI
Institute of Zoology, University of Siena, Siena, Italy Received September 23, 1970 In this paper the authors observe that the limiting membrane of the insect spermatozoon is asymmetrical. There is a "unit membrane" of the classical type, displaying an intense phosphatase activity, on the outer surface of which is a glycoprotein coat occurring in three structural patterns exhibiting progressive structural complexity. In Ceratitis and Drosophila ("fruit-fly model"), this coat is very thin and apparently amorphous; in Ctenocephalus ("flea" model), it is thicker and composed of transverse fibres; in Pezotettix and Aiolopus (the "locust" model), the coat is very thick and made up of short filaments or rodlets which are usually oriented normal to the sperm surface and grouped in tetrads with a rhomboidal arrangement. In the literature on the submicroscopic morphology of spermatozoa, a considerable number of published micrographs suggest the presence of an asymmetrical plasma membrane. In the insects, membrane asymmetry is particularly evident; it has been examined closely by some workers (3, 18), whereas others have simply illustrated it without commenting upon its significance. Membrane asymmetry is now of particular interest because of its possible implications for the specificity of the cell surface. The most recent reviews of membrane structure (10, 27, 29) have stressed the need for consideration of models other than the classic ones of Danielli and Davson (8) and Robertson (24), but they nevertheless constantly reaffirm that the two dense layers of the "unit membrane" are not identical. This is to say that membrane asymmetry depends on extraneous substances tightly adhering to one of its surfaces. These are usually thought to consist either of protein globules representing enzymes associated with the cytoplasmic aspect of the membrane (25, 26) or carbohydrates forming the so-called "glycocalyx" (6, 16) from a layer on the outer aspect of the plasma membrane. Carbohydrates form rather amorphous layers, even when the glycocalyx consists of filaments in disorderly array composing the "fuzzy" layer of intestinal epithelium or amoebae (22, 23). On the other hand, the plasma membrane of cell junctions in surface view and after negative staining, shows
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a m o s a i c of h e x a g o n a l facets with a center-to-center distance of a b o u t 90 ~ (5, 12). I n view of the extreme specialization of s p e r m a t o z o a , it was expected t h a t their p l a s m a m e m b r a n e m i g h t show noticeable differences f r o m t h a t of other cells in the p a t t e r n a n d degree of symmetry. I n the present p a p e r , three m a i n p a t t e r n s of organiz a t i o n of s p e r m p l a s m a m e m b r a n e s are described in some detail.
M A T E R I A L S A N D METHODS The present investigations were carried out on Ceratitis capitata and Drosophila melanogaster (Diptera); Pezotettix giornai and Aiolopus strepens (Orthoptera); Ctenocephalus canis (Aphaniptera). Comparatively some representative species of more primitive families of Orthoptera (from Phaneropteridae to Eumastacidae) were examined. Small pieces of testes and seminal vesicles were fixed in vivo for 20 minutes to 2 hours at 0°C in 2.5 % glutaraldehyde in Hoyle's buffer at p H 7.2. The specimens were postfixed in OsO4 solution for 10 minutes, dehydrated in a graded series of ethyl alcohol, and embedded in Epon 812. Sections, cut on LKB or Reichert ultramicrotomes, were collected on grids and stained with 1.5 % uranyl acetate and lead citrate. F o r investigation of proteins, ultrathin sections were treated for 20 minutes with 5 % periodic acid, rinsed in distilled water, and then incubated for 15-40 minutes in 0.1% pepsin solution in 0.1 N HC1. F o r the study of carbohydrates, ultrathin sections were treated for 20 minutes with 5 % periodic acid, rinsed in distilled water, and incubated for 10-90 minutes at 37°C in 0.1% amylase solution in 0.1 M phosphate buffer at p H 6.9. Ultrathin sections of the same material were stained by Thi6ry's thiosemicarbazide-proteinate method. Glycoproteins were demonstrated in material fixed in glutaraldehyde alone, embedded in glycol-methacrylate, sectioned and stained by the Rambourg, Hernandez, and Leblond's method (20) with 0.1% PTA in 10 % chromic acid for 2-5 minutes counterstaining. The submicroscopical topochemical localization of several enzymes was also studied. F o r demonstration of acid phosphatase activity, unfixed and 2.5% cacodylate-buffered glutaraldehyde-fixed spermatozoa were used. The specimens were incubated at 37 ° for 30-60 minutes in Gomori's acid phosphatase medium. Control samples were incubated in a substrate-free medium. After incubation, the specimens were rinsed for 10-15 minutes in 0.1 M sodium cacodylate buffer (pH 7.2), postfixed for 10 minutes in OsO4 solution, dehydrated, and embedded in Epon 812. Alkaline phosphatase, glucose-6-phosphatase and ATPase activities were assayed with the methods described in a previous work (7). Sperm suspension obtained by squeezing the testes and seminal vesicles of Ceratitis, Pezotettix, and Ctenocephalus were prepared by mechanical fragmentation followed by ultrasonication for several minutes. After being repeatedly washed and centrifuged, this material was negatively stained with 5 % PTA. The specimens were mounted on Formvar-coated grids. The electron microscopes used were the Siemens Elmiskop I (with tilting stage) and II. Observations with the stereoscopic electron microscope were carried out on briefly fixed sperms obtained by squeezing the testes and seminal vesicles. Gold palladium-coated specimens were observed and photographed using a Jeol scanning electron microscope. Some electron micrographs were analyzed by the optical diffraction technique. F o r freeze etching technique, small pieces of testes and seminal vesicles were immersed for
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30 minutes at 4°C in Hoyle buffer containing 25 % glycerol. The specimens were then transferred into small copper disks that were rapidly plunged into freezing liquid Freon 22 cooled with liquid nitrogen for 4 minutes. After such "rapid freezing," the disks were immersed into liquid nitrogen for a period of 10 minutes to 3 days. Freeze-etching was carried out with the apparatus manufactured by Balzers. The technique followed there was that of Moor et aL (15). The carbon-platinum replica was removed from the specimen holder and treated with chromic acid followed by several rinses with distilled water in order to remove cellular and other debris. The floating replica was then picked up on copper grids and examined in a Siemens Elmiskop I electron microscope.
OBSERVATIONS
The fruit fly type of membrane In both Ceratitis capitata and Drosophila melanogaster (Insecta, Diptera), the sperm plasma membrane is clearly triple-layered, 100-110 A thick, and definitely asymmetrical throughout (Fig. 1). The innermost leaflet is moderately electron dense and about 20 A thick; the intermediate layer, completely transparent to the electron beam, is some 30 N thick. When sectioned perpendicular to the plane of the membrane, the outer layer consistently appears as an opaque line ranging from 50 to 60 A in thickness. In some sections, the light interspace is traversed by slender electron-opaque septa giving the appearance of an array of globules within the plane of the membrane. The nature of the material responsible for the asymmetry of the layers was studied by various histochemical techniques. Pepsin, trypsin, or amylase digestion failed to show a differential effect upon the inner and the outer opaque layers. Thi6ry's thiosemicarbazide method with the time extended to 24 hours results in diffuse staining in the form of fine electron-opaque granules over the thick outermost layer of the plasma membrane, while the other two layers are completely negative (Fig. 3). The results yielded by application of the 0.1% PTA in 10 % chromic acid method for glycoproteins, according to Rambourg, Hernandez, and Leblond (20), appear to be significant. By this method (Fig. 4), a continuous electron opaque halo up to 90 A thick can be seen external to the outer leaflet of the sperm plasma membrane. This layer is thicker than the membrane proper and often merges with a similar coat on neighboring spermatozoa. It is quite evident where heads or tails of spermatozoa are closely apposed (Fig. 5). This selective staining with the Rambourg, Hernandez, and Leblond method suggests the presence of glycoprotein.
FIG. 1. Cross section of Ceratitis sperm tail showing the asymmetrical trilaminar plasma membrane (arrow). x 180 000. FIG. 2. Cross section of Ceratitis sperm tail showing the localization of acid phosphatase among the fibrillar elements on the plasma membrane (arrow). × 110 000. FIG. 3. Cross section of Ceratitis sperm tails treated with Thi6ry's method: A positive reaction is localized at the level of the relatively thick outer layer of the plasma membrane (arrows). x 120 000.
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FIcs. 4 and 5. Cross section through the tail (Fig. 4) and the head (Fig. 5) of Ceratitis sperms treated with the PTA method of Rambourg, Hernandez, and Leblond (20). A positive reaction is seen at the level of the plasma membrane and in the interspaces among the sperm cells. × 120 000.
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FIGS. 6-8. Freeze-etched preparations of Ceratitis spermatozoa. The outer sperm surface (O) appears smooth; below it, a longitudinally oriented granular component (GR) is evident, x 60 000.
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The plasma membrane was negative after histochemical reactions. Most of the histochemical assays performed by us were for enzyme activities for ATPase, glucose6-phosphatase, and peroxidase. However, with the Gomori method for acid phosphatase, a heavy deposit was formed on both the outer and the inner electron opaque layers (Fig. 2). As a rule, the light interspace was unreactive. The outer surface of the spermatozoon appears quite smooth under the scanning electron microscope and in freeze-etched preparations (Figs. 6 and 7). In the latter, a granular component is clearly demonstrated beneath the outer surface (Figs. 6 and 7), but separated from the sperm organelles by a thin membrane. We assume that this material is located between the two limiting layers of the cell membrane, which according to Meyer and Winkelmann's (14) interpretation become separated from each other by the etching processes. In some of our replicas, this is quite evident, the sperm membrane having been fractured in profile (Fig. 8). In this case, amorphous material is observed upon the membrane, which corresponds to the glycoprotein coat. The foregoing observations reveal the plasma membrane of fruit fly spermatozoon as a typical cell membrane, with its outer layer slightly thickened by an overlying glycoprotein coat.
The flea type membrane The flea (Ctenocephalus canis; Insecta, Aphaniptera) spermatozoon is limited by a m e m b r a n e about 2130 A thick in which two sharply differing layers can be observed (Fig. 9). The innermost one is a typical plasma membrane with an overall thickness of 70 ~ . External to this is a layer about 130 A thick which has different appearances depending upon the plane of section. In sections transverse to the long axis of the spermatozoon, it appears amorphous. In midline longitudinal sections, it displays an array of globules 130 A in diameter with an electron-opaque cortex and a light core. In oblique or parasagittal longitudinal sections, it is clearly striated, being made up of short, parallel electron-opaque bars about 70 N thick, separated by light interspaces of the same thickness. Where the section just grazes the sperm surface, this outer coating appears cross-striated throughout its width. The flea spermatozoon is helically coiled with a pitch of 1.7 # (1), resulting in totFIGS. 9-12. Longitudinal sections of the Ctenoeephalus sperm membrane. FIG. 9. The two layers forming this type of membrane are evident; the typical plasma membrane (p) and the outer array of cross-sectioned fibers (f) with an electron-opaque wall and a light core. x 150 000. FIG. 10. Acid phosphatase activity (arrows) appears localized both on the inner plasma membrane and on the outer cell coat. x 80 000. FIG. 11. The treatment with Thi6ry's method reveals a slight deposit around the fibers of the surface coat (arrows). x 120 000. FIG. 12. The method of Rambourg, Hernandez, and Leblond (20) reveals a strongly positive reaction at the level outer cell coat (arrows). x 120 000.
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FIG. 13. Sperm tail of Ctenocephalus photographed by the scanning electron microscope. A striated pattern cannot be resolved, x 60 000. FI~. 14. Cell membrane of Ctenoeephalus spermatozoa fragmented and negatively stained. The cross striation (arrows) is evident. × 90 000. F ~ . 15. Freeze-etched preparation of Ctenoeephalus sperms. The striations of the outer sperm surface (O) and the granulated space of the cell membrane (GR) are evident, x 60 000.
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Fio. 16. Cross section of Pezotettix sperm tail showing the two layers forming the sperm membrane: an inner trilaminar plasma membrane (P)and the outer coat formed by closely aggregated rods (C). x 120 0 0 0 .
sion of the v a r i o u s c o m p o n e n t s of the flagellum, b u t these can never be seen simultan e o u s l y in the same transverse section (19). Therefore, the cross striations of the c o a t o v e r l y i n g the p l a s m a m e m b r a n e are never at right angles to the l o n g i t u d i n a l axis of the s p e r m since they are n o r m a l to the axis of the i n d i v i d u a l gyres of the helix. This i n t e r p r e t a t i o n is c o n f i r m e d b y observations on m e m b r a n e fragments negatively
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FIG. 17. Cross section of Pezotettix sperm tails treated with Thi6ry's method. The trilaminar plasma membrane appears positive with this method (arrow). x 120 000. F i t . 18. Pezotettix sperms treated with the PTA method of Rambourg, Hernandez, and Leblond (20). The reaction is positive on the outer coat and around the coarse fibers and inner central pair. x 120 000.
stained with potassium phosphotungstate, where the cross-striations stand out very clearly (Fig. 14). However, the PTA-infiltrated interspaces look narrower (under 50 A) while the unstained bars are wider (about 100 A) than in sectioned material. Nonetheless, the period resulting from the sum of a bar and an interspace is the same in both cases, that is, little below 140 A. This structure, however, is not very well resolved with the scanning electron microscope, for the limited power of resolution of this instrument. More information is given by the freeze-etching method. With this method, the striation of the outer surface of the sperm cell is quite evident, and the limiting membrane is resolved as two juxtaposed components (Fig. 15). The granular component lying subjacent to the cell membrane has definite orientation. Its subunits are arranged in lines that run helically in a direction opposite to that of the outer striae but coinciding with that of the mitochondrial helix, and it is related to the coiled configuration of the whole spermatozoon. Information available about the chemical nature of the two different components making up the membrane of this sperm type is scant but nevertheless significant. Neither layer is preferentially digested by amylase, pepsin or trypsin, even when other components of the same spermatozoon are eliminated by these enzymes. When Thi6ry's method is applied for as long as 24 hours, a slight deposit is seen to form around the striae of the surface coating (Fig. 11).
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FIG. 19. Acid phosphatase activity in Pezotettix sperm tails. The reaction appears positive on the plasma membrane (arrows). x 120 000. P T A treatment at p H 3 according to Rambourg, Hernandez, and Leblond (20) for demonstration of glycoproteins gives a strongly positive reaction of the level of the electron opaque bars (Fig. 12). In longitudinal sections where this layer appears to be composed of globular subunits (each bar being transversely cut), positive reaction
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BACCETTI, BIGLIARDI AND ROSATI
takes the form of a wide halo surrounding each globular profile. Among the enzymes sought by us, only acid phosphatase was found to occur in these layers. With the Gomori method, in fact, a heavy electron opaque deposit is found both on the inner cell membrane and over the outermost striated glycoprotein layer (Fig. 10). From these observations, it may be inferred that in Ctenoeephalus spermatozoon, the cell periphery is made up of a typical plasma membrane coated by a thick glycoprotein layer which consists of filaments of uniform caliber and orientation, and rich in acid phosphatase activity, like the plasma membrane.
The locust type of membrane Spermatozoa of Pezotettix giornai and Aiolopus strepens (Insecta, Orthoptera) are bounded by a membrane about 400/~ thick in which two layers can be identified: an inner unit membrane about 100 A in thickness overlain by a conspicuous coating, 300 A thick (Fig. 16). The inner plasma membrane is of the classical asymmetric triple-layered structure described above for Ceratitis. Proceeding from the inside to the outside, there is an inner electron opaque layer 20 ~ thick, a light interspace 30 ,~ thick, and a highly electron dense outer layer some 40-50 A thick. On the surface of this latter layer, a slight deposit is revealed by Thi6ry's thiosemicarbazide method applied for 24 hours (Fig. 17), and a heavy reaction for glycoproteins is given by PTA, pH 0.3 according to the Rambourg, Hernandez, and Leblond method (20) (Fig. 18). The Gomori method for acid phosphatase is also strongly positive at this level (Fig. 19). The 300 A thick coating rests upon this glycoprotein layer of the plasma membrane. In both transverse and longitudinal sections, this coating is seen to be composed of electron dense rodlets about 70 A thick and 300 A long oriented radially and closely packed about 70 A apart (Figs. 20 and 21). These rodlets can be seen in their entirety only in favorably oriented sections where they may occupy a long stretch of the spermatozoon edge. In cross sections they are detectable only on the two sides of the sperm, those lying against the flagellum and the two mitochondrial derivatives (Fig. 16). The optical diffraction pattern of this material (Fig. 22) confirms the presence of periodic rodlike units perpendicularly arranged on a basal surface. Under the scanning electron microscope, these rodlets appear not resolved, but the study of sections suggests that the rodlets are slanted in an anterior direction on one side and in a posterior direction on the other side, and perpendicular to the longitudinal axis of the sperm in the intervening regions. If we designate as the basal plane of the sperm that upon which the two mitochondrial derivatives lie, and as the axis of symmetry the median plane normal to it, it may be observed that the rodlet arrangement is oriented with respect to these two planes rather than to the plane containing the two central tubules (9). The plane of the central pair of tubules is tilted by about
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FIG. 20. Cross section of P e z o t e t t i x sperm membrane (at left) and tangential section at the level of'the outer coat (at right). The trilaminar plasma membrane (P) and outer coat (C) are evident. × 120 000. FIG. 21. Transverse section of P e z o t e t t i x sperm membrane at higher magnification; P, plasma membrane; C, outer coat. x 180 000. FIG. 22. Optical diffraction pattern of the same material. 39 - 711838 J . Ultrastructure Research
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20 ° with respect to the normal one. The rodlets composing the outer coating are devoid of enzymatic activity and are resistant to digestion with amylase or pepsin. The study of replicas obtained by the freeze-etching method lends support to the above described structure (Figs. 27 and 28). The plasma membrane is 100 A thick and dotted with minute globular granules about 50 A in diameter on both the inner and outer surfaces. The outermost layer is generally visualized in cross section alone, where it merely displays its 300 A thickness without revealing the rodlet pattern. Examination of flakes of membrane with their coating negatively stained by potassium phosphotungstate (Fig. 24) is also rewarding. With this procedure, the rodlets are seen from above and appear to be arranged in tetrads. Each rodlet is about 70 A thick. They lie at distances varying from less than 20 A apart, in the case of the four units of a single tetrad, to about 70 A between one tetrad and another. In each tetrad, the four rodlets are arranged in a rhomboidal configuration. The various tetrads are also oriented along lines forming a rhomboidal lattice. By tilting the specimen (Figs. 23 and 25) the four units of each tetrad, appearing as spots in frontal view, appear clearly elongated. The optical diffraction pattern (Fig. 26) of the same specimen confirms that the lattice units in frontal view are regularly disposed in tetragonal arrangement. Pezotettix and Aiolopus spermatozoa therefore appear coated by a typical plasma membrane which is made asymmetrical by an amorphous outer coating of glycoprotein and further protected by an array of closely packed glycoprotein filament on rodlets which are radially arranged or slanting obliquely forward on one side and backward on the other. It is of interest to trace the origin of this patterned coating. Spermatids are provided with a conspicuous glycocalyx (Fig. 29). During their evolution this glycoprotein coating grows thicker and begins to differentiate into filamentous or rodlike units which tend to be arranged perpendicular to the plasma membrane, but they do not acquire their individuality until the spermatozoon is almost mature (Figs. 30-32.) P The "locust type" model of sperm membrane is not common to all the Orthoptera species. The most primitive families (as Phaneropteridae, Tettigoniidae, Gryllidae, Gryllotalpidae) examined by us have the usual "fruit fly" model membrane. The "locust" model is therefore restricted to acridoids, and it is important to note that it is well developed only in the highest families examined, i.e., Catantopidae (Pezotettix) and Acrididae (Aiolopus), whereas in the most primitive acridoids, the Australian group Morabinae, the characters appear less evident. The rodlets (Fig. 34) are in fact only 100 A high. FIGS. 23-25. Cell membrane of Pezotettix sperm fragmented and negatively stained. The rodlet projections seem to be arranged in tetrads (arrow), and appear as spots in frontal view (Fig. 24), but become elongated if the specimen is tilted + or -20 ° (Figs. 23 and 25). x 360 000.
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FI~. 26. Optical diffraction pattern of Fig. 24. DISCUSSION F r o m all the observations reported here, the limiting membrane of the insect spermatozoon clearly emerges as being of an asymmetric type. There is in fact a "unit m e m b r a n e " of the classical type, displaying intense phosphatase activity, on which is superimposed a glycoprotein coating which has evolved in different ways in different species, and which we have classified in three patterns of progressive structural complexity. The first (the "fruit fly" type) in which the glycoprotein coating is very thin (40 A_) and amorphous, the second (the "flea" model) where it is thicker and formed of transverse fibres displaying phosphatase activity, and the third (the "locust" type) in which the extracellular glycoprotein coat is very thick (about 300 A_) and made u p of rodlets which are grouped in tetrads in a rhomboidal arrangement. The orientaFIGS. 27 and 28. Samples of Pezotettix sperms obtained by the freeze-etching method. The cell coat (C) disposed upon the granular space of the cell membrane (GR) is clearly seen. x 120 000.
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FIG. 29. Cross section of Pezotettix giornai spermatids. Note the conspicuous glycocalyx around the cell (arrow). M, mitochondria, F, flagellum, x 60 000.
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FIGS. 30-32. Stages of evolution of glycocalyx in Pezotettix spermatids. The glycocalyx first (Fig. 30) differentiates filamentous units (arrows) which tend to be arranged perpendicularly to the plasma membrane (Fig. 31). Only in almost mature spermatozoa (Fig. 32) do these rodlets acquire their final arrangement and become embedded in a rich glycoprotein material. P, Plasma membrane; C, coat of rodlets. Figs. 30 and 31, x 120 000; Fig. 32, x 60 000.
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tion of these rodlets is usually normal to the spermatozoon surface; on one side of the sperm, however, they are slanting in a forward direction and on the opposite side in a backward direction, at an angle of some 40 ° to the perpendicular plane. The increasing structural complexity of the spermatozoon plasma membrane is probably related to the biology of the particular sperm. In some spermatozoa (spiders, pseudoscorpions, opilionids, etc.), which are ejaculated within cysts, even thicker walls are known (2, 21, 28). The nature of these has yet to be investigated. Myriapod sperms are even enclosed in pairs in individual spermathecae (11) whose nature and origin are as yet unknown. Nearly all cells have a glycoprotein coating, the "glycocalyx," which is endowed with special properties, particularly at the cell-to-cell junctions. At the free cell sur-
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Fro. 33. Schematic drawing of a segment of Pezotettix sperm tail. faces, the glycocalyx merely thickens (as on microvilli) or gives rise to filaments arranged in a disorderly fashion (as on protozoa), but at junctions it forms specialized dense patches occupying the central part of desmosomes. Recent investigations (17) have suggested that the insect septate desmosomes are glycoprotein laminae oriented transversely between adjacent cells. It is tempting to relate the possible presence of septate desmosomes to the thickened glycoprotein coverings found around the spermatozoa of the same animal groups. A convincing parallelism between the extracellular coating of the locust type of spermatozoa and the structure of septate desmosomes can be established by comparing the images of negatively stained preparations reported here with tangential sections of desmosomes photographed by Locke (13). The significance of this desmosome-like specialization of the membrane surrounding the spermatozoa is rather obscure. There is possibly some relationship between their occurrence and the way these spermatozoa are anchored to one another in animals that ejaculate spermatozeugma or spermodesmata. The most intriguing aspect of these studies lies in the striking symmetry and ordered structure of the glycoprotein layer overlying the unit membrane. Some images bring to mind the hexagonal subunits of the negatively stained plasma membrane of mammalian cells described by Benedetti and Emmelot (5). There are, however, conspicuous differences between these two membranes in the center-to-center spacing of the subunits. Therefore, it appears unlikely that a pattern within the plasma membrane induces or imposes a similar
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FIG. 34. Cross section of Moraba viatica sperm tail, showing the limited height of the outer coat. x 120 000. pattern on the overlying glycoprotein material. The spatial arrangement of subunits in the latter is evidently acquired independently and is specific for each type. The authors thank Professor Don Fawcett for the help in editing the manuscript; Professor E. L. Benedetti for discussing the results; Professor D. Bocciarelli Steve for the opportunity to use the optical diffraction apparatus of the Istituto Superiore di Sanit~t, and Franco Tangucci and Carlo Ramoni for the excellent technical assistance. This research was supported by a grant from Consiglio Nazionale delle Ricerche.
REFERENCES 1. 2. 3. 4. 5.
BACCETTI,B., Redia 51, 153 (1968). BACCETTI,B., DALLAI,R. and ROSATI,F., Or. Cell Biol. 44, 3 (1970). BAIRATI,A. and BAIRATI,A., JR., Protoplasma 63, 283 (1967). BENEDETTI,E. L. and EMMELOT,P., J. Cell Biol. 38, 15 (1968). -it/DALTON, A. J. and HAGENAU,F. (Eds.), The Membranes, Academic Press, New York, 1968.
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6. BENNET,H. S., J. Histochem. Cytochem. 11, 14 (1963). 7. BIGUARDI,E., BACCETXI,B. BURRINI,A. G. and PALLINg,V. In BACCETT~,B. (Ed.), Comparative Spermatology. Accademia dei Lincei, Rome-Siena, t970. 8. DANIELLI,J. R. and DAVSON, H. A., J. Cell Comp. Physiol. 5, 495 (1935). 9. FAWCETT,D. W., J. Cell Sci. 3, 187 (1968). 10. FINEAN,J. B., Quart. Rev. Biophys. 2, 1 (1969). 11. HORSTMANY,E. and BREUCKER,H., Z. Zellforsch. Mikrosk. Anat. 96, 505 (1969). 12. KAVANAtr,J. L., Structure and Function in Biological Membranes, Vol. II, HoldenDay, San Francisco 1965. 13. LO¢~,_E,M., J. CellBiol, 25, 166 (1965). 14. MEYER, H. W. and WINKELMANN,H., Protoplasma 68, 253 (1969). 15. MooR, H., Mi2HLETHALER,K., WALDNER, H. and FREY-WYsSLrNG, A. J., Biophys. Biochem. Cytol. 10, 1 (1961). 16. MORRIS, B., CANDIOTTr,A. and FABRO, J., Z. Zellforsch. Mikrosk. Anat. 99, 64 (1969). 17. NOmOT, C. and NOmOT TIMOXH~E,C., J. Microsc. (Paris) 8, 72a (1969). 18. PEROTTI,M. E., J. Submicrosc. Cytol. 1, 171 (1969). 19. PmLLIPS, D. M., J. Cell Biol. 40, 28 (1969). 20. RAMBOURG,A., HERNANDEZ,W. and LEBLOND,C. P., J. Cell Biol. 40, 395 (1969). 21. REGER, J. F., J. Ultrastruct. Res. 28, 422 (1969). 22. REVEL, J. P., J. Microscop. (Paris) 3, 535 (1964). 23. REVEL, J. P. and Ixo, S., in DAvis, B. D. and WARREN, L. (Eds.), The Specificity of Cell Surfaces, p. 211. Prentice-Hall, Englewood Cliffs, New Jersey, 1967. 24. ROBERTSON, J. D., in NACHMANSOHN, D. (Ed.), Molecular Biology. Academic Press, New York, 1960. 25. SJ6STRANO,F. S., Electron microscopy of myelin and nerve cells and tissues. In Ct~MrNGS, J. N. (Ed.), Modern Scientific Aspects of Neurology, p. 188. Arnold, London, 1960. 26. - in DALTON, A. J. and HAGENAU, F. (Eds.) Ultrastructure in Biological Systems, p. 151. Academic Press, New York, 1968. 27. STOECKENmS,W. and EN~ELMAN,D. M., J. Cell BioL 42, 613 (1969). 28. TUZET, O., MANIER, J. F. and BOISStN, L., C. R. Acad. ScL Ser. D. 262, 376 (1966). 29. WEiss, L., lnt. Rev. Cytol. 26, 63 (1969).
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J. ULTRASTRtlCrURERESEARCH35, 582-605 (1971)
The Spermatozoon of Arthropoda X I I I . The Cell Surface BACCIO BACCETTI. ELISA BIGLIARDI AND FLORIANA ROSATI
Institute of Zoology, University of Siena, Siena, Italy Received September 23, 1970 In this paper the authors observe that the limiting membrane of the insect spermatozoon is asymmetrical. There is a "unit membrane" of the classical type, displaying an intense phosphatase activity, on the outer surface of which is a glycoprotein coat occurring in three structural patterns exhibiting progressive structural complexity. In Ceratitis and Drosophila ("fruit-fly model"), this coat is very thin and apparently amorphous; in Ctenocephalus ("flea" model), it is thicker and composed of transverse fibres; in Pezotettix and Aiolopus (the "locust" model), the coat is very thick and made up of short filaments or rodlets which are usually oriented normal to the sperm surface and grouped in tetrads with a rhomboidal arrangement. In the literature on the submicroscopic morphology of spermatozoa, a considerable number of published micrographs suggest the presence of an asymmetrical plasma membrane. In the insects, membrane asymmetry is particularly evident; it has been examined closely by some workers (3, 18), whereas others have simply illustrated it without commenting upon its significance. Membrane asymmetry is now of particular interest because of its possible implications for the specificity of the cell surface. The most recent reviews of membrane structure (10, 27, 29) have stressed the need for consideration of models other than the classic ones of Danielli and Davson (8) and Robertson (24), but they nevertheless constantly reaffirm that the two dense layers of the "unit membrane" are not identical. This is to say that membrane asymmetry depends on extraneous substances tightly adhering to one of its surfaces. These are usually thought to consist either of protein globules representing enzymes associated with the cytoplasmic aspect of the membrane (25, 26) or carbohydrates forming the so-called "glycocalyx" (6, 16) from a layer on the outer aspect of the plasma membrane. Carbohydrates form rather amorphous layers, even when the glycocalyx consists of filaments in disorderly array composing the "fuzzy" layer of intestinal epithelium or amoebae (22, 23). On the other hand, the plasma membrane of cell junctions in surface view and after negative staining, shows
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a m o s a i c of h e x a g o n a l facets with a center-to-center distance of a b o u t 90 ~ (5, 12). I n view of the extreme specialization of s p e r m a t o z o a , it was expected t h a t their p l a s m a m e m b r a n e m i g h t show noticeable differences f r o m t h a t of other cells in the p a t t e r n a n d degree of symmetry. I n the present p a p e r , three m a i n p a t t e r n s of organiz a t i o n of s p e r m p l a s m a m e m b r a n e s are described in some detail.
M A T E R I A L S A N D METHODS The present investigations were carried out on Ceratitis capitata and Drosophila melanogaster (Diptera); Pezotettix giornai and Aiolopus strepens (Orthoptera); Ctenocephalus canis (Aphaniptera). Comparatively some representative species of more primitive families of Orthoptera (from Phaneropteridae to Eumastacidae) were examined. Small pieces of testes and seminal vesicles were fixed in vivo for 20 minutes to 2 hours at 0°C in 2.5 % glutaraldehyde in Hoyle's buffer at p H 7.2. The specimens were postfixed in OsO4 solution for 10 minutes, dehydrated in a graded series of ethyl alcohol, and embedded in Epon 812. Sections, cut on LKB or Reichert ultramicrotomes, were collected on grids and stained with 1.5 % uranyl acetate and lead citrate. F o r investigation of proteins, ultrathin sections were treated for 20 minutes with 5 % periodic acid, rinsed in distilled water, and then incubated for 15-40 minutes in 0.1% pepsin solution in 0.1 N HC1. F o r the study of carbohydrates, ultrathin sections were treated for 20 minutes with 5 % periodic acid, rinsed in distilled water, and incubated for 10-90 minutes at 37°C in 0.1% amylase solution in 0.1 M phosphate buffer at p H 6.9. Ultrathin sections of the same material were stained by Thi6ry's thiosemicarbazide-proteinate method. Glycoproteins were demonstrated in material fixed in glutaraldehyde alone, embedded in glycol-methacrylate, sectioned and stained by the Rambourg, Hernandez, and Leblond's method (20) with 0.1% PTA in 10 % chromic acid for 2-5 minutes counterstaining. The submicroscopical topochemical localization of several enzymes was also studied. F o r demonstration of acid phosphatase activity, unfixed and 2.5% cacodylate-buffered glutaraldehyde-fixed spermatozoa were used. The specimens were incubated at 37 ° for 30-60 minutes in Gomori's acid phosphatase medium. Control samples were incubated in a substrate-free medium. After incubation, the specimens were rinsed for 10-15 minutes in 0.1 M sodium cacodylate buffer (pH 7.2), postfixed for 10 minutes in OsO4 solution, dehydrated, and embedded in Epon 812. Alkaline phosphatase, glucose-6-phosphatase and ATPase activities were assayed with the methods described in a previous work (7). Sperm suspension obtained by squeezing the testes and seminal vesicles of Ceratitis, Pezotettix, and Ctenocephalus were prepared by mechanical fragmentation followed by ultrasonication for several minutes. After being repeatedly washed and centrifuged, this material was negatively stained with 5 % PTA. The specimens were mounted on Formvar-coated grids. The electron microscopes used were the Siemens Elmiskop I (with tilting stage) and II. Observations with the stereoscopic electron microscope were carried out on briefly fixed sperms obtained by squeezing the testes and seminal vesicles. Gold palladium-coated specimens were observed and photographed using a Jeol scanning electron microscope. Some electron micrographs were analyzed by the optical diffraction technique. F o r freeze etching technique, small pieces of testes and seminal vesicles were immersed for
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30 minutes at 4°C in Hoyle buffer containing 25 % glycerol. The specimens were then transferred into small copper disks that were rapidly plunged into freezing liquid Freon 22 cooled with liquid nitrogen for 4 minutes. After such "rapid freezing," the disks were immersed into liquid nitrogen for a period of 10 minutes to 3 days. Freeze-etching was carried out with the apparatus manufactured by Balzers. The technique followed there was that of Moor et aL (15). The carbon-platinum replica was removed from the specimen holder and treated with chromic acid followed by several rinses with distilled water in order to remove cellular and other debris. The floating replica was then picked up on copper grids and examined in a Siemens Elmiskop I electron microscope.
OBSERVATIONS
The fruit fly type of membrane In both Ceratitis capitata and Drosophila melanogaster (Insecta, Diptera), the sperm plasma membrane is clearly triple-layered, 100-110 A thick, and definitely asymmetrical throughout (Fig. 1). The innermost leaflet is moderately electron dense and about 20 A thick; the intermediate layer, completely transparent to the electron beam, is some 30 N thick. When sectioned perpendicular to the plane of the membrane, the outer layer consistently appears as an opaque line ranging from 50 to 60 A in thickness. In some sections, the light interspace is traversed by slender electron-opaque septa giving the appearance of an array of globules within the plane of the membrane. The nature of the material responsible for the asymmetry of the layers was studied by various histochemical techniques. Pepsin, trypsin, or amylase digestion failed to show a differential effect upon the inner and the outer opaque layers. Thi6ry's thiosemicarbazide method with the time extended to 24 hours results in diffuse staining in the form of fine electron-opaque granules over the thick outermost layer of the plasma membrane, while the other two layers are completely negative (Fig. 3). The results yielded by application of the 0.1% PTA in 10 % chromic acid method for glycoproteins, according to Rambourg, Hernandez, and Leblond (20), appear to be significant. By this method (Fig. 4), a continuous electron opaque halo up to 90 A thick can be seen external to the outer leaflet of the sperm plasma membrane. This layer is thicker than the membrane proper and often merges with a similar coat on neighboring spermatozoa. It is quite evident where heads or tails of spermatozoa are closely apposed (Fig. 5). This selective staining with the Rambourg, Hernandez, and Leblond method suggests the presence of glycoprotein.
FIG. 1. Cross section of Ceratitis sperm tail showing the asymmetrical trilaminar plasma membrane (arrow). x 180 000. FIG. 2. Cross section of Ceratitis sperm tail showing the localization of acid phosphatase among the fibrillar elements on the plasma membrane (arrow). × 110 000. FIG. 3. Cross section of Ceratitis sperm tails treated with Thi6ry's method: A positive reaction is localized at the level of the relatively thick outer layer of the plasma membrane (arrows). x 120 000.
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FIcs. 4 and 5. Cross section through the tail (Fig. 4) and the head (Fig. 5) of Ceratitis sperms treated with the PTA method of Rambourg, Hernandez, and Leblond (20). A positive reaction is seen at the level of the plasma membrane and in the interspaces among the sperm cells. × 120 000.
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FIGS. 6-8. Freeze-etched preparations of Ceratitis spermatozoa. The outer sperm surface (O) appears smooth; below it, a longitudinally oriented granular component (GR) is evident, x 60 000.
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The plasma membrane was negative after histochemical reactions. Most of the histochemical assays performed by us were for enzyme activities for ATPase, glucose6-phosphatase, and peroxidase. However, with the Gomori method for acid phosphatase, a heavy deposit was formed on both the outer and the inner electron opaque layers (Fig. 2). As a rule, the light interspace was unreactive. The outer surface of the spermatozoon appears quite smooth under the scanning electron microscope and in freeze-etched preparations (Figs. 6 and 7). In the latter, a granular component is clearly demonstrated beneath the outer surface (Figs. 6 and 7), but separated from the sperm organelles by a thin membrane. We assume that this material is located between the two limiting layers of the cell membrane, which according to Meyer and Winkelmann's (14) interpretation become separated from each other by the etching processes. In some of our replicas, this is quite evident, the sperm membrane having been fractured in profile (Fig. 8). In this case, amorphous material is observed upon the membrane, which corresponds to the glycoprotein coat. The foregoing observations reveal the plasma membrane of fruit fly spermatozoon as a typical cell membrane, with its outer layer slightly thickened by an overlying glycoprotein coat.
The flea type membrane The flea (Ctenocephalus canis; Insecta, Aphaniptera) spermatozoon is limited by a m e m b r a n e about 2130 A thick in which two sharply differing layers can be observed (Fig. 9). The innermost one is a typical plasma membrane with an overall thickness of 70 ~ . External to this is a layer about 130 A thick which has different appearances depending upon the plane of section. In sections transverse to the long axis of the spermatozoon, it appears amorphous. In midline longitudinal sections, it displays an array of globules 130 A in diameter with an electron-opaque cortex and a light core. In oblique or parasagittal longitudinal sections, it is clearly striated, being made up of short, parallel electron-opaque bars about 70 N thick, separated by light interspaces of the same thickness. Where the section just grazes the sperm surface, this outer coating appears cross-striated throughout its width. The flea spermatozoon is helically coiled with a pitch of 1.7 # (1), resulting in totFIGS. 9-12. Longitudinal sections of the Ctenoeephalus sperm membrane. FIG. 9. The two layers forming this type of membrane are evident; the typical plasma membrane (p) and the outer array of cross-sectioned fibers (f) with an electron-opaque wall and a light core. x 150 000. FIG. 10. Acid phosphatase activity (arrows) appears localized both on the inner plasma membrane and on the outer cell coat. x 80 000. FIG. 11. The treatment with Thi6ry's method reveals a slight deposit around the fibers of the surface coat (arrows). x 120 000. FIG. 12. The method of Rambourg, Hernandez, and Leblond (20) reveals a strongly positive reaction at the level outer cell coat (arrows). x 120 000.
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FIG. 13. Sperm tail of Ctenocephalus photographed by the scanning electron microscope. A striated pattern cannot be resolved, x 60 000. FI~. 14. Cell membrane of Ctenoeephalus spermatozoa fragmented and negatively stained. The cross striation (arrows) is evident. × 90 000. F ~ . 15. Freeze-etched preparation of Ctenoeephalus sperms. The striations of the outer sperm surface (O) and the granulated space of the cell membrane (GR) are evident, x 60 000.
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Fio. 16. Cross section of Pezotettix sperm tail showing the two layers forming the sperm membrane: an inner trilaminar plasma membrane (P)and the outer coat formed by closely aggregated rods (C). x 120 0 0 0 .
sion of the v a r i o u s c o m p o n e n t s of the flagellum, b u t these can never be seen simultan e o u s l y in the same transverse section (19). Therefore, the cross striations of the c o a t o v e r l y i n g the p l a s m a m e m b r a n e are never at right angles to the l o n g i t u d i n a l axis of the s p e r m since they are n o r m a l to the axis of the i n d i v i d u a l gyres of the helix. This i n t e r p r e t a t i o n is c o n f i r m e d b y observations on m e m b r a n e fragments negatively
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FIG. 17. Cross section of Pezotettix sperm tails treated with Thi6ry's method. The trilaminar plasma membrane appears positive with this method (arrow). x 120 000. F i t . 18. Pezotettix sperms treated with the PTA method of Rambourg, Hernandez, and Leblond (20). The reaction is positive on the outer coat and around the coarse fibers and inner central pair. x 120 000.
stained with potassium phosphotungstate, where the cross-striations stand out very clearly (Fig. 14). However, the PTA-infiltrated interspaces look narrower (under 50 A) while the unstained bars are wider (about 100 A) than in sectioned material. Nonetheless, the period resulting from the sum of a bar and an interspace is the same in both cases, that is, little below 140 A. This structure, however, is not very well resolved with the scanning electron microscope, for the limited power of resolution of this instrument. More information is given by the freeze-etching method. With this method, the striation of the outer surface of the sperm cell is quite evident, and the limiting membrane is resolved as two juxtaposed components (Fig. 15). The granular component lying subjacent to the cell membrane has definite orientation. Its subunits are arranged in lines that run helically in a direction opposite to that of the outer striae but coinciding with that of the mitochondrial helix, and it is related to the coiled configuration of the whole spermatozoon. Information available about the chemical nature of the two different components making up the membrane of this sperm type is scant but nevertheless significant. Neither layer is preferentially digested by amylase, pepsin or trypsin, even when other components of the same spermatozoon are eliminated by these enzymes. When Thi6ry's method is applied for as long as 24 hours, a slight deposit is seen to form around the striae of the surface coating (Fig. 11).
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FIG. 19. Acid phosphatase activity in Pezotettix sperm tails. The reaction appears positive on the plasma membrane (arrows). x 120 000. P T A treatment at p H 3 according to Rambourg, Hernandez, and Leblond (20) for demonstration of glycoproteins gives a strongly positive reaction of the level of the electron opaque bars (Fig. 12). In longitudinal sections where this layer appears to be composed of globular subunits (each bar being transversely cut), positive reaction
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BACCETTI, BIGLIARDI AND ROSATI
takes the form of a wide halo surrounding each globular profile. Among the enzymes sought by us, only acid phosphatase was found to occur in these layers. With the Gomori method, in fact, a heavy electron opaque deposit is found both on the inner cell membrane and over the outermost striated glycoprotein layer (Fig. 10). From these observations, it may be inferred that in Ctenoeephalus spermatozoon, the cell periphery is made up of a typical plasma membrane coated by a thick glycoprotein layer which consists of filaments of uniform caliber and orientation, and rich in acid phosphatase activity, like the plasma membrane.
The locust type of membrane Spermatozoa of Pezotettix giornai and Aiolopus strepens (Insecta, Orthoptera) are bounded by a membrane about 400/~ thick in which two layers can be identified: an inner unit membrane about 100 A in thickness overlain by a conspicuous coating, 300 A thick (Fig. 16). The inner plasma membrane is of the classical asymmetric triple-layered structure described above for Ceratitis. Proceeding from the inside to the outside, there is an inner electron opaque layer 20 ~ thick, a light interspace 30 ,~ thick, and a highly electron dense outer layer some 40-50 A thick. On the surface of this latter layer, a slight deposit is revealed by Thi6ry's thiosemicarbazide method applied for 24 hours (Fig. 17), and a heavy reaction for glycoproteins is given by PTA, pH 0.3 according to the Rambourg, Hernandez, and Leblond method (20) (Fig. 18). The Gomori method for acid phosphatase is also strongly positive at this level (Fig. 19). The 300 A thick coating rests upon this glycoprotein layer of the plasma membrane. In both transverse and longitudinal sections, this coating is seen to be composed of electron dense rodlets about 70 A thick and 300 A long oriented radially and closely packed about 70 A apart (Figs. 20 and 21). These rodlets can be seen in their entirety only in favorably oriented sections where they may occupy a long stretch of the spermatozoon edge. In cross sections they are detectable only on the two sides of the sperm, those lying against the flagellum and the two mitochondrial derivatives (Fig. 16). The optical diffraction pattern of this material (Fig. 22) confirms the presence of periodic rodlike units perpendicularly arranged on a basal surface. Under the scanning electron microscope, these rodlets appear not resolved, but the study of sections suggests that the rodlets are slanted in an anterior direction on one side and in a posterior direction on the other side, and perpendicular to the longitudinal axis of the sperm in the intervening regions. If we designate as the basal plane of the sperm that upon which the two mitochondrial derivatives lie, and as the axis of symmetry the median plane normal to it, it may be observed that the rodlet arrangement is oriented with respect to these two planes rather than to the plane containing the two central tubules (9). The plane of the central pair of tubules is tilted by about
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FIG. 20. Cross section of P e z o t e t t i x sperm membrane (at left) and tangential section at the level of'the outer coat (at right). The trilaminar plasma membrane (P) and outer coat (C) are evident. × 120 000. FIG. 21. Transverse section of P e z o t e t t i x sperm membrane at higher magnification; P, plasma membrane; C, outer coat. x 180 000. FIG. 22. Optical diffraction pattern of the same material. 39 - 711838 J . Ultrastructure Research
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20 ° with respect to the normal one. The rodlets composing the outer coating are devoid of enzymatic activity and are resistant to digestion with amylase or pepsin. The study of replicas obtained by the freeze-etching method lends support to the above described structure (Figs. 27 and 28). The plasma membrane is 100 A thick and dotted with minute globular granules about 50 A in diameter on both the inner and outer surfaces. The outermost layer is generally visualized in cross section alone, where it merely displays its 300 A thickness without revealing the rodlet pattern. Examination of flakes of membrane with their coating negatively stained by potassium phosphotungstate (Fig. 24) is also rewarding. With this procedure, the rodlets are seen from above and appear to be arranged in tetrads. Each rodlet is about 70 A thick. They lie at distances varying from less than 20 A apart, in the case of the four units of a single tetrad, to about 70 A between one tetrad and another. In each tetrad, the four rodlets are arranged in a rhomboidal configuration. The various tetrads are also oriented along lines forming a rhomboidal lattice. By tilting the specimen (Figs. 23 and 25) the four units of each tetrad, appearing as spots in frontal view, appear clearly elongated. The optical diffraction pattern (Fig. 26) of the same specimen confirms that the lattice units in frontal view are regularly disposed in tetragonal arrangement. Pezotettix and Aiolopus spermatozoa therefore appear coated by a typical plasma membrane which is made asymmetrical by an amorphous outer coating of glycoprotein and further protected by an array of closely packed glycoprotein filament on rodlets which are radially arranged or slanting obliquely forward on one side and backward on the other. It is of interest to trace the origin of this patterned coating. Spermatids are provided with a conspicuous glycocalyx (Fig. 29). During their evolution this glycoprotein coating grows thicker and begins to differentiate into filamentous or rodlike units which tend to be arranged perpendicular to the plasma membrane, but they do not acquire their individuality until the spermatozoon is almost mature (Figs. 30-32.) P The "locust type" model of sperm membrane is not common to all the Orthoptera species. The most primitive families (as Phaneropteridae, Tettigoniidae, Gryllidae, Gryllotalpidae) examined by us have the usual "fruit fly" model membrane. The "locust" model is therefore restricted to acridoids, and it is important to note that it is well developed only in the highest families examined, i.e., Catantopidae (Pezotettix) and Acrididae (Aiolopus), whereas in the most primitive acridoids, the Australian group Morabinae, the characters appear less evident. The rodlets (Fig. 34) are in fact only 100 A high. FIGS. 23-25. Cell membrane of Pezotettix sperm fragmented and negatively stained. The rodlet projections seem to be arranged in tetrads (arrow), and appear as spots in frontal view (Fig. 24), but become elongated if the specimen is tilted + or -20 ° (Figs. 23 and 25). x 360 000.
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FI~. 26. Optical diffraction pattern of Fig. 24. DISCUSSION F r o m all the observations reported here, the limiting membrane of the insect spermatozoon clearly emerges as being of an asymmetric type. There is in fact a "unit m e m b r a n e " of the classical type, displaying intense phosphatase activity, on which is superimposed a glycoprotein coating which has evolved in different ways in different species, and which we have classified in three patterns of progressive structural complexity. The first (the "fruit fly" type) in which the glycoprotein coating is very thin (40 A_) and amorphous, the second (the "flea" model) where it is thicker and formed of transverse fibres displaying phosphatase activity, and the third (the "locust" type) in which the extracellular glycoprotein coat is very thick (about 300 A_) and made u p of rodlets which are grouped in tetrads in a rhomboidal arrangement. The orientaFIGS. 27 and 28. Samples of Pezotettix sperms obtained by the freeze-etching method. The cell coat (C) disposed upon the granular space of the cell membrane (GR) is clearly seen. x 120 000.
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FIG. 29. Cross section of Pezotettix giornai spermatids. Note the conspicuous glycocalyx around the cell (arrow). M, mitochondria, F, flagellum, x 60 000.
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FIGS. 30-32. Stages of evolution of glycocalyx in Pezotettix spermatids. The glycocalyx first (Fig. 30) differentiates filamentous units (arrows) which tend to be arranged perpendicularly to the plasma membrane (Fig. 31). Only in almost mature spermatozoa (Fig. 32) do these rodlets acquire their final arrangement and become embedded in a rich glycoprotein material. P, Plasma membrane; C, coat of rodlets. Figs. 30 and 31, x 120 000; Fig. 32, x 60 000.
602
BACCETT1, BIGLIARDI AND ROSATI
tion of these rodlets is usually normal to the spermatozoon surface; on one side of the sperm, however, they are slanting in a forward direction and on the opposite side in a backward direction, at an angle of some 40 ° to the perpendicular plane. The increasing structural complexity of the spermatozoon plasma membrane is probably related to the biology of the particular sperm. In some spermatozoa (spiders, pseudoscorpions, opilionids, etc.), which are ejaculated within cysts, even thicker walls are known (2, 21, 28). The nature of these has yet to be investigated. Myriapod sperms are even enclosed in pairs in individual spermathecae (11) whose nature and origin are as yet unknown. Nearly all cells have a glycoprotein coating, the "glycocalyx," which is endowed with special properties, particularly at the cell-to-cell junctions. At the free cell sur-
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Fro. 33. Schematic drawing of a segment of Pezotettix sperm tail. faces, the glycocalyx merely thickens (as on microvilli) or gives rise to filaments arranged in a disorderly fashion (as on protozoa), but at junctions it forms specialized dense patches occupying the central part of desmosomes. Recent investigations (17) have suggested that the insect septate desmosomes are glycoprotein laminae oriented transversely between adjacent cells. It is tempting to relate the possible presence of septate desmosomes to the thickened glycoprotein coverings found around the spermatozoa of the same animal groups. A convincing parallelism between the extracellular coating of the locust type of spermatozoa and the structure of septate desmosomes can be established by comparing the images of negatively stained preparations reported here with tangential sections of desmosomes photographed by Locke (13). The significance of this desmosome-like specialization of the membrane surrounding the spermatozoa is rather obscure. There is possibly some relationship between their occurrence and the way these spermatozoa are anchored to one another in animals that ejaculate spermatozeugma or spermodesmata. The most intriguing aspect of these studies lies in the striking symmetry and ordered structure of the glycoprotein layer overlying the unit membrane. Some images bring to mind the hexagonal subunits of the negatively stained plasma membrane of mammalian cells described by Benedetti and Emmelot (5). There are, however, conspicuous differences between these two membranes in the center-to-center spacing of the subunits. Therefore, it appears unlikely that a pattern within the plasma membrane induces or imposes a similar
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BACCETTI~ BIGLIARDI AND ROSATI
FIG. 34. Cross section of Moraba viatica sperm tail, showing the limited height of the outer coat. x 120 000. pattern on the overlying glycoprotein material. The spatial arrangement of subunits in the latter is evidently acquired independently and is specific for each type. The authors thank Professor Don Fawcett for the help in editing the manuscript; Professor E. L. Benedetti for discussing the results; Professor D. Bocciarelli Steve for the opportunity to use the optical diffraction apparatus of the Istituto Superiore di Sanit~t, and Franco Tangucci and Carlo Ramoni for the excellent technical assistance. This research was supported by a grant from Consiglio Nazionale delle Ricerche.
REFERENCES 1. 2. 3. 4. 5.
BACCETTI,B., Redia 51, 153 (1968). BACCETTI,B., DALLAI,R. and ROSATI,F., Or. Cell Biol. 44, 3 (1970). BAIRATI,A. and BAIRATI,A., JR., Protoplasma 63, 283 (1967). BENEDETTI,E. L. and EMMELOT,P., J. Cell Biol. 38, 15 (1968). -it/DALTON, A. J. and HAGENAU,F. (Eds.), The Membranes, Academic Press, New York, 1968.
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6. BENNET,H. S., J. Histochem. Cytochem. 11, 14 (1963). 7. BIGUARDI,E., BACCETXI,B. BURRINI,A. G. and PALLINg,V. In BACCETT~,B. (Ed.), Comparative Spermatology. Accademia dei Lincei, Rome-Siena, t970. 8. DANIELLI,J. R. and DAVSON, H. A., J. Cell Comp. Physiol. 5, 495 (1935). 9. FAWCETT,D. W., J. Cell Sci. 3, 187 (1968). 10. FINEAN,J. B., Quart. Rev. Biophys. 2, 1 (1969). 11. HORSTMANY,E. and BREUCKER,H., Z. Zellforsch. Mikrosk. Anat. 96, 505 (1969). 12. KAVANAtr,J. L., Structure and Function in Biological Membranes, Vol. II, HoldenDay, San Francisco 1965. 13. LO¢~,_E,M., J. CellBiol, 25, 166 (1965). 14. MEYER, H. W. and WINKELMANN,H., Protoplasma 68, 253 (1969). 15. MooR, H., Mi2HLETHALER,K., WALDNER, H. and FREY-WYsSLrNG, A. J., Biophys. Biochem. Cytol. 10, 1 (1961). 16. MORRIS, B., CANDIOTTr,A. and FABRO, J., Z. Zellforsch. Mikrosk. Anat. 99, 64 (1969). 17. NOmOT, C. and NOmOT TIMOXH~E,C., J. Microsc. (Paris) 8, 72a (1969). 18. PEROTTI,M. E., J. Submicrosc. Cytol. 1, 171 (1969). 19. PmLLIPS, D. M., J. Cell Biol. 40, 28 (1969). 20. RAMBOURG,A., HERNANDEZ,W. and LEBLOND,C. P., J. Cell Biol. 40, 395 (1969). 21. REGER, J. F., J. Ultrastruct. Res. 28, 422 (1969). 22. REVEL, J. P., J. Microscop. (Paris) 3, 535 (1964). 23. REVEL, J. P. and Ixo, S., in DAvis, B. D. and WARREN, L. (Eds.), The Specificity of Cell Surfaces, p. 211. Prentice-Hall, Englewood Cliffs, New Jersey, 1967. 24. ROBERTSON, J. D., in NACHMANSOHN, D. (Ed.), Molecular Biology. Academic Press, New York, 1960. 25. SJ6STRANO,F. S., Electron microscopy of myelin and nerve cells and tissues. In Ct~MrNGS, J. N. (Ed.), Modern Scientific Aspects of Neurology, p. 188. Arnold, London, 1960. 26. - in DALTON, A. J. and HAGENAU, F. (Eds.) Ultrastructure in Biological Systems, p. 151. Academic Press, New York, 1968. 27. STOECKENmS,W. and EN~ELMAN,D. M., J. Cell BioL 42, 613 (1969). 28. TUZET, O., MANIER, J. F. and BOISStN, L., C. R. Acad. ScL Ser. D. 262, 376 (1966). 29. WEiss, L., lnt. Rev. Cytol. 26, 63 (1969).