J O U R N A L OF U L T R A S T R U C T U R E R E S E A R C H
67, 180-193 (1979)
A Thin Section and Freeze-Fracture Study on Membrane Specializations
in Spermatozoa of the Isopod, A r m a d i l l i d i u m v u l g a t e 1 JAMES F.
R E G E R , PATRICIA W . ITAYA, 2 A N D MALINDA E . FITZGERALD
Department of Anatomy, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Received January 3, 1979 Mature spermatozoa of the Isopod, Armadillidium vulgare, were examined following tannic acid processing, thin section, and freeze-fracture techniques. They reveal a moderately thick glycocalyx and a typical random distribution of 5- to 10-nm-size, plasmalemmal, PF-face particles. Seven distinct areas of particulate membrane specializations occur in the spermatozoa. Five regions occur in the plasmalemma and two occur in the acrosomal membrane. The plasmalemmal specializations include: (1) clusters of 8- to 10-nm-size, PF-face particles found along a line made by the posterior margin of the acrosomal membrane; (2) orthogonal arrays of 4- to 6-nm-size EFface particles similarly disposed; (3} a linear array of 8- to 10-nm-size, PF-face particles extending the length of the acrosome, along the region where the paddle-shaped acrosome becomes thinner; (4) a cluster of 8- to 10-nm-size, PF-face particles on the cytoplasmic hood, which surrounds the insertion region of the cross-striated, taft-like appendage; and (5) a cross-linear array of 6- to 8nm-size, PF-face particles which repeat every 67 nm along the cross-striated taft-like appendage, mimicking the cross-striated repeat period exhibited by the filamentous component as seen in thin sections. The acrosomal membrane specializations include: (1) tightly packed, 8- to 10-nm-size, EF-face particles limited to the thin portion of the paddle-shaped acrosome; and (2) linear arrays of periodically repeating (25-26 nm), 8- to 10-nm-size, PF face particles limited to the thick portion of the acrosome whose periodic repeat mimics the paracrystalline order of the internally directed portions of the unit membrane in this region. The results of this study are discussed in terms of the functional significance of such specializations. It is concluded that the function of each group of particle arrays is unique, and is in fact a reflection of either their topographical relationships with other membrane specializations or their possible role in fertilization. Particular attention is drawn to the functional significance of the following groups of particles: (1) the double set of particle arrays in the postacrosomal region, (2) the cross-linear arrays which mimic the crossstriated repeat pattern of longitudinally oriented filaments in the cross-striated appendage seen in thin sections, and (3) the two sets of particles in the PF and EF faces of the acrosomal membrane.
Since the initial freeze-fracture studies on membrane structure of bovine spermatozoa (Blom and Birch-Andersen, 1965; Koehler, 1966) several studies have appeared on vertebrate sperm. These include Spermatozoa from rabbit (Koehler, 1970a, b; Flechon, 1974), rainbow trout (Koehler, 1970b), human (Koehler, 1970a, 1972), bull (Plattner, 1971), water buffalo (Koehler, 1973), guinea pig (Friend and Fawcett, 1974; Friend and Rudolf, 1974; Koehler and Gaddum-Rosse, 1975; Fawcett, 1977), mouse (Stackpole and Devorkin, 1974), rat (Friend
and Fawcett, 1974; Friend and Rudolf, 1974), and opossum (Olson et al., 1977; Fawcett, 1977). The findings in these various animals demonstrate the following. First, a series of double or multiple rows of longitudinally arranged, approximately 9-nmsize, plasmalemmal PF-face particles occurs in the principle piece. Second, circumferentially or helically arranged rows of 6to 8-nm-size plasmalemmal PF-face particles occur in the midpiece. Third, paracrystalline arrays of larger, 10- to 25-nm-size, particles occur in the acrosomal membrane EF face of rabbit (Koehler, 1970b), guinea pig (Friend and Rudolf, 1974; Friend and Fawcett, 1974), rat (Friend and Fawcett,
Supported by NSF Grant No. PCM 76-16961. 2 Present address: Department of Anatomy, University of Iowa, Iowa City, Iowa 52240. 180 0022-5320/79/050180-14502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
FREEZE-FRACTURE OF ISOPOD SPERMATOZOA 1974), a n d h u m a n ( K o e h l e r , 1972) s p e r m a tozoa, in t h e n u c l e a r m e m b r a n e o f g u i n e a pig a n d r a t ( F r i e n d a n d F a w c e t t , 1974), a n d in c e r t a i n r e g i o n s o f t h e p l a s m a l e m m a o f guinea p i g a n d r a t ( F r i e n d a n d F a w c e t t , 1974). F o r a m o r e c o m p l e t e e v a l u a t i o n o f c o m p a r i s o n s of f r e e z e - f r a c t u r e c o m p o s i t i o n in g u i n e a pig a n d r a t e p i d i d y m a l s p e r m , see F r i e n d a n d F a w c e t t (1974). C o n c l u s i o n s about t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e s e various p a r t i c u l a t e o r g a n i z a t i o n s a r e of necessity l a r g e l y s p e c u l a t i v e a t t h i s t i m e . However, c o m p a r a t i v e o r e x p e r i m e n t a l studies o n v a r i o u s t y p e s of s p e r m a t o z o a should b e of s o m e a s s i s t a n c e in e x p a n d i n g our i n s i g h t s i n t o t h e f u n c t i o n s o f t h e s e v a r ious p a r t i c u l a t e a r r a y s . Only a v e r y l i m i t e d n u m b e r o f s t u d i e s , b y correlative t h i n s e c t i o n a n d f r e e z e - f r a c t u r e technique, h a v e b e e n c a r r i e d o u t o n i n v e r tebrate spermatozoa. These include the e a r t h w o r m , Lumbricus terrestris (Bergstrom a n d H e n l e y , 1973); t w o s p e c i e s of D i p l o p o d a , Polyxenus lagurus a n d Pachyjulus sp. ( B a c c e t t i et al., 1974; 1977); t h e tick, Amblyomma hebraeum ( W u e s t et al., 1978); a n d t h e D i p l o p o d a n , Spirobolus sp. (Reger a n d F i t z g e r a l d , 1979). T h e l a t t e r is the o n l y c o m p l e t e s t u d y on all m e m b r a n e s of an i n v e r t e b r a t e s p e r m a t o z o o n . T h e following s t u d y o n all m e m b r a n e s o f a r e p r e sentative i s o p o d , ArmadiUidium vulgare, was u n d e r t a k e n n o t o n l y t o fill t h e g a p in such s t u d i e s o f i n v e r t e b r a t e s p e r m a t o z o a , but m o r e i m p o r t a n t l y , t o d e m o n s t r a t e s o m e of t h e r a t h e r u n i q u e s p e c i a l i z a t i o n s in t h i s type of c r u s t a c e a n s p e r m a t o z o o n . W h e r e possible t h e d a t a will b e c o m p a r e d to t h o s e a r r a y s f o u n d in m o r e t y p i c a l v e r t e b r a t e s p e r m a t o z o a in a n e f f o r t to d r a w f u n c t i o n a l correlates. MATERIALS AND METHODS Following ventral orientation of male isopods, Armadillidium vulgare, an incision was made in the thoracoabdominal cavity, and testes were removed and placed in cold (0-5°C) 4% glutaraldehyde (pH 7.6, 0.2 M sodium cacodylate buffer). Tissue was then dissected into small pieces and fixed for an hour. After fLxation in glutaraldehyde, tissue was processed for
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either thin sectioning or freeze-fracture techniques as follows. Tissue for thin sectioning was washed for an hour in cold (0-5°C) sodium cacodylate buffer (pH 7.6, 0.4 M) and postfixed for an hour in cold (0-5°C) 2% OsO4 (pH 7.6, 0.4 M cacodylate buffer ). Some tissues were then processed for an hour in 1% tannic acid (TA; Mallinkroff, AR, code No. 1764) in cacodylate buffer (pH. 7.6, 0.4 M; Simioneseu and Simionescu, 1976). Subsequently all tissue was dehydrated in 5-rain changes of successive grades of methanol (beginning with 50%) and embedded in Epon 812. Sections, 1-2 #m thick, were made of the Epon-embedded tissue and stained with Mallory azure II-methylene blue, for purposes of locating particular stages of spermiogenesis and mature spermatozoa in various regions of the vas deferens prior to thin sectioning. Thin sections were cut with a diamond knife fitted to an LKB Ultratome II, floated on distilled water, mounted on carbon-coated grids, and stained with uranyl acetate and lead citrate. Tissue for freeze-fracture was also fixed in 4% glutaraldehyde (pH 7.6, 0.2 M sodium eacodylate buffer) as above and subsequently equilibrated in a series of 10, 15, and 25% glycerols in buffer (0.2 M sodium cacodylate). Following 1 to 2 hr in 25% glycerol, tissue was mounted preparatory to being introduced into a Balzers 301 freeze-fracture device for fracturing and replication. Following replication, the replicas were cleaned with either 50% chromic acid or purex to digest tissue. Replicas were then washed with distilled H20 and mounted on copper grids for observation. Thin sections and replicas were examined with a Hitachi HU l l A electron microscope. Micrographs were taken at initial magnifications of 5000 to 50 000 × at exposures of 2 to 4 sec and were made on Cronar, Ortholitho Type A sheet film. Negatives were enlarged up to 10 times with a Durst S-45 enlarger equipped with a 250-W mercury vapor point source. OBSERVATIONS The morphology of the mature spermat o z o o n o f t h e i s o p o d , Armadillidium vulgare (Fig. 1), is s i m i l a r to t h a t o f o t h e r p e r i c a r i d e s . T h e s p e r m a t o z o o n is a n elong a t e cell, a t o n e e n d of w h i c h a r e s i t u a t e d t h e a c r o s o m e (A, Fig. 1), t h e n u c l e u s (N, Fig. 1), t w o c e n t r i o l e s (C, Fig. 1), a n d a r o d s h a p e d p e r f o r a t o r i u m (P, Fig. 1). A n e l o n gate, c r o s s - s t r i a t e d a p p e n d a g e (TA, Fig. 1) extends from the base of the acrosome near its i n d e n t a t i o n b y t h e p e r f o r a t o r i u m (P, Fig. 1). W h e n o b s e r v e d in vivo t h e e l o n g a t e , c r o s s - s t r i a t e d a p p e n d a g e ( T A , Fig. 1) a p p e a r s stiff a n d inflexible, w h i l e t h e a c r o s o -
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FIG. 1. Schematic diagram of a mature spermatozoon of Armadillidium vulgare demonstrating the acrosome (A), perforatorium (P), centrioles (C), nucleus (N), and cross-striated, tail-like appe~adage(TA). Cross-sectional levels and regions identified in subsequent electron micrographs are appropriately labeled. (1, 2, 3) Section levels seen in Figs. 5 and 6.
mal and nuclear regions appear flexible. Thus the spermatozoon resembles a whip, with a cross-striated appendage serving as the handle, and the acrosomal-nuclear region serving as the lash of the whip. The acrosomal region is paddle shaped and, when seen in cross section, has a thick and a thin portion (Tk, Th, Fig. 6). The nucleus exhibits no unusual characteristics and contains the usual longitudinally oriented DNA protein filaments. The details of the fine structure of spermatozoa from several species of pericarides have previously been
described (Reger, 1964, 1966, 1969, 1970, 1971; Reger et al., 1977; Fain-Maurel, 1966, 1969; Fain-Maurel et al., 1975a, 1975b; Cotelli et al., 1976) and, except for the details of plasmalemmal and acrosomal membrane specializations, will not be dealt with further here. The plasmalemma is characterized by a typical, unit membrane (P1, Fig. 2), a moderately thick glycocalyx (G1, Figs. 5 and 6), and scattered 8- to 10-nm-size, PF-face particles (PPF, Fig. 7) (Branton et al., 1975). The glycocalyx, which is highlighted by tannic acid (G1, Figs. 5, 14, and 15), is characterized by homogeneously distributed, electron-opaque particles. Unique, particulate patterns occur in the plasmalemma of the acrosomal region (Figs. 7 and 8) and along the entire length of the cross-striated appendage (Figs. 8 and 13). Plasmalemmal specializations in the acrosomal region include four groups of particles. The first is a linear array of 8- to 10-nm-size, PF-face particles (arrows, Fig. 7) situated the length of the transition area between the thick (Tk, Fig. 6) and thin (Th, Fig. 6) portions of the paddle-shaped acrosome. The second is a linear array of 4- to 6-nm-size, EF-face particles grouped in square lattices of various sizes (arrows, Fig. 9; arrows, inset, Fig. 9) which extend the length of a line made by the caudally curved acrosomal membrane (see also dotted arrows, Fig. 4). The third is a linear array of 8- to 10-nm-size, PF-face particles (thin arrows, Fig. 8) following the same line made by the caudally curved, acrosomal membrane. Some of these mimic the paracrystalline order (thick arrows, inset, Fig. 8) of the smallersized paracrystalline EF-face particle aggregates. The fourth consists of grouped, 8to 10-nm-size, PF-face particles which occur on the sleeve which wraps around the base of the cross-striated appendage (GP, Fig. 8) at its point of origin from the acroo somal region of the spermatozoon (TA, Fig. 8). The particulate, plasmalemmal specializations of the cross-striated appendage
FREEZE-FRACTURE OF ISOPOD SPERMATOZOA consist of cross-linear a r r a y s of 6- to 8-nmsize, P F - f a c e particles ( P P F , Fig. 13). Laterally t h e y are s p a c e d 7-10 n m apart, a n d each row of particles r e p e a t s at approximately 67 n m intervals, reflecting t h e repeat p a t t e r n exhibited b y t h e filamentous, cross-banded a p p e n d a g e as seen in t h i n sections (arrows, Figs. 14 a n d 15). T h e glycocalyx (GL, Fig. 14) p r e s e n t s a granular, stippled a p p e a r a n c e (GL, Fig. 15) following tannic acid processing. The a c r o s o m a l m e m b r a n e (A, Figs. 4, 6, and 7) also exhibits specializations seen in both thin section (Figs. 3, 4, a n d 6) a n d freeze-fracture (Figs. 7-10) images. B o t h longitudinal (Figs. 3 a n d 4) a n d cross-sectional (Fig. 6) views of t h i n sections d e m onstrate a striated p a t t e r n . W h e n this region is seen in t a n g e n t i a l section (Fig. 4), it exhibits a p a r a c r y s t a l l i n e order. T h e paracrystalline o r d e r is c o n f i n e d to t h e t h i c k e r portion of t h e a c r o s o m e (arrows, Fig. 6). The a c r o s o m a l m e m b r a n e in t h e t h i n n e r portion of t h e p a d d l e - s h a p e d a c r o s o m e practically fuses w i t h t h e p l a s m a l e m m a (arrows, inset, Fig. 2). F r e e z e - f r a c t u r e images of t h e a c r o s o m a l m e m b r a n e in t h e thick a n d t h i n p o r t i o n s reveal two sets of particles (Figs. 9, 10, a n d 11). One set consists of linear a r r a y s of periodically r e p e a t ing (25-26 nm), 8- to 10-nm-size, P F - f a c e particles (P, Fig. 11) o c c u r r i n g in the thicker p o r t i o n of t h e a c r o s o m e , w h o s e re-
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p e a t period laterally a n d longitudinally m i m i c s t h a t of t h e i n n e r l a m i n a r projections (28-30 n m ) of t h e a c r o s o m a l u n i t m e m b r a n e seen in t h i n sections (see Figs. 3 a n d 4). T h e s e c o n d set of particles consists of a h e a v y c o n c e n t r a t i o n of 8- to 10-nmsize, E F - f a c e particles (Figs. 9 a n d 10) limited to t h e t h i n p o r t i o n of t h e paddles h a p e d acrosome. DISCUSSION The randomly distributed plasmalemmal P F - f a c e particles in t h e a c r o s o m a l a n d nuclear regions of t h e s p e r m a t o z o o n of Arr n a d i l l i d i u m vulgare are similar in size a n d relative d i s t r i b u t i o n to similar particles f o u n d in o t h e r cells. A l t h o u g h t h e precise b i o c h e m i c a l c o m p o s i t i o n a n d functions of t h e s e particles are still o p e n to question, it is b e c o m i n g increasingly e v i d e n t t h a t t h e y represent intercalated membrane proteins w i t h or w i t h o u t associated lipids or carboh y d r a t e s (Pollack, 1978). F o r example, certain b i o c h e m i c a l c h a r a c t e r i s t i c s h a v e b e e n described for e r y t h r o c y t e s (Bretscher, 1971; P i n t o da Silva et al., 1971, 1973; Guidotti, 1972; M a r c h e s i et al., 1972; Tillack et al., 1972; R u b i n et al., 1973) a n d in t h e sarcop l a s m i c r e t i c u l u m of c e r t a i n v e r t e b r a t e skeletal m u s c l e s ( D e a m e r a n d Baskin, 1969; S t e w a r t a n d M a c L e n n a n , 1974; Tillack et al., 1974; M a c L e n n a n et al., 1971). I n all p r o b a b i l i t y several kinds of p r o t e i n com-
FIa. 2. This longitudinal to slightly oblique section through several spermatozoa demonstrates the unit membranes of the plasmalemma (P1) and acrosome (A) as well as the practically Complete fusion (arrows, inset) of the outer lamina of the acrosomal with the inner lamina of the plasmalemmal unit membranes. One percent tannic acid post-OsO4, 1 hr. × 64 000; inset, × 156 000. FIGS. 3 AND4. Cross-sectional (Fig. 3) and longitudinally tangential (Fig. 4) views of the thick portion of the acrosome (A, Figs. 3 and 4) (see also Figs. 1 and 6) to demonstrate projecting repeat components from the acrosomal inner lamina (arrows, Fig. 3) which, when seen tangentially, appear to have inner connecting elements (arrows, Fig. 4), thus presenting an appearance of paracrystalline order. (dotted arrows, Fig. 4) Posterior curved end of the acrosomal membrane. Late spermatids. Fig. 3, x 48 000; Fig. 4, x 70 000. Fro. 5. Low-power, cross-sectional view of spermatozoa sectioned at different levels. For example, compare levels of sections 1, 2, and 3 with levels 1, 2, and 3 seen in Figs. 1 and 6. Gl, glycocalyx. One percent tannic acid post-OsO4, 1 hr. x 32 000. FIG. 6. Higher-magnification view of spermatozoa cross sections. Note that the region of origin of the taillike appendage (TA) at the outer edge of the appendage is infolded within an indentation of the adjacent portion of the cell (arrows). Note also the faintly striated pattern along the inner acrosomal membraile (A) (see also Figs. 3 and 4). G1, glycocalyx; Tk and Th, thick and thin portions of the paddle-shaped acrosomal region. (1, 2, 3) Section levels corresponding to those seen in Figs. 1 and 5. One percent tannic acid post-OsO4, 1 hr. × 64 000.
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FIG. 7. A freeze-fracture image at the acrosomal region near its juncture with the tail-like appendage (TA) demonstrating, from the top to the bottom, the plasmalemmal PF face (PPF), acrosomal PF face (APF), acrosomal EF face (AEF), and plasmalemmal EF face (PEF). Arrows, linear array of particles; A, acrosome. × 64 000.
plexes, and therefore more than one function, m a y be ascribed to the P F particles of various cell types, as is becoming increasingly clear for the e r y t h r o c y t e (Pinto da Silva and Nicolson, 1974). It is of some interest to note here t h a t the randomly distributed, 8- to 10-nm-size, particles seen in this study were n o t found along the length of the cross-striated appendage. T h e significance of such a difference in particle distribution is impossible to precisely assess. It has been assumed t h a t such particles are A T P a s e or ATPase-associated protein complexes, as suggested for erythrocytes (Marchesi et al., 1972) and the sarcoplasmic reticulum of muscle (Deamer and Baskin, 1969). Therefore, their absence in the cross-striated appendage m a y reflect a difference in energy requirements.
T h e m o d e r a t e l y thick glycocalyx, highlighted in the spermatozoa of this study by tannic acid, demonstrates no particular morphological or regional specializations as sometimes found in other spermotozoa (see Baccetti and Afzelius, 1976). T h e glycocalyx m a y be classified as the "fruitfly" type (Baccetti et al., 1971). T h e tannic acid reaction appears as discrete, homogeneously distributed granules in the glycocalyx t h r o u g h o u t the cell surface. Although studies by Kalina and Pease (1971a, b) demonstrated t h a t tannic acid utilized in combination with osmium tetroxide reacts with phosphatidyl cholines, its mode of reaction with the glycocalyx is unknown. It may react with negative charges such as the acidic mucopolysaccharides, as in the case of r u t h e n i u m red (Luft, 1971).
FREEZE-FRACTURE OF ISOPOD SPERMATOZOA
The more highly ordered and specifically distributed plasmalemmal particles observed here include: (1) clusters of 8- to 10nm-size, PF-face particles found along a line made by the posterior margin of the acrosomal membrane; (2) orthogonal arrays of 4- to 6-nm-size, EF-face particles similarly disposed; (3) a linear array of 8- to 10nm-size, PF-face particles extending the length of the acrosome, along the region where the paddle-shaped acrosome becomes thinner; (4) a cluster of 8- to 10-nmsize, PF-face particles on the cytoplasmic hood which surrounds the insertion region of the cross-striated, tail-like appendage; and (5) a cross-linear array of 6- to 8-nmsize, PF-face particles which repeat every 67 nm along the cross-striated, tail-like appendage mimicking the cross-striated repeat period exhibited by the filamentous component as seen in thin sections. Although it is impossible to draw direct conclusions about the functional significance of these various arrays, certain indirectly derived conclusions are appropriate. The most intriguing arrays include the two juxtaposed 8- to 10-nm-size, PF-face and 4- to 6-nm-size, EF-face particles found in the plasmalemma along the posterior margin of the termination of the acrosome.
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Not only were the two sets of particles aligned in two faces of the same membrane along the same line, but suggestive evidence demonstrated that both sets are orthogonally arrayed particles. Definitive conclusions about the functional significance of these arrays or their interrelationship cannot be made. However, this region of the plasmalemma overlies the acrosomal membrane which will fuse with the oolemma during fertilization. It therefore may have unique properties contributing to the fertilization process. Whether or not this region ultimately becomes incorporated into the fertilized oolemma is unknown, but would certainly be worth investigating in view of the particle arrays and their possible significance in the fertilization process. The locale of these arrays is reminiscent of particle specializations found in postacrosomal and equatorial regions of certain vertebrate spermatozoa (Koehler, 1970b; Friend and Fawcett, 1974). The plasmalemma of spermatozoa has been seen to exhibit specializations, such as a serrated pattern in the case of guinea pig spermatozoa (Friend and Fawcett, 1974) and a highly ordered "fine periodic substructure" in rabbit spermatozoa (Koehler, 1972). It is more difficult to assess the functional
FIG. 8. Freeze-fracture replica showing portions of four spermatozoa. The plasmalemmal PF face in the acrosomal region is characterized by randomly distributed particles (P); a linear array of particles situated the length of the acrosomal level at the juncture of the thick and thin portions of the paddle-shaped acrosome (dotted arrows); a group of linearly arrayed particles which follow the posterior level of the acrosome (thin arrows), some of which (thick arrows, enlarged inset) take on a paracrystalline order; and a group of particles which are situated on the apron-like portion of the spermatozoon which envelops the tail-like appendage (TA) at its insertion point (GP). APF, acrosomal PF face. x 32 000; inset, x 64 000. FIos. 9 AND 10. These two freeze-fracture images demonstrate a series of small, 4- to 6-nm-size, paracrystalline-ordered, plasmalemmal EF-face particles (arrows, Fig. 9) which follow the line of the posterior edge of the acrosome as it skirts the edge of the point of the tail-like appendage insertion point (see also arrows, inset). Note also the particle-studded acrosomal EF face (AEF, Figs. 9 and 10) in the flat portion of the paddle-shaped acrosome and the corresponding pitted PF face (APF, Fig. 10). × 64 000; inset, x 64 000. FIG. 11. A freeze-fracture image of portions of the acrosomal PF face (APF) demonstrating that the acrosomal PF-face particles (P) in the thick portion of the paddle-shaped acrosome exhibit a paracrystalline order which mimics the paracrystalline order of the projections from the inner lamina of the acrosomal membrane (See Figs. 3 and 4), while the PF face of the thinner portion of the acrosome (arrows) is pitted corresponding to the distribution of EF-face particles in this area (also see Fig. 9). x 64 000. FIG. 12. A freeze-fracture replica through the nuclear region to demonstrate the sparseness of particles in the plasmalemmal PF face (PPF) and a practically complete lack of particles in the nuclear membranes (NM). x 64 000.
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FREEZE-FRACTURE OF ISOPOD SPERMATOZOA significance of both the linear arrays along the length of the acrosome and the particles grouped along the cytoplasmic hood, which partially enfolds the cross-striated appendage at its point of origin. T h e r e have thus fax been no similarly organized particle arrays described for other spermatozoa. Since both of these sets of particles occur along regions of plasmalemmal curvatures, their presence m a y reflect a function in the maintenance of m e m b r a n e conformation. Previous suggestions along these lines have been made for guinea pig and rat spermatozoa (Friend and Fawcett, 1974) and other cell types (Pollack, 1978). An interesting example of such a possibility m a y be drawn from studies on the midpiece of opossum spermatozoa (Fawcett, 1977). In this study rows of particles are longitudinally directed in the wall of a highly scalloped membrane. Suggestions t h a t the particles alone function in m e m b r a n e conformation are highly speculative. However, a role in m e m b r a n e conformational characteristics cannot be ruled out. The last group of plasmalemmal patticles, the cross-linear arrays of 6- to 8-nmsize particles which periodically r e p e a t along the length of the cross-striated appendage, is highly unique and of particular interest in this study. T h e i r precise register with the major, 67-nm, r e p e a t p a t t e r n exhibited by the longitudinally directed fflaments seen in thin sections is an excellent example of particle association with internal i n t r a m e m b r a n o u s filament components. Although no direct link of particles with f i a m e n t s was observed in the crossstriated appendage, the repeat p a t t e r n is
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highly suggestive of such an association. Other examples of particle-filament associations include the ciliary necklace of quail oviduct cells (Bouisvieux-Ulrich et al., 1977), where the particles are seen associated with fibrillar linkers joining microtubules of the cilium; the Xenopus oocyte plasmalemma, where filaments are seen to be directly linked to particles (Bluemink and Tertoolen, 1978); and elsewhere (Weiss et al., 1977; M c N u t t , 1978). Two groups of acrosomal m e m b r a n e particles were observed in this study and include: (1) the tightly packed, 8- to 10-nmsize, EF-face particles limited to the thin portion of the paddle-shaped acrosome; and (2) a more highly ordered linear array of periodically repeating, 8- to 10-nm-size, PFface particles. T h e latter one is found in the thick portion of the paddle-shaped acrosome, whose r e p e a t of 25-26 n m mimics the paracrystalline order of 28-30 n m exhibited by the internally directed portions of the unit m e m b r a n e within this region of the acrosome. T h e finding of two groups of particles in the acrosome is unique when c o m p a r e d to previous freeze-facture studies on spermatozoa of any animal. T h e association of one highly ordered group o f patticles with the internal acrosomal paracrystaUine region is reminiscent of a similar association observed in rat sPermatozoa (Friend and Fawcett, 1974). In terms of its linear r e p e a t order, it is also similar to the particle organization reported in the acrosome of bull sperm (Plattner, 1971). T h e presence of the two sets of particles in two different faces of the m e m b r a n e has not been described in other spermatozoa. It is
FIGS. 13 AND 14, Freeze-fracture (Fig. 13) and thin section (Fig. 14) images of the cross-striated, tail-like appendage to demonstrate the precise register of the 6- to 8-nm-size, PF-face particles (PPF, Fig, 13) with the 67-nm major repeat period (arrows, Fig. 14) found in the tail-like appendage. GL, glycocalyx.Fig. 14, 1% tannic acid post-OsO4, 1 hr. × 64 000. FIG. 15. High-power-magnificationview of a thin section through the tail-like appendage to demonstrate the longitudinal striations (arrows) exhibited by the cross-striated bands, as well as the homogeneously distributed dense granules of the glycocalyx (GL) enhanced by tannic acid (1% tannic acid post-OsO4, 1 hr). × 160000.
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REGER, ITAYA, AND FITZGERALD
of interest to note t h a t the EF-face particles occur in a region where the a c r o s o m a l m e m b r a n e and p l a s m a l e m m a come into very close apposition. It is possible t h a t this association causes the particles to adhere to the E F face. P a r t i c u l a t e organization in a c r o s o m a l m e m b r a n e s , as observed following freezefracture, was first described b y B l o m and Birch-Andersen {1965) in bull spermatozoa. Since then, studies of particulate organization in acrosomes h a v e b e e n m a d e on bovine (Plattner, 1971), r a b b i t (Koehler, 1970a, b), h u m a n (Koehler, 1972), guinea pig (Friend and Fawcett, 1974), and r a t {Friend a n d Fawcett, 1974). As pointed out b y F a w c e t t (1970), K o e h l e r (1972), and Friend and F a w c e t t (1974), such paracrystalline arrays of particles occur with or without associated intraacrosomal, inner unit m e m b r a n e paracrystalline order. Friend and R u d o l f (1974) h a v e demons t r a t e d a "disintegration of geometric patterns" of these paracrystalline arrays in such a c r o s o m a l m e m b r a n e s following capacitation in r a t and guinea pig s p e r m a t o zoa. Paracrystalline order m a y be a reflection of the presence in these m e m b r a n e s of crystalline orders of enzymes such as hyaluronidase or a trypsin-like protease which p r e s u m a b l y helps the s p e r m to penetrate the zona pellucida ( S t a m b a u g h and Buckley, 1969; Gould, 1973). Correlative structural and biochemical studies are needed before such conclusions b e c o m e plausible. A n o t h e r suggested function for these particles has b e e n t h a t t h e y m a y reflect a "tight packing of m e m b r a n e constituents, which t h e n rapidly m o v e a p a r t for m e m b r a n e expansion" (Friend and Fawcett, 1974). W h e t h e r or not either of these two speculations applies to the particles observed here in the s p e r m a t o z o o n of A r madillidium v u l g a r e is open to question. T h i s is particularly true since no studies h a v e yet a p p e a r e d on fertilization or enzym a t i c characteristics in a n y isopod spermatozoa.
REFERENCES BACCETTI, B., BURRINI, A. G., DALLAI, R., PALLINI, V., CAMATINI, M., FRANCHI, E., AND PAOLETT, L.
(1977) J. Submicrosc. Cytol. 9, 187-219. BACCETTI, B., DALLAI, F., BERNINI, F., AND MAZZINI,
M. (1974) J. Morphol. 143, 187-245. BACCETTI, B., BIGLIARDI, E., AND ROSATI, F. (1971) J. Ultrastruct. Res. 35, 582-605.
BACCETTI,B., AND AFZELIUS,B. A. (1976) The Biology
of the Sperm Cell, Monographs in Developmental Biology, Vol. 10, S. Karger Press, New York. BERGSTROM, B. H., AND HENLEY C. (1973) J. Ultrastruct. Res. 42, 551-553.
BLOM, E., AND BIRCH-ANDERSEN, A. (1965) Nord. Vet. Med. 17, 193-200.
BLUEMINK, J. G., AND TERTOOLEN, L. G. J. (1978) Cytobiologie 16, 358-366:
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WEINSTEIN,R. S. (1975) Science 190, 54-56. BRETSCHER, M. S. (1971) J. Mol. Biol. 71, 523-528. COTELLI, F., FERRAGUTI,M., LANZAVECCHIA,G., AND
LORALAMIADONIN, C. (1976} J. Ultrastruct. Res. 55, 378-390. DEAMER, D. W., AND BASKIN, R. J. (1969) J. Cell Biol. 42, 296-307.
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(1975b) J. Ultrastruct. Res. 51, 269-280. FAWCETT,D. W. (1970) Biol. Reprod. 2, Suppl. 2, 90127. FAWCETT, D. W. (1977) in B. R. BRINKLEYAND K. R.
PORTER (Eds.), International Cell Biology, pp. 588601, The Rockefeller University Press, New York. FLECHON, J. E. {1974). J. Microsc. 19, 59-64. FRIEND, D. S., AND RUDOLF, I. (1974) J. CeUBiol. 63,
466-479. FRIEND, D. S., AND FAWCETT, D. W. (1974) J. Cell Biol. 63, 641-664.
GOULD, K. G. {1973) Fed. Proc. 32, 2069-2074. GUIDOTTI, G. {1972) Annu. Rev. Biochem. 41, 731-
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FREEZE-FRACTURE OF ISOPOD SPERMATOZOA KOEHLER, J. K. (1970a) /n B. BACCETTI (Ed.), Comparative Spermatology, pp. 515-522, Academic Press, New York and London. KOEHLER, J. K. (1970b) J. Ultrastruct. Res. 33, 598614. KOEHLER, J. K. (1972) J. Ultrastruct. Res. 39, 520539. KOEHLER, J. K. {1973) J. Ultrastruct. Res. 44, 355368. KOEHLER, J. K., AND GADDUM-ROSSE, R. (1975) J. Ultrastruct Res. 51, 106-118. LUFT, J. (1971) Anat. Rec. 171, 369-416. MACLENNAN, D. H., SEEMAN, P., ILES, G. H., AND VIP, C. C. (1971) J. Biol. Chem. 246, 2702-2710. MARCHESI, V. T., TILLACK, T. W., JACKSON, R. L., SEGREST, J. P., AND SCOTT, R. E. (1972) Proc. Nat. Acad. Sci. USA 69, 1445-1449. McNUTT, N. S. (1978) J. Cell Biol. 79, 774-787. 0LSON, G. E., LIFSICS, M., FAWCETT, D. W., AND HAMILTON, D. W. (1977) J. Ultrastruct. Res. 59, 207-221. PINTO DA SILVA, P., BRANTON, P. D., AND DOUGLAS, S. A. (1971) Nature (London) 232, 194-196. PINTO DA SILVA, P., Moss, P. S., AND FUDENBERG, H. H. (1973) Exp. Cell Res. 81, 127-138. PINTO DA SILVA, P., AND NICOLSON, G. (1974) Biochim. Biophys. Acta 363, 311-319. PLATTNER, H. (1971) J. Submicrosc. Cytol. 3, 19-32. POLLACK, E. G. (1978) Amer. Zool. 18, 25-69.
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REGER, J. F. (1964) J. Microsc. 3, 559-572. REGER, J. F. (1966) Z. Zellforsch. Mikr. Anat. 75, 579590. REGER, J. F. (1969) J. Ultrastruct. Res. 28, 422-435. REGER, J. F. (1970} J. Morphol. 132, 89-99. REGER, J. F. (1971) J. Submicrosc. Cytol. 3, 33-44. REGER, J. F., AND COOPER, D. P. (1968) J. Ultrastruct. Res. 23, 60-70. REGER, J. F., FAIN-MAUREL, M. A., AND CASSIER, P. (1977) J. Ultrastruct. Res. 60, 84-94. REGER, J. F., AND FITZGERALD,M. E. (1979) J. Ultrastruct. Res. 67, 95-108. RUBIN, C. S., ROSENFELD, R. D., AND ROSEN, A. M. (1973) Proc. Nat. Acad. Sci. USA 79, 3537-3738. SIMIONESCU, N., AND SIMIONESCU, M. (1976) J. Cell Biol. 70, 608-621. STACKPOLE, C. W., AND DEVORKIN, D. (1974) J. Ultrastruct. Res. 49, 167-187. STAMBAUGH, R., AND BUCKLEY, J. (1969) J. Reprod. Fertil. 18, 423-432. STEWART, P. S., AND MACLENNAN, D. H. (1974) J. Biol. Chem. 249, 985-993. TILLACK, T. W., SCOTT, R. E., AND MARCHESI, V. T. (1972) J. Exp. Med. 135, 1209-1227. TILLACK, T. W., BOLAND, R., AND MARTONOSI, A. (1974) J. Biol. Chem. 249, 624-633. WEISS, R. L., GOODENOUGH, D. A., AND GOODENOUGH, J. W. (1977) J. Cell Biol. 72, 133-143. WUEST, J., EL SAID, A., SWIDERSKI, Z., AND AESCHLIMANN, A. (1978) Z. Parasitenkd. 55, 91-99.
67, 180-193 (1979)
A Thin Section and Freeze-Fracture Study on Membrane Specializations
in Spermatozoa of the Isopod, A r m a d i l l i d i u m v u l g a t e 1 JAMES F.
R E G E R , PATRICIA W . ITAYA, 2 A N D MALINDA E . FITZGERALD
Department of Anatomy, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Received January 3, 1979 Mature spermatozoa of the Isopod, Armadillidium vulgare, were examined following tannic acid processing, thin section, and freeze-fracture techniques. They reveal a moderately thick glycocalyx and a typical random distribution of 5- to 10-nm-size, plasmalemmal, PF-face particles. Seven distinct areas of particulate membrane specializations occur in the spermatozoa. Five regions occur in the plasmalemma and two occur in the acrosomal membrane. The plasmalemmal specializations include: (1) clusters of 8- to 10-nm-size, PF-face particles found along a line made by the posterior margin of the acrosomal membrane; (2) orthogonal arrays of 4- to 6-nm-size EFface particles similarly disposed; (3} a linear array of 8- to 10-nm-size, PF-face particles extending the length of the acrosome, along the region where the paddle-shaped acrosome becomes thinner; (4) a cluster of 8- to 10-nm-size, PF-face particles on the cytoplasmic hood, which surrounds the insertion region of the cross-striated, taft-like appendage; and (5) a cross-linear array of 6- to 8nm-size, PF-face particles which repeat every 67 nm along the cross-striated taft-like appendage, mimicking the cross-striated repeat period exhibited by the filamentous component as seen in thin sections. The acrosomal membrane specializations include: (1) tightly packed, 8- to 10-nm-size, EF-face particles limited to the thin portion of the paddle-shaped acrosome; and (2) linear arrays of periodically repeating (25-26 nm), 8- to 10-nm-size, PF face particles limited to the thick portion of the acrosome whose periodic repeat mimics the paracrystalline order of the internally directed portions of the unit membrane in this region. The results of this study are discussed in terms of the functional significance of such specializations. It is concluded that the function of each group of particle arrays is unique, and is in fact a reflection of either their topographical relationships with other membrane specializations or their possible role in fertilization. Particular attention is drawn to the functional significance of the following groups of particles: (1) the double set of particle arrays in the postacrosomal region, (2) the cross-linear arrays which mimic the crossstriated repeat pattern of longitudinally oriented filaments in the cross-striated appendage seen in thin sections, and (3) the two sets of particles in the PF and EF faces of the acrosomal membrane.
Since the initial freeze-fracture studies on membrane structure of bovine spermatozoa (Blom and Birch-Andersen, 1965; Koehler, 1966) several studies have appeared on vertebrate sperm. These include Spermatozoa from rabbit (Koehler, 1970a, b; Flechon, 1974), rainbow trout (Koehler, 1970b), human (Koehler, 1970a, 1972), bull (Plattner, 1971), water buffalo (Koehler, 1973), guinea pig (Friend and Fawcett, 1974; Friend and Rudolf, 1974; Koehler and Gaddum-Rosse, 1975; Fawcett, 1977), mouse (Stackpole and Devorkin, 1974), rat (Friend
and Fawcett, 1974; Friend and Rudolf, 1974), and opossum (Olson et al., 1977; Fawcett, 1977). The findings in these various animals demonstrate the following. First, a series of double or multiple rows of longitudinally arranged, approximately 9-nmsize, plasmalemmal PF-face particles occurs in the principle piece. Second, circumferentially or helically arranged rows of 6to 8-nm-size plasmalemmal PF-face particles occur in the midpiece. Third, paracrystalline arrays of larger, 10- to 25-nm-size, particles occur in the acrosomal membrane EF face of rabbit (Koehler, 1970b), guinea pig (Friend and Rudolf, 1974; Friend and Fawcett, 1974), rat (Friend and Fawcett,
Supported by NSF Grant No. PCM 76-16961. 2 Present address: Department of Anatomy, University of Iowa, Iowa City, Iowa 52240. 180 0022-5320/79/050180-14502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
FREEZE-FRACTURE OF ISOPOD SPERMATOZOA 1974), a n d h u m a n ( K o e h l e r , 1972) s p e r m a tozoa, in t h e n u c l e a r m e m b r a n e o f g u i n e a pig a n d r a t ( F r i e n d a n d F a w c e t t , 1974), a n d in c e r t a i n r e g i o n s o f t h e p l a s m a l e m m a o f guinea p i g a n d r a t ( F r i e n d a n d F a w c e t t , 1974). F o r a m o r e c o m p l e t e e v a l u a t i o n o f c o m p a r i s o n s of f r e e z e - f r a c t u r e c o m p o s i t i o n in g u i n e a pig a n d r a t e p i d i d y m a l s p e r m , see F r i e n d a n d F a w c e t t (1974). C o n c l u s i o n s about t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e s e various p a r t i c u l a t e o r g a n i z a t i o n s a r e of necessity l a r g e l y s p e c u l a t i v e a t t h i s t i m e . However, c o m p a r a t i v e o r e x p e r i m e n t a l studies o n v a r i o u s t y p e s of s p e r m a t o z o a should b e of s o m e a s s i s t a n c e in e x p a n d i n g our i n s i g h t s i n t o t h e f u n c t i o n s o f t h e s e v a r ious p a r t i c u l a t e a r r a y s . Only a v e r y l i m i t e d n u m b e r o f s t u d i e s , b y correlative t h i n s e c t i o n a n d f r e e z e - f r a c t u r e technique, h a v e b e e n c a r r i e d o u t o n i n v e r tebrate spermatozoa. These include the e a r t h w o r m , Lumbricus terrestris (Bergstrom a n d H e n l e y , 1973); t w o s p e c i e s of D i p l o p o d a , Polyxenus lagurus a n d Pachyjulus sp. ( B a c c e t t i et al., 1974; 1977); t h e tick, Amblyomma hebraeum ( W u e s t et al., 1978); a n d t h e D i p l o p o d a n , Spirobolus sp. (Reger a n d F i t z g e r a l d , 1979). T h e l a t t e r is the o n l y c o m p l e t e s t u d y on all m e m b r a n e s of an i n v e r t e b r a t e s p e r m a t o z o o n . T h e following s t u d y o n all m e m b r a n e s o f a r e p r e sentative i s o p o d , ArmadiUidium vulgare, was u n d e r t a k e n n o t o n l y t o fill t h e g a p in such s t u d i e s o f i n v e r t e b r a t e s p e r m a t o z o a , but m o r e i m p o r t a n t l y , t o d e m o n s t r a t e s o m e of t h e r a t h e r u n i q u e s p e c i a l i z a t i o n s in t h i s type of c r u s t a c e a n s p e r m a t o z o o n . W h e r e possible t h e d a t a will b e c o m p a r e d to t h o s e a r r a y s f o u n d in m o r e t y p i c a l v e r t e b r a t e s p e r m a t o z o a in a n e f f o r t to d r a w f u n c t i o n a l correlates. MATERIALS AND METHODS Following ventral orientation of male isopods, Armadillidium vulgare, an incision was made in the thoracoabdominal cavity, and testes were removed and placed in cold (0-5°C) 4% glutaraldehyde (pH 7.6, 0.2 M sodium cacodylate buffer). Tissue was then dissected into small pieces and fixed for an hour. After fLxation in glutaraldehyde, tissue was processed for
181
either thin sectioning or freeze-fracture techniques as follows. Tissue for thin sectioning was washed for an hour in cold (0-5°C) sodium cacodylate buffer (pH 7.6, 0.4 M) and postfixed for an hour in cold (0-5°C) 2% OsO4 (pH 7.6, 0.4 M cacodylate buffer ). Some tissues were then processed for an hour in 1% tannic acid (TA; Mallinkroff, AR, code No. 1764) in cacodylate buffer (pH. 7.6, 0.4 M; Simioneseu and Simionescu, 1976). Subsequently all tissue was dehydrated in 5-rain changes of successive grades of methanol (beginning with 50%) and embedded in Epon 812. Sections, 1-2 #m thick, were made of the Epon-embedded tissue and stained with Mallory azure II-methylene blue, for purposes of locating particular stages of spermiogenesis and mature spermatozoa in various regions of the vas deferens prior to thin sectioning. Thin sections were cut with a diamond knife fitted to an LKB Ultratome II, floated on distilled water, mounted on carbon-coated grids, and stained with uranyl acetate and lead citrate. Tissue for freeze-fracture was also fixed in 4% glutaraldehyde (pH 7.6, 0.2 M sodium eacodylate buffer) as above and subsequently equilibrated in a series of 10, 15, and 25% glycerols in buffer (0.2 M sodium cacodylate). Following 1 to 2 hr in 25% glycerol, tissue was mounted preparatory to being introduced into a Balzers 301 freeze-fracture device for fracturing and replication. Following replication, the replicas were cleaned with either 50% chromic acid or purex to digest tissue. Replicas were then washed with distilled H20 and mounted on copper grids for observation. Thin sections and replicas were examined with a Hitachi HU l l A electron microscope. Micrographs were taken at initial magnifications of 5000 to 50 000 × at exposures of 2 to 4 sec and were made on Cronar, Ortholitho Type A sheet film. Negatives were enlarged up to 10 times with a Durst S-45 enlarger equipped with a 250-W mercury vapor point source. OBSERVATIONS The morphology of the mature spermat o z o o n o f t h e i s o p o d , Armadillidium vulgare (Fig. 1), is s i m i l a r to t h a t o f o t h e r p e r i c a r i d e s . T h e s p e r m a t o z o o n is a n elong a t e cell, a t o n e e n d of w h i c h a r e s i t u a t e d t h e a c r o s o m e (A, Fig. 1), t h e n u c l e u s (N, Fig. 1), t w o c e n t r i o l e s (C, Fig. 1), a n d a r o d s h a p e d p e r f o r a t o r i u m (P, Fig. 1). A n e l o n gate, c r o s s - s t r i a t e d a p p e n d a g e (TA, Fig. 1) extends from the base of the acrosome near its i n d e n t a t i o n b y t h e p e r f o r a t o r i u m (P, Fig. 1). W h e n o b s e r v e d in vivo t h e e l o n g a t e , c r o s s - s t r i a t e d a p p e n d a g e ( T A , Fig. 1) a p p e a r s stiff a n d inflexible, w h i l e t h e a c r o s o -
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REGER, ITAYA, AND FITZGERALD
FIG. 1. Schematic diagram of a mature spermatozoon of Armadillidium vulgare demonstrating the acrosome (A), perforatorium (P), centrioles (C), nucleus (N), and cross-striated, tail-like appe~adage(TA). Cross-sectional levels and regions identified in subsequent electron micrographs are appropriately labeled. (1, 2, 3) Section levels seen in Figs. 5 and 6.
mal and nuclear regions appear flexible. Thus the spermatozoon resembles a whip, with a cross-striated appendage serving as the handle, and the acrosomal-nuclear region serving as the lash of the whip. The acrosomal region is paddle shaped and, when seen in cross section, has a thick and a thin portion (Tk, Th, Fig. 6). The nucleus exhibits no unusual characteristics and contains the usual longitudinally oriented DNA protein filaments. The details of the fine structure of spermatozoa from several species of pericarides have previously been
described (Reger, 1964, 1966, 1969, 1970, 1971; Reger et al., 1977; Fain-Maurel, 1966, 1969; Fain-Maurel et al., 1975a, 1975b; Cotelli et al., 1976) and, except for the details of plasmalemmal and acrosomal membrane specializations, will not be dealt with further here. The plasmalemma is characterized by a typical, unit membrane (P1, Fig. 2), a moderately thick glycocalyx (G1, Figs. 5 and 6), and scattered 8- to 10-nm-size, PF-face particles (PPF, Fig. 7) (Branton et al., 1975). The glycocalyx, which is highlighted by tannic acid (G1, Figs. 5, 14, and 15), is characterized by homogeneously distributed, electron-opaque particles. Unique, particulate patterns occur in the plasmalemma of the acrosomal region (Figs. 7 and 8) and along the entire length of the cross-striated appendage (Figs. 8 and 13). Plasmalemmal specializations in the acrosomal region include four groups of particles. The first is a linear array of 8- to 10-nm-size, PF-face particles (arrows, Fig. 7) situated the length of the transition area between the thick (Tk, Fig. 6) and thin (Th, Fig. 6) portions of the paddle-shaped acrosome. The second is a linear array of 4- to 6-nm-size, EF-face particles grouped in square lattices of various sizes (arrows, Fig. 9; arrows, inset, Fig. 9) which extend the length of a line made by the caudally curved acrosomal membrane (see also dotted arrows, Fig. 4). The third is a linear array of 8- to 10-nm-size, PF-face particles (thin arrows, Fig. 8) following the same line made by the caudally curved, acrosomal membrane. Some of these mimic the paracrystalline order (thick arrows, inset, Fig. 8) of the smallersized paracrystalline EF-face particle aggregates. The fourth consists of grouped, 8to 10-nm-size, PF-face particles which occur on the sleeve which wraps around the base of the cross-striated appendage (GP, Fig. 8) at its point of origin from the acroo somal region of the spermatozoon (TA, Fig. 8). The particulate, plasmalemmal specializations of the cross-striated appendage
FREEZE-FRACTURE OF ISOPOD SPERMATOZOA consist of cross-linear a r r a y s of 6- to 8-nmsize, P F - f a c e particles ( P P F , Fig. 13). Laterally t h e y are s p a c e d 7-10 n m apart, a n d each row of particles r e p e a t s at approximately 67 n m intervals, reflecting t h e repeat p a t t e r n exhibited b y t h e filamentous, cross-banded a p p e n d a g e as seen in t h i n sections (arrows, Figs. 14 a n d 15). T h e glycocalyx (GL, Fig. 14) p r e s e n t s a granular, stippled a p p e a r a n c e (GL, Fig. 15) following tannic acid processing. The a c r o s o m a l m e m b r a n e (A, Figs. 4, 6, and 7) also exhibits specializations seen in both thin section (Figs. 3, 4, a n d 6) a n d freeze-fracture (Figs. 7-10) images. B o t h longitudinal (Figs. 3 a n d 4) a n d cross-sectional (Fig. 6) views of t h i n sections d e m onstrate a striated p a t t e r n . W h e n this region is seen in t a n g e n t i a l section (Fig. 4), it exhibits a p a r a c r y s t a l l i n e order. T h e paracrystalline o r d e r is c o n f i n e d to t h e t h i c k e r portion of t h e a c r o s o m e (arrows, Fig. 6). The a c r o s o m a l m e m b r a n e in t h e t h i n n e r portion of t h e p a d d l e - s h a p e d a c r o s o m e practically fuses w i t h t h e p l a s m a l e m m a (arrows, inset, Fig. 2). F r e e z e - f r a c t u r e images of t h e a c r o s o m a l m e m b r a n e in t h e thick a n d t h i n p o r t i o n s reveal two sets of particles (Figs. 9, 10, a n d 11). One set consists of linear a r r a y s of periodically r e p e a t ing (25-26 nm), 8- to 10-nm-size, P F - f a c e particles (P, Fig. 11) o c c u r r i n g in the thicker p o r t i o n of t h e a c r o s o m e , w h o s e re-
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p e a t period laterally a n d longitudinally m i m i c s t h a t of t h e i n n e r l a m i n a r projections (28-30 n m ) of t h e a c r o s o m a l u n i t m e m b r a n e seen in t h i n sections (see Figs. 3 a n d 4). T h e s e c o n d set of particles consists of a h e a v y c o n c e n t r a t i o n of 8- to 10-nmsize, E F - f a c e particles (Figs. 9 a n d 10) limited to t h e t h i n p o r t i o n of t h e paddles h a p e d acrosome. DISCUSSION The randomly distributed plasmalemmal P F - f a c e particles in t h e a c r o s o m a l a n d nuclear regions of t h e s p e r m a t o z o o n of Arr n a d i l l i d i u m vulgare are similar in size a n d relative d i s t r i b u t i o n to similar particles f o u n d in o t h e r cells. A l t h o u g h t h e precise b i o c h e m i c a l c o m p o s i t i o n a n d functions of t h e s e particles are still o p e n to question, it is b e c o m i n g increasingly e v i d e n t t h a t t h e y represent intercalated membrane proteins w i t h or w i t h o u t associated lipids or carboh y d r a t e s (Pollack, 1978). F o r example, certain b i o c h e m i c a l c h a r a c t e r i s t i c s h a v e b e e n described for e r y t h r o c y t e s (Bretscher, 1971; P i n t o da Silva et al., 1971, 1973; Guidotti, 1972; M a r c h e s i et al., 1972; Tillack et al., 1972; R u b i n et al., 1973) a n d in t h e sarcop l a s m i c r e t i c u l u m of c e r t a i n v e r t e b r a t e skeletal m u s c l e s ( D e a m e r a n d Baskin, 1969; S t e w a r t a n d M a c L e n n a n , 1974; Tillack et al., 1974; M a c L e n n a n et al., 1971). I n all p r o b a b i l i t y several kinds of p r o t e i n com-
FIa. 2. This longitudinal to slightly oblique section through several spermatozoa demonstrates the unit membranes of the plasmalemma (P1) and acrosome (A) as well as the practically Complete fusion (arrows, inset) of the outer lamina of the acrosomal with the inner lamina of the plasmalemmal unit membranes. One percent tannic acid post-OsO4, 1 hr. × 64 000; inset, × 156 000. FIGS. 3 AND4. Cross-sectional (Fig. 3) and longitudinally tangential (Fig. 4) views of the thick portion of the acrosome (A, Figs. 3 and 4) (see also Figs. 1 and 6) to demonstrate projecting repeat components from the acrosomal inner lamina (arrows, Fig. 3) which, when seen tangentially, appear to have inner connecting elements (arrows, Fig. 4), thus presenting an appearance of paracrystalline order. (dotted arrows, Fig. 4) Posterior curved end of the acrosomal membrane. Late spermatids. Fig. 3, x 48 000; Fig. 4, x 70 000. Fro. 5. Low-power, cross-sectional view of spermatozoa sectioned at different levels. For example, compare levels of sections 1, 2, and 3 with levels 1, 2, and 3 seen in Figs. 1 and 6. Gl, glycocalyx. One percent tannic acid post-OsO4, 1 hr. x 32 000. FIG. 6. Higher-magnification view of spermatozoa cross sections. Note that the region of origin of the taillike appendage (TA) at the outer edge of the appendage is infolded within an indentation of the adjacent portion of the cell (arrows). Note also the faintly striated pattern along the inner acrosomal membraile (A) (see also Figs. 3 and 4). G1, glycocalyx; Tk and Th, thick and thin portions of the paddle-shaped acrosomal region. (1, 2, 3) Section levels corresponding to those seen in Figs. 1 and 5. One percent tannic acid post-OsO4, 1 hr. × 64 000.
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FIG. 7. A freeze-fracture image at the acrosomal region near its juncture with the tail-like appendage (TA) demonstrating, from the top to the bottom, the plasmalemmal PF face (PPF), acrosomal PF face (APF), acrosomal EF face (AEF), and plasmalemmal EF face (PEF). Arrows, linear array of particles; A, acrosome. × 64 000.
plexes, and therefore more than one function, m a y be ascribed to the P F particles of various cell types, as is becoming increasingly clear for the e r y t h r o c y t e (Pinto da Silva and Nicolson, 1974). It is of some interest to note here t h a t the randomly distributed, 8- to 10-nm-size, particles seen in this study were n o t found along the length of the cross-striated appendage. T h e significance of such a difference in particle distribution is impossible to precisely assess. It has been assumed t h a t such particles are A T P a s e or ATPase-associated protein complexes, as suggested for erythrocytes (Marchesi et al., 1972) and the sarcoplasmic reticulum of muscle (Deamer and Baskin, 1969). Therefore, their absence in the cross-striated appendage m a y reflect a difference in energy requirements.
T h e m o d e r a t e l y thick glycocalyx, highlighted in the spermatozoa of this study by tannic acid, demonstrates no particular morphological or regional specializations as sometimes found in other spermotozoa (see Baccetti and Afzelius, 1976). T h e glycocalyx m a y be classified as the "fruitfly" type (Baccetti et al., 1971). T h e tannic acid reaction appears as discrete, homogeneously distributed granules in the glycocalyx t h r o u g h o u t the cell surface. Although studies by Kalina and Pease (1971a, b) demonstrated t h a t tannic acid utilized in combination with osmium tetroxide reacts with phosphatidyl cholines, its mode of reaction with the glycocalyx is unknown. It may react with negative charges such as the acidic mucopolysaccharides, as in the case of r u t h e n i u m red (Luft, 1971).
FREEZE-FRACTURE OF ISOPOD SPERMATOZOA
The more highly ordered and specifically distributed plasmalemmal particles observed here include: (1) clusters of 8- to 10nm-size, PF-face particles found along a line made by the posterior margin of the acrosomal membrane; (2) orthogonal arrays of 4- to 6-nm-size, EF-face particles similarly disposed; (3) a linear array of 8- to 10nm-size, PF-face particles extending the length of the acrosome, along the region where the paddle-shaped acrosome becomes thinner; (4) a cluster of 8- to 10-nmsize, PF-face particles on the cytoplasmic hood which surrounds the insertion region of the cross-striated, tail-like appendage; and (5) a cross-linear array of 6- to 8-nmsize, PF-face particles which repeat every 67 nm along the cross-striated, tail-like appendage mimicking the cross-striated repeat period exhibited by the filamentous component as seen in thin sections. Although it is impossible to draw direct conclusions about the functional significance of these various arrays, certain indirectly derived conclusions are appropriate. The most intriguing arrays include the two juxtaposed 8- to 10-nm-size, PF-face and 4- to 6-nm-size, EF-face particles found in the plasmalemma along the posterior margin of the termination of the acrosome.
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Not only were the two sets of particles aligned in two faces of the same membrane along the same line, but suggestive evidence demonstrated that both sets are orthogonally arrayed particles. Definitive conclusions about the functional significance of these arrays or their interrelationship cannot be made. However, this region of the plasmalemma overlies the acrosomal membrane which will fuse with the oolemma during fertilization. It therefore may have unique properties contributing to the fertilization process. Whether or not this region ultimately becomes incorporated into the fertilized oolemma is unknown, but would certainly be worth investigating in view of the particle arrays and their possible significance in the fertilization process. The locale of these arrays is reminiscent of particle specializations found in postacrosomal and equatorial regions of certain vertebrate spermatozoa (Koehler, 1970b; Friend and Fawcett, 1974). The plasmalemma of spermatozoa has been seen to exhibit specializations, such as a serrated pattern in the case of guinea pig spermatozoa (Friend and Fawcett, 1974) and a highly ordered "fine periodic substructure" in rabbit spermatozoa (Koehler, 1972). It is more difficult to assess the functional
FIG. 8. Freeze-fracture replica showing portions of four spermatozoa. The plasmalemmal PF face in the acrosomal region is characterized by randomly distributed particles (P); a linear array of particles situated the length of the acrosomal level at the juncture of the thick and thin portions of the paddle-shaped acrosome (dotted arrows); a group of linearly arrayed particles which follow the posterior level of the acrosome (thin arrows), some of which (thick arrows, enlarged inset) take on a paracrystalline order; and a group of particles which are situated on the apron-like portion of the spermatozoon which envelops the tail-like appendage (TA) at its insertion point (GP). APF, acrosomal PF face. x 32 000; inset, x 64 000. FIos. 9 AND 10. These two freeze-fracture images demonstrate a series of small, 4- to 6-nm-size, paracrystalline-ordered, plasmalemmal EF-face particles (arrows, Fig. 9) which follow the line of the posterior edge of the acrosome as it skirts the edge of the point of the tail-like appendage insertion point (see also arrows, inset). Note also the particle-studded acrosomal EF face (AEF, Figs. 9 and 10) in the flat portion of the paddle-shaped acrosome and the corresponding pitted PF face (APF, Fig. 10). × 64 000; inset, x 64 000. FIG. 11. A freeze-fracture image of portions of the acrosomal PF face (APF) demonstrating that the acrosomal PF-face particles (P) in the thick portion of the paddle-shaped acrosome exhibit a paracrystalline order which mimics the paracrystalline order of the projections from the inner lamina of the acrosomal membrane (See Figs. 3 and 4), while the PF face of the thinner portion of the acrosome (arrows) is pitted corresponding to the distribution of EF-face particles in this area (also see Fig. 9). x 64 000. FIG. 12. A freeze-fracture replica through the nuclear region to demonstrate the sparseness of particles in the plasmalemmal PF face (PPF) and a practically complete lack of particles in the nuclear membranes (NM). x 64 000.
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FREEZE-FRACTURE OF ISOPOD SPERMATOZOA significance of both the linear arrays along the length of the acrosome and the particles grouped along the cytoplasmic hood, which partially enfolds the cross-striated appendage at its point of origin. T h e r e have thus fax been no similarly organized particle arrays described for other spermatozoa. Since both of these sets of particles occur along regions of plasmalemmal curvatures, their presence m a y reflect a function in the maintenance of m e m b r a n e conformation. Previous suggestions along these lines have been made for guinea pig and rat spermatozoa (Friend and Fawcett, 1974) and other cell types (Pollack, 1978). An interesting example of such a possibility m a y be drawn from studies on the midpiece of opossum spermatozoa (Fawcett, 1977). In this study rows of particles are longitudinally directed in the wall of a highly scalloped membrane. Suggestions t h a t the particles alone function in m e m b r a n e conformation are highly speculative. However, a role in m e m b r a n e conformational characteristics cannot be ruled out. The last group of plasmalemmal patticles, the cross-linear arrays of 6- to 8-nmsize particles which periodically r e p e a t along the length of the cross-striated appendage, is highly unique and of particular interest in this study. T h e i r precise register with the major, 67-nm, r e p e a t p a t t e r n exhibited by the longitudinally directed fflaments seen in thin sections is an excellent example of particle association with internal i n t r a m e m b r a n o u s filament components. Although no direct link of particles with f i a m e n t s was observed in the crossstriated appendage, the repeat p a t t e r n is
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highly suggestive of such an association. Other examples of particle-filament associations include the ciliary necklace of quail oviduct cells (Bouisvieux-Ulrich et al., 1977), where the particles are seen associated with fibrillar linkers joining microtubules of the cilium; the Xenopus oocyte plasmalemma, where filaments are seen to be directly linked to particles (Bluemink and Tertoolen, 1978); and elsewhere (Weiss et al., 1977; M c N u t t , 1978). Two groups of acrosomal m e m b r a n e particles were observed in this study and include: (1) the tightly packed, 8- to 10-nmsize, EF-face particles limited to the thin portion of the paddle-shaped acrosome; and (2) a more highly ordered linear array of periodically repeating, 8- to 10-nm-size, PFface particles. T h e latter one is found in the thick portion of the paddle-shaped acrosome, whose r e p e a t of 25-26 n m mimics the paracrystalline order of 28-30 n m exhibited by the internally directed portions of the unit m e m b r a n e within this region of the acrosome. T h e finding of two groups of particles in the acrosome is unique when c o m p a r e d to previous freeze-facture studies on spermatozoa of any animal. T h e association of one highly ordered group o f patticles with the internal acrosomal paracrystaUine region is reminiscent of a similar association observed in rat sPermatozoa (Friend and Fawcett, 1974). In terms of its linear r e p e a t order, it is also similar to the particle organization reported in the acrosome of bull sperm (Plattner, 1971). T h e presence of the two sets of particles in two different faces of the m e m b r a n e has not been described in other spermatozoa. It is
FIGS. 13 AND 14, Freeze-fracture (Fig. 13) and thin section (Fig. 14) images of the cross-striated, tail-like appendage to demonstrate the precise register of the 6- to 8-nm-size, PF-face particles (PPF, Fig, 13) with the 67-nm major repeat period (arrows, Fig. 14) found in the tail-like appendage. GL, glycocalyx.Fig. 14, 1% tannic acid post-OsO4, 1 hr. × 64 000. FIG. 15. High-power-magnificationview of a thin section through the tail-like appendage to demonstrate the longitudinal striations (arrows) exhibited by the cross-striated bands, as well as the homogeneously distributed dense granules of the glycocalyx (GL) enhanced by tannic acid (1% tannic acid post-OsO4, 1 hr). × 160000.
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of interest to note t h a t the EF-face particles occur in a region where the a c r o s o m a l m e m b r a n e and p l a s m a l e m m a come into very close apposition. It is possible t h a t this association causes the particles to adhere to the E F face. P a r t i c u l a t e organization in a c r o s o m a l m e m b r a n e s , as observed following freezefracture, was first described b y B l o m and Birch-Andersen {1965) in bull spermatozoa. Since then, studies of particulate organization in acrosomes h a v e b e e n m a d e on bovine (Plattner, 1971), r a b b i t (Koehler, 1970a, b), h u m a n (Koehler, 1972), guinea pig (Friend and Fawcett, 1974), and r a t {Friend a n d Fawcett, 1974). As pointed out b y F a w c e t t (1970), K o e h l e r (1972), and Friend and F a w c e t t (1974), such paracrystalline arrays of particles occur with or without associated intraacrosomal, inner unit m e m b r a n e paracrystalline order. Friend and R u d o l f (1974) h a v e demons t r a t e d a "disintegration of geometric patterns" of these paracrystalline arrays in such a c r o s o m a l m e m b r a n e s following capacitation in r a t and guinea pig s p e r m a t o zoa. Paracrystalline order m a y be a reflection of the presence in these m e m b r a n e s of crystalline orders of enzymes such as hyaluronidase or a trypsin-like protease which p r e s u m a b l y helps the s p e r m to penetrate the zona pellucida ( S t a m b a u g h and Buckley, 1969; Gould, 1973). Correlative structural and biochemical studies are needed before such conclusions b e c o m e plausible. A n o t h e r suggested function for these particles has b e e n t h a t t h e y m a y reflect a "tight packing of m e m b r a n e constituents, which t h e n rapidly m o v e a p a r t for m e m b r a n e expansion" (Friend and Fawcett, 1974). W h e t h e r or not either of these two speculations applies to the particles observed here in the s p e r m a t o z o o n of A r madillidium v u l g a r e is open to question. T h i s is particularly true since no studies h a v e yet a p p e a r e d on fertilization or enzym a t i c characteristics in a n y isopod spermatozoa.
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