Surface of the Equatorial Segment of the

Transcription

1 BIOLOGY OF REPRODUCTION 16, (1977) Surface of the Equatorial Segment of the Mammalian Acrosome DAVID M. PHILLIPS Population Rockefeller Council, University, New York, N.Y ABSTRACT Surface replicas were made of spermatozoa of 8 mammalian species, which had been fixed after either brief washing in isotonic Hank s BSS or exposure to hypotonic Hank s for several minutes. In bull, rabbit, hamster, guinea pig, degu and Rhesus monkey the surface of the outer acrosomal membrane in the equatorial region was found to be composed of regular rows of hexagonally packed particles with center-to-center spacing of 170A. These particles are limited to the equatorial segment. The majority of the surface of the outer acrosomal membrane in mouse and rat was composed of similar 170A spaced particles suggesting that the major portion of the acrosomal surface in mouse and rat is analogous to the equatorial segment of other species. The postacrosomal region in several species was found to be composed of material arranged in parallel ridges with center-to-center spacing of 1 50A. INTRODUCTION During the acrosome reaction, in spermatozoa of mammalian species which have so far been studied, most of the contents of the acrosome are lost. In vivo, the acrosome reaction is believed to occur during passage through the cumulus. The enzymes which are thereby released from the acrosome presumably hydrolyze material between cumulus cells which would otherwise impede passage to the zona (Bedford, 1972a, b; Zamboni, 1971). The inner acrosomal membrane with associated enzymes, the equatorial segment of the acrosome, and the postacrosomal sheath apparently maintain their integrity during the acrosome reaction, and subsequently the plasma membrane is seen to have merged with the acrosomal membrane overlying the equatorial segment. Other functional differences appear to exist between the equatorial segment and the main body of the acrosome. Cytochemical studies have shown that some enzymes present in the anterior portion of the acrosome do not occur in the equatorial segment (Morton, 1975; Yanagimachi and Teichman, 1971; Allison and Hartree, 1970). In mammalian species which have been studied, the equatorial segment remains intact after the spermatozoon passes through the zona pellucida and, along with the post- Accepted September 7, Received August 5, acrosomal region, is incorporated into the ooplasm where it degenerates (Bedford, 1972a, b; Zamboni, 1971; Yanagimachi and Noda, 1970). Using surface replicas, we have examined the surface of the equatorial segment in 8 mammalian species. The surface in this region, but not elsewhere, is consistently composed of hexagonally packed particles with center-to-center spacing of 170A. These particles are limited to the equatorial segment of the acrosomal surface. MATERIALS AND METHODS Spermatozoa of bull, rabbit, mouse, rat, golden hamster (/t4esocricetus auratus), guinea pig and degu (Octodon degus) were obtained from the cauda epididymis. Rhesus monkey spermatozoa were obtained from semen. Spermatozoa were washed briefly in Hank s BSS. Some spermatozoa were treated from 30 seconds to 5 mm in 1/3 to 1/2 strength (hypotonic) I-lank s BSS to remove some of the plasma membrane. Other spermatozoa were untreated. Subsequently spermatozoa were fixed for 1 h in 3 percent collidine-buffered glutaraldehyde and pelleted by low speed centrifugation. The fixative was aspirated off and the spermatozoa were resuspended in water and again pelleted. Spermatozoa were then suspended in about 1 ml of water, placed on a poly-l-lysine coated coverslip and allowed several minutes to settle and attach. (The poly-l-lysine coated coverslip was previously prepared by placing a coverslip in a solution of 2 mg/mi poly-l-lysine [Sigmal for a few minutes and washing the coverslip in water.) Coverslips with attached sperm were dehydrated through graded alcohols to acetone and were critical point dried with a Sorvall critical point drying apparatus. Replicas were 128

2 EQUATORIAL SEGMENT: ACROSOME 129 made with an Edwards 306 coater by shadowing at 40#{176}with platinum and 90#{176} with carbon. Replicas were removed by carefully inserting the coverslip into concentrated (48#{176})hydrofluoric acid. (If the coverslip is inserted slowly at an acute angle, the replica floats onto the surface of the hydrofluoric acid in the same way that formvar floats from a glass slide onto a water surface. Sometimes the replica does not detach from the slide. In this case the coverslip can be left to dissolve in the hydrofluoric acid.) The replicas were transferred with a glass rod to the surface of water (briefly) and then to 5 percent aqueous sodium hypochlorite (or clorox) for 5 to 30 mm. Subsequently the replicas were transferred with a glass rod to water for about 30 mm. Replicas are picked up on uncoated 200 mesh grids and viewed in a Phillips 300 microscope. RESULTS Images seen in surface replica preparations contain information which is different from the FIGS. 1 and 2. Replica of the surface of rabbit spermatozoon which had been exposed to hypotonic flanks BSS for several minutes before fixation. The surface of most of the equatorial segment of the acrosome is covered with particles with center-to-center spacing of 170A. Fig. 1 X7000. Fig. 2 (same cell) X20,000. FIGS. 3 and 4. Spermatozoa fixed after brief washing in Hank s occasionally have holes in the plasma membrane. Through such windows, 170A spaced particles may be observed on the surface of the outer acrosomal membrane in the equatorial segment. Fig. 3 >( Fig. 4 (same cell) X 25,000.

3 130 PHILLIPS FIGS. 5 and 6. Bull spermatozoa. This spermatozoon is from a sample which was fixed after brief washing in isotonic Hank s BSS. Some spermatozoa have holes in the plasmalemma through the surface of the outer acrosomal membrane may be observed. Rows of particles with 1 70A spacing occur on the equatorial segment. Larger irregularly spaced particles are seen on the surface of the rest of the acrosome. Fig. 5 X Fig. 6 (same spermatozoa) X60,000. information obtainable from micrographs of freeze cleaved material. In surface replicas one can observe the entire surface of many sperm heads and readily obtain data about the location of surface structures and the degree of variation among cells. Surface replicas differ from replicas of freeze cleaved preparations in that they reflect the topography of the outer membrane surface rather than the topography of structures within the plane of the membrane. FIGS. 7 and 8. Guinea pig spermatozoon treated with hypotonic saline before fixation. Rows of 170A spaced particles are observed in the thin equatorial segment immediately anterior to the postacrosomal region. The postacrosomal region is characterized by parallel ridges with 1 50A spacing. Fig. 7 X 9,000. Fig. 8 (same cell) X45,000.

4 EQUATORIAL SEGMENT: ACROSOME 131 In addition, some differences in images obtained with the two procedures may be caused by differences in preparative technique. Freeze cleaved specimens are passed through a cryoprotective agent whereas surface replicas are made from specimens previously dehydrated in alcohol and acetone. Rabbit In low magnification micrographs of replicas of rabbit spermatozoa which have been exposed to hypotonic medium, a relatively smooth, cup-shaped region is observed in the equatorial segment of the sperm head (Fig. 1). Under higher magnification this region appears as hexagonally packed particles with center-tocenter spacing of 170A (Fig. 2). Characteristically, there is a semicircular space inside the cup just anterior to the postacrosomal region which is devoid of the regular 170A particles (Fig. 2). Spermatozoa fixed after a brief washing in isotonic Hank s BSS often had holes in the plasmalemma (Fig. 3). Holes may occur naturally in some spermatozoa or may be fixation, dehydration, or drying artifact. In any event, the holes often provide a window to the underlying surface of the outer acrosomal membrane (Fig. 4). Through such windows, 1 70A particles are observed on the acrosomal membrane in the region of the equatorial segment just as in spermatozoa fully stripped of the plasmalemma by hypotonic treatment. FIGS Replicas of degu sperm heads. The acrosome is characterized by a number of bumps. The number and location of the bumps varies among cells, but they are consistently absent from the equatorial segment. X FIG. 12. Enlargement of equatorial segment of Fig. 10 showing rows of particles and finger-like processes extending from the border between the postacrosomal region and the equatorial segment. X60,000.

5 132 PHILLIPS Therefore, there are two lines of evidence that the particles are located on the outside surface of the outer acrosomal membrane. Bull After brief exposure to hypotonic Hank s BSS, bull sperm display holes in the plasma membrane (Fig. 5). An equatorial range similar in shape to the equatorial region in rabbit spermatozoa likewise appears as hexagonally packed particles with center-to-center spacing of 170A (Fig. 6). Adjacent to the postacrosomal region there is a zone devoid of the 170A spaced particles containing larger irregular shaped material. Anterior to the equatorial segment the surface of the acrosome generally appears irregularly granular. Guinea Pig When guinea pig spermatozoa are placed in hypotonic Hank s BSS they undergo a false acrosome reaction. The equatorial segment, apparently without the overlying plasmalemma, remains (Fig. 7). The equatorial segment is FIGS. 13 and 14. Replica of hamster spermatozoon treated with hypotonic Hank s BSS. The equatorial segment is characterized by rows of particles. Fig. 13 X5000. Fig. 14 (same cell) X33,000. FIGS. 15 and 16. Replica of a spermatozoon in which an acrosome reaction has apparently been brought about by hypotonic treatment. The postacrosomal region is characterized by rows of 180A spaced particles. Fig, 15 X7,000. Fig. 16 X36,000.

6 EQUATORIAL SEGMENT: ACROSOME 133 characterized by 170A spaced particles like those observed in rabbit or bull (Fig. 8). Finger-like processes extend from the region between the equatorial segment and postacrosomal region and overlie the equatorial segment. The surface of the postacrosomal region is characterized by ridges with 150A spacing (Fig. 8). The ridges are oriented roughly parallel to the flagellum. Large irregular particles are often observed overlying the ridges of the postacrosomal region. Similar ridges are observed in other species, however, they are much more obvious in some preparations. This may be due to difficulty in removing the plasmalemma from the postacrosomal region or lability of the material which composes the ridges. Degu Degus are small rodents native to costal regions of Peru and Chile. The heads of degu spermatozoa are broad and do not display the hooked-shape asymmetry which characterize sperm heads of some other species of rodents (Figs. 9-11). The surface of the anterior portion of the acrosome is characterized by a number of large protrusions which vary in size and shape among spermatozoa (Figs. 9-11). The equatorial segment of the acrosome is lacking protrusions. As in other species, the equatorial segment is characterized by rows of particles with 170A spacing (Fig. 12). Fingerlike processes extend over the caudal portion of the equatorial segment (Fig. 12). The anterior portion of the postacrosomal region is marked by a saw tooth pattern (Figs. 9, 10). Golden Hamster As in the case of guinea pig spermatozoa, hamster sperm sometime undergo a false acrosome reaction when they are put into hypotonic medium as result of which the major portion of the acrosome is lost (Fig. 15). In some cells the acrosome appears to remain intact (Fig. 13). Whether or not the major portion of the acrosome is lost, the equatorial segment remains and the plasma membrane overlying the entire acrosome is lost. In a true acrosome reaction this segment of the plasma membrane would remain and becomes fused with the outer acrosomal membrane. The outer acrosomal membrane in the equatorial segment contains rows of 1 70A particles similar to those which characterize the membrane of the equatorial segment in other species (Figs. 14, 16). Rhesus Monkey As in other species, brief hypotonic treatment of Rhesus sperm appears to remove FIGS. 17 and 18. Replica of monkey spermatozoon treated with hypotonic I-lank s BSS. A portion of the plasrnalemma has been removed revealing the surface of the underlying outer acrosomal membrane. As in sperm of other species, the surface of the outer acrosomal membrane in the equatorial region is covered with rows of 180A-spaced particles. In the Rhesus monkey, however, the particles appear to be more irregular in size and spacing than in other species. Fig. 17 X10,000. Fig. 18 (same cell) X55,000.

7 134 PHILLIPS portions of the plasmalemma overlying the acrosome (Fig. 17). Rows of 170A spaced particles are observed in the equatorial region of the acrosome (Fig. 18). These particles are somewhat more irregular in size and shape than in other species. The anterior portion of the acrosome surface appear as irregular particles. In preparations where spermatozoa were treated with hypotonic Hank s BSS containing 0.5 percent MgC12, we observed 150A ridges in the postacrosomal region (Fig. 19). This suggests that these ridges may be a feature of the postacrosomal region of many species but that we do not observe them because the plasmalemma in the postacrosomal region is resistant to disruption by brief hypotonic treatment alone. Rat Surface replicas of rat spermatozoa treated briefly with hypotonic Hank s BSS reveal rows of 170A spaced particles over most of the surface of the outer acrosomal membrane (Figs. 20, 21). Only a thin rim along the convex surface of the acrosome is devoid of particles. Therefore, structurally the majority of the acrosome surface resembles the thin equatorial segment which we observed in other mammalian species. A narrow region covering the convex surface of the sperm head appears to correspond to the major region of the acrosome of other Mouse species. After hypotonic treatment, the surface of mouse sperm head is similar to rat (Phillips, 1975) (Fig. 22). Rows of 170A spaced particles cover most of the surface of the outer acrosomal membrane. A region considerably more broad than in the rat on the convex surface of the mouse spermatozoa is devoid of 170A particles. The region immediately along the convex surface appears rougher than the zone between this area and the zone containing the 170A spaced particles (Fig. 22). DISCUSSION We have examined the surface of the outer acrosomal membrane in spermatozoa of 8 mammalian species. In 6 of the eight, rabbit, bull, guinea pig, degu, hamster and monkey, rows of evenly spaced hexagonally packed particles with center-to-center spacing of 1 70A occur on the surface of the outer acrosomal FIG. 19. Surface of the postacrosomal region of a Rhesus monkey spermatozoa. This preparation was treated for about 15 minutes in 0.5 percent MgCl2. The surface of this region is characterized by parallel ridges with 1 50A center-to-center spacing. X 80,000.

8 EQUATORIAL SEGMENT: ACROSOME 135.i_ FIG. 20. Surface of the anterior region of a rat spermatozoa subjected briefly to hypotonic treatment before fixation. Through large holes in the lysed plasmalemma, rows of 1 70A spaced particles can be observed extending to the anteriormost tip of the sperm. Arrows indicate a thin region on the convex surface of the spermatozoa where the evenly spaced particles are not observed. X 55,000. FIG. 21. Surface of the posterior region of the rat sperm acrosomal surface. The surface of the acrosome is characterized by rows of 1 70A particles. The convex surface of the acrosome is characterized by larger irregular particles (arrows). X 36,000.

9 136 PHILLIPS FIG. 22. Mouse spermatozoon after brief hypotonic treatment. Rows of 170A are present in a region of the outer acrosomal membrane indicated by arrows. X 27,000. membrane in the equatorial segment. Such particles are not observed on the major portion of the surface of the acrosome. In the other two species, rat and mouse, 170A spaced particles cover a greater portion of the acrosome surface. In the rat only a narrow zone on the convex surface is devoid of the particles. These results suggest that an extensive region of the acrosomal surface in rat and mouse might correspond to the equatorial segment of other mammalian sperm. This structural evidence is in agreement with cytochemical studies of Yanagimachi and Teichman (1972). Using the silver proteinate method for demonstrating acid proteases, these workers found reaction product over the major portion of the acrosome, but absent from the equatorial segment in golden hamster, guinea pig, dog, bull and human. However, in the rat and mouse the major portion of the acrosome was acid protease negative. Acid proteases occurred only on the convex surface of the acrosome. The procedure for making replicas involves prior fixation, dehydration and critical point drying of sperm. Any of these treatments could presumably produce artifacts. Similar structures have, however, been observed after other methods of tissue preparation. In freeze cleave preparations Koehler (1970, 1972) has observed rows of particles with similar spacing in what he believed to be the outer acrosomal membrane of human and rabbit spermatozoa. Friend and Fawcett (1974) also described such structures in outer acrosomal membrane of guinea pig and rat sperm. Periodic structures we observed in the postacrosomal region are also seen with other techniques. They can be observed in both freeze cleave preparations and in this section of rabbit spermatozoa (Koehler, 1970; Phillips, 1972). The finger-like processes which we observed extending over the equatorial segment in guinea pig and degu have been observed in guinea pig in freeze cleave and sectioned material (Koehler, 1974; Friend and Fawcett, 1974). Since all structures described here have been observed in at least one other species, by other techniques, it is likely that our method for preparing surface replicas accurate-

10 EQUATORIAL SEGMENT: ACROSOME 137 ly preserved many surface structures. It is interesting that surface replicas reveal the same structures observed in freeze cleave preparations since replicas presumably facilitate visualization of surface structures and freeze cleave preparations reveal structures in the plane of the membrane. There could be several interpretations of this. The structure of the outer acrosomal membrane may be such that it cleaves along the surface rather than in the membrane plane; particles in the plane of the membrane may protrude through the surface, or our solvent extraction may extract membrane material to reveal intramembrane structures. Using surface replica technique, Koo et al. (1973) attempted to localize H-Y antigen on the surface of mouse spermatozoa. The figure presented in their paper, however, shows the H-Y antigen to be located on the region of 170A spaced particles which we have found to be characteristic of the outer acrosomal membrane, not the plasma membrane. Therefore, the H-Y antigen may be characteristic of the outer acrosomal membrane in the equatorial segment rather than the plasma membrane as interpreted by these workers. Our results emphasize the importance of controlling tonicity in studies which attempt to localize substances cytochemically on sperm surfaces. To determine if the plasma membiane has maintained its integrity throughout preparative procedures, surface replicas could be examined for the presence of characteristic particles of the residual segment of the acrosomal membrane. The presence of particles would indicate that the plasma membrane had been removed. REFERENCES Allison, A. C. and Hartree, E. F. (1970). Lysomal enzymes in the acrosome and their possible role in fertilization. J. Reprod. Fert. 21, 501. Bedford, J. M. (1972). An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat. 133, 213. Bedford, J. M. (1972). Sperm transport, capacitation and fertilization. Excerpta Medica, Amsterdam (Eds. H. Balin and S. Glasser). Friend, D. S. and Fawcett, D. W. (1974). Membrane differentiations in freeze-fractured mammalian sperm. J. Cell Biol. 63, 641. Koehler, J. K. (1970). A freeze-etch study of rabbit spermatozoa with particular reference to head structures. J. Ultrastr. Res. 33, 598. Koehler, J. K. (1972). Human sperm head ultrastructure A freeze-etching study. J. Ultrastr. Res. 39, 520. Koehler, J. K. (1974). Changes in the fine structure of guinea pig sperm heads following experimental treatment. In Functional Anatomy of the Spermatozoon (Ed. Afzelius), Pergamon Press. Koo, G. G., Stackpole, C. W., Boyse, E. A., Hammerling, U. and Lardes, M. P. (1973). Topographical localization of H-Y antigens on mouse spermatozoa by immunoelectron-microscopy. Proc. Nail. Acad. Sci. 70, Morton, D. B. (1975). Acrosomal enzymes: Immunechemical localization of acrosin and hyaluronidase in ram spermatozoa. J. Reprod. Fert. 45, 375. Phillips, D. M. (1972). Substructure of the mammalian acrosome. J. Ultrastruct. Res. 38, 591. Yanagimachi, R. and Nods, Y. D. (1970). Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastr. Res. 31, 465. Yanagimachi, R. and Teichman, R. J. (1972). Chemical demonstration of acrosomal proteinase in mammalian and avian spermatozoa by a silver proteinate method. Biol. of Reprod. 6, 87. Zamboni, L. (1971). Fine Morphology of Mammalian Fertilization. Harper & Row publishers.