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Ultrastructural Study of Separated Cell and Acrosomal Membranes from Bull Spermatozoa
Vol. 26. No.9. September 1975 Printed in U.SA.
FERTILITY AND STERILITY Copyright ~ 1975 The American Fertility Society
ULTRASTRUCTURAL STUDY OF SEPARATED CELL AND ACROSOMAL MEMBRANES FROM BULL SPERMATOZOA SEPPO MULTAMAKI, M.D., LAURI J. PELLINIEMI, M.D.,
AND
JYRKI SUOMINEN, PH.D.
Department of Anatomy and Laboratory of Electron Microscopy, University of Turku, SF-20520 Turku 52, Finland
The mammalian acrosome contains hydrolytic enzymes which are obviously necessary when spermatozoa penetrate the investments around the ovum. Therefore, several methods have been developed for removal and solubilization of the acrosomal components of mammalian spermatozoa. The cationic detergent Hyamine 2389, alone or in combination with the nonionic detergent Triton X-100, is widely used for this purpose. 1-4 By this method several enzymes not originating from the acrosome are also removed and thus contaminate the acrosomal extract.4-6 This can at least partly be avoided ifthe sperm heads are first removed by sonication and collected by centrifugation. 7 When the Hyamine extract of bull spermatozoa was fractionated on a two-step sucrose gradient, four different bands consisting of membrane material were obtained. 8 For better separation, a third sucrose step was added, and one additional band and a better biochemical separation of the acrosomal enzymes were obtained. 9 This work describes the ultrastructure of the fractions obtained by the improved sucrose gradient centrifugation of the Hyamine extract of bull spermatozoa.
were motile. The spermatozoa were washed and incubated for 90 minutes with 0.05% Hyamine 2389 (British Drug Houses, Poole, England), as described earlier.9 After the Hyamine treatment, 8 ml of the supernatant were layered on a sucrose gradient, which consisted of three steps: 4 ml of 2.0 M sucrose, 6 ml of 1.57 M sucrose, and 4 ml of 1.0 M sucrose. The tubes were centrifuged at 56,000 x g for 2 hours with an MSE Superspeed centrifuge using a swingout head. After the centrifugation, five clearly separated bands could be seen (Fig. 1). The bottom of the tube was pierced, and fractions containing 20 drops were collected. The fractions corresponding to the visible bands in the tube 9 were pooled; fixed with modified Karnovsky's fixative containing 1.33 M paraformaldehyde, 0.5 M glutaraldehyde, and 4.0 mM CaCl2 in a
Bond
ClI
1.0
IV III upper lower
molll
VI
MATERIALS AND METHODS
0 ~
Ejaculated spermatozoa were obtained from Finnish Ayrshire bulls; the average concentration of spermatozoa was 2 to 5 x 109/ml, of which at least 50%
u
1.57 molll
~
(j)
2.0
molll
\
"
FIG. 1. A schematic representation of the bands to V) obtained by centrifugation of Hyamine extract on a three-step sucrose density gradient.
(J
Received September 6, 1974.
-v
Sample
932
Vol. 26, No.9
ULTRASTRUCTURE OF SPERMATOZOAL MEMBRANE FRACTIONS
933
0.15 M sodium cacodylate buffer (pH 7.4); this paper. Compared with the two-step and prepared further for electron micros- gradient, the content of the upper part of copy as described earlier.s Thin sections the third band was, on the contrary, were cut with a diamond knife on an LKB- clearly different, and an additional band Huxley ultramicrotome and observed on a (band IV) was obtained (Fig. 1). The ultrastructure of these two latter bands JEM-T 8 electron microscope. and that of the unsedimented material in RESULTS the fifth band are thus described. The upper part of the third band, just The general outlines of the Hyamine effect on the ultrastructure of bovine above the surface of the 1.57 M sucrose spermatozoa have been described by layer (Fig. 1), consisted of separate vesiO'Donnel et a1.10 and Multamaki and cles of medium (150 to 200 nm diameter) Pelliniemi. S Their observations were also and large (500 to 700 nm) size and of confirmed in this work. The cell mem- acrosomal vesicles in the form of random brane was removed as a separate entity, aggregates or of an acrosomal cap (Figs. generally in the form of whorls or vesicles 2 to 5). One half of the acrosomal vesicle of different sizes, or it was fused with the was composed of the cell membrane and outer acrosomal membrane to form small the other half was composed of the outer vesicles (Figs. 2 and 3). The vesicles acrosomal membrane with acrosomal maappeared in random clusters or retained terial tightly bound to its outer surface the acrosomal cap form (Fig. 3). In both (Fig. 4). The separate large- and mediumcases, acrosomal material was left at- sized vesicles (Figs. 3 and 5) had a typical tached to that part of the vesicles which unit membrane structure; some of them originated from the outer acrosomal mem- were cut in oblique planes and were brane (Fig. 4). When the cell membrane therefore thickwalled. These mediumwas removed as a separate unit, the outer and large-sized vesicles were not coated by acrosomal membrane was detached as a the fuzzy material that was always whole cap and it glided unbroken over attached to that part of the acrosomal the sperm head. Also in this case, acro- vesicle originating from the outer acrosomal material was attached to the inner somal membrane. Separate, nonvesicusurface of the outer acrosomal membrane. lated, outer acrosomal membranes were By the three-step gradient, four dif- never observed in the upper part of the ferent bands within the sucrose and a fifth third band, but were left in the lower part band representing the layered sample of the same band inside the 1.57 M sucrose above the sucrose were obtained and layer. s The fourth band (Figs. 6 and 7) conanalyzed biochemically. 9 These bands are presented schematically in Figure 1. The tained large, medium, and small (30 to pellets obtained after the pooling of the 50 nm) vesicles and some membrane fractions from each band were submitted profiles; each of these structures had a for electron microscopic examination. trilaminar unit membrane. Within this Consistent with earlier results, the first fourth band, there were no membranes or and second band and the lower part of the vesicles with any attached fuzzy material third band showed the same fine structure on one side, which was characteristic as that observed earlier in fractions from a of the acrosomal vesicles in the upper two-step sucrose gradient because they part of the third band and of the separate were all below the upper surface of the 1.57 acrosomal caps in the lower part of the M sucrose, and this part of the gradient third band. The fifth band represented the unsediwas left unchanged (Fig. 1). Therefore, their ultrastructures is not presented in mented or dissolved material of the sam-
934
MULTAMAKI ET AL.
September 1975
Vol. 26, No.9
ULTRASTRUCTURE OF SPERMATOZOAL MEMBRANE FRACTIONS
pIe placed on the sucrose gradient. The sections made from the pellets of the pooled fractions of this band consisted mainly of small vesicles and small membrane fragments (Figs. 8 and 10). A narrow margin of the pellet also contained medium-sized vesicles similar to those found in band IV (Figs. 9 and 11). The main part (Fig. 8) did not contain acrosomal caps or vesicles in cap form. Even though the size of the small vesicles in the main part was equal to that of the acrosomal vesicles, they were clearly different because the whole circle of the vesicle wall had a uniform structure and no acrosomal material was seen bound to the membranes. DISCUSSION
These results confirm the advantageous effect of the third additional sucrose step on the separation of the cell membrane and acrosomal components, also noted biochemically earlier.9 The main part of the smooth membrane vesicles of different sizes was separated as a new additional zone (band IV), and the acrosomal vesicles, either in the form of aggregates or acrosomal caps, were left in the upper part of band III. The formation of these acrosomal vesicles has been reported earlier,S and it is similar to the acrosome reactionY On the other hand, separate outer acrosomal membranes sedimented in the lower part of band III, which was just below the surface of the 1.57 M sucrose step. The main part of the smooth membrane vesicles of different sizes was found in band IV, and those found in the upper part of band III were apparently a marginal contamination.
935
On the basis of these findings it can be concluded that the density of the outer acrosomal membrane, or that of the acrosomal material bound to it, is higher than the density of the cell membrane alone. If the membranous particles were totally composed of the outer acrosomal membrane, they were sedimented in the 1.57 M sucrose; however, if they also contained cell membrane, like the acrosomal vesicles, they were settled just above the surface of the 1.57 M surcrose (upper part of band III). If the vesicles were formed solely from the cell membrane, they were left in the upper part of the 1.0 M sucrose (band IV). The vesicles found in band IV did not contain any material bound to their surface, like the acrosomal vesicles or outer acrosomal membranes in the third band. The trilaminar unit membrane structure was also visible in the vesicles of this band. Therefore, it seems obvious that these vesicles originate from the cell membrane; the material in this band is thus very suitable for studies in which reasonably pure cell membranes are needed. The fifth band, the saline portion with material unsedimented in the sucrose, contained mainly small uniform vesicles and small membrane fragments, both with a trilaminar structure. In one part of the thin section, medium-sized vesicles were noted. They probably represented a contamination from the fourth band, and were sedimented in the bottom of the block during the preparation process. On a morphologic basis, the origin of the small vesicles within the fifth band is not clear. Their size was similar to that of the acrosomal vesicles, but they were without the attached acrosomal material which
FIGs. 2 TO 5. Upper part of band III. FIG. 2. A survey picture showing various vesicles and aggregates of acrosomal vesicles (thin arrow) and one vesiculated acrosomal cap (thick arrow). F, Fibrous sheath (x 7,000). FIG. 3. Vesiculated acrosomal cap (A V) and large (LV)- and medium-sized (MV) vesicles (x 30,000). FIG. 4. AV, A portion of a vesiculated acrosomal cap; AD, outer acrosomal membrane with attached acrosomal material; M, cell membrane (x 70,000). FIG. 5. Large, smooth membrane vesicles (x 70,000).
936
MULTAMAKI ET AL.
September 1975
FIG. 6. A survey picture of band IV containing various vesicles (x 7,000). FIG.7. Medium (MV)- and small-sized (BV) vesicles in band IV (x 70,000).
was always seen in the acrosomal vesicles. On the other hand, they also looked quite similar to the vesicles and bulges formed from the inner acrosomal membrane by a prolonged treatment with Hyamine or by a subsequent treatment with Triton X-IOO.12 The fifth band also demonstrated lactate dehydrogenase and isocitrate dehydrogenase activites. 9 On this basis it was assumed that both the material visualized in the picture of
the pellet from this band and the solubilized components of the same band originated from various parts rather than from a single component of the spermatozoa. The present results indicate that the cell and outer acrosomal membrane of bull spermatozoa can be separated by centrifugation on a sucrose gradient, depending on the density differences of these two structures, but the vesicular acrosomal
Vol. 26, No.9
ULTRASTRUCTURE OF SPERMATOZOAL MEMBRANE FRACTIONS
FIGs. 8 TO 11. Supernatant material (band V). FIG. 8. A survey picture of the main part of the pellet composed ofsmall vesicles (x 7,000). FIG. 9. A survey picture of the marginal part of the pellet showing small vesicles on the left and medium vesicles on the right (x 7,000). FIG. 10. Main part of the pellet containing various sections of small vesicles (BV) and some membrane fragments (arrows) (x 70,000). FIG. 11. Marginal part of the pellet composed of medium vesicles (MV) and some small vesicles (BV) (x 70,000).
937
938
MULTAMAKI ET AL.
fractions always contain some cell membrane as a component of the vesicle wall. SUMMARY
Ejaculated bull spermatozoa were washed and incubated with a cationic detergent, Hyamine 2389, and the material removed was centrifuged on a threestep sucrose gradient. Five different bands were obtained, three of which contained cell and acrosomal membranes and also acrosomal material. The first of these, band III, was composed of two parts. The lower part contained V-shaped membrane profiles originating from the outer acrosomal membrane, acrosomal material attached to them, and vesicles of different sizes. The upper part, in addition to various vesicles (probably formed from cell membrane), contained acrosomal vesicles arranged in the form of aggregates or caps. The latter vesicles were formed by fusion and vesiculation of the cell and outer acrosomal membranes with tightly bound acrosomal material. Band IV was uniform and comprised various round vesicles with a smooth membrane, which had a typical unit-membrane structure. There were obviously formed from the cell membrane. The fifth band, in the saline portion with material unsedimented in the sucrose, included small vesicles and membrane fragments, the origin of which was obscure. The solubilized material was also left in this fraction. Acknowledgments. The authors thank Mrs. Marita Aaltonen, Mrs. Sirpa From, Miss Ulla Miintylii, Mr. Mauno Lehtimiiki, and Mr. Urpo Reunanen for their skillful technical assistance, and Mrs. Airi Ketola for secretarial work.
September 1975 REFERENCES
1. Hartree EF, Srivastava PN: Chemical composition of the acrosomes of ram spermatozoa. J Reprod Fertil 9:47, 1965 2. Zaneveld LJD, Srivastava PN, Williams WL: Relationship of a trypsinlike enzyme in rabbit spermatozoa to capacitation. J Reprod Fertil 20:337, 1969 3. Polakoski KL, Zaneveld LJD, Williams WL: Purification of a proteolytic enzyme from rabbit acrosome. Bioi Reprod 6:23, 1972 4. Stambaugh R, Smith M: An evaluation of several extraction procedures for acrosomal enzymes. Bioi Reprod 7:100, 1972 5. Multamiiki S, Niemi M: Trypsin-like proteolytic activity in an acrosomal extract of bull spermatozoa. Int J Fertil17:43, 1942 6. Stambaugh R, Smith M: A comparison of several extraction procedures for rabbit acrosomal enzymes. J Reprod Fertil 35:127, 1973 7. Stambaugh R, Buckley J: Identification and subcellular localization of the enzymes effecting penetration of the zona pellucida by rabbit spermatozoa. J Reprod Fertil 19:423, 1969 8. Multamiiki S, Pelliniemi LJ: Ultrastructure of subcellular fractions of bull spermatozoa after Hyamine treatment. Z Zellforsch Mikrosk Anat 144:395, 1973 9. Multamiiki S: Isolation of pure acrosomes by subcellular fractionation of bull spermatozoa. Int J Fertil 18:193, 1973 10. O'Donnel JM, Symons DBA, Wooding FBP: Immunofluorescence and electron microscopy of acrosomes of bull spermatozoa. J Physiol (Lond) 210:120P, 1970 11. Bedford JM: Sperm capacitation and fertilization in mammals. BioI Reprod [Suppl] 2:128, 1970 12. Multamiiki S, Suominen J: Distribution and removal of acrosin of bull spermatozoa. Int J Fertil, submitted for publication
FERTILITY AND STERILITY Copyright ~ 1975 The American Fertility Society
ULTRASTRUCTURAL STUDY OF SEPARATED CELL AND ACROSOMAL MEMBRANES FROM BULL SPERMATOZOA SEPPO MULTAMAKI, M.D., LAURI J. PELLINIEMI, M.D.,
AND
JYRKI SUOMINEN, PH.D.
Department of Anatomy and Laboratory of Electron Microscopy, University of Turku, SF-20520 Turku 52, Finland
The mammalian acrosome contains hydrolytic enzymes which are obviously necessary when spermatozoa penetrate the investments around the ovum. Therefore, several methods have been developed for removal and solubilization of the acrosomal components of mammalian spermatozoa. The cationic detergent Hyamine 2389, alone or in combination with the nonionic detergent Triton X-100, is widely used for this purpose. 1-4 By this method several enzymes not originating from the acrosome are also removed and thus contaminate the acrosomal extract.4-6 This can at least partly be avoided ifthe sperm heads are first removed by sonication and collected by centrifugation. 7 When the Hyamine extract of bull spermatozoa was fractionated on a two-step sucrose gradient, four different bands consisting of membrane material were obtained. 8 For better separation, a third sucrose step was added, and one additional band and a better biochemical separation of the acrosomal enzymes were obtained. 9 This work describes the ultrastructure of the fractions obtained by the improved sucrose gradient centrifugation of the Hyamine extract of bull spermatozoa.
were motile. The spermatozoa were washed and incubated for 90 minutes with 0.05% Hyamine 2389 (British Drug Houses, Poole, England), as described earlier.9 After the Hyamine treatment, 8 ml of the supernatant were layered on a sucrose gradient, which consisted of three steps: 4 ml of 2.0 M sucrose, 6 ml of 1.57 M sucrose, and 4 ml of 1.0 M sucrose. The tubes were centrifuged at 56,000 x g for 2 hours with an MSE Superspeed centrifuge using a swingout head. After the centrifugation, five clearly separated bands could be seen (Fig. 1). The bottom of the tube was pierced, and fractions containing 20 drops were collected. The fractions corresponding to the visible bands in the tube 9 were pooled; fixed with modified Karnovsky's fixative containing 1.33 M paraformaldehyde, 0.5 M glutaraldehyde, and 4.0 mM CaCl2 in a
Bond
ClI
1.0
IV III upper lower
molll
VI
MATERIALS AND METHODS
0 ~
Ejaculated spermatozoa were obtained from Finnish Ayrshire bulls; the average concentration of spermatozoa was 2 to 5 x 109/ml, of which at least 50%
u
1.57 molll
~
(j)
2.0
molll
\
"
FIG. 1. A schematic representation of the bands to V) obtained by centrifugation of Hyamine extract on a three-step sucrose density gradient.
(J
Received September 6, 1974.
-v
Sample
932
Vol. 26, No.9
ULTRASTRUCTURE OF SPERMATOZOAL MEMBRANE FRACTIONS
933
0.15 M sodium cacodylate buffer (pH 7.4); this paper. Compared with the two-step and prepared further for electron micros- gradient, the content of the upper part of copy as described earlier.s Thin sections the third band was, on the contrary, were cut with a diamond knife on an LKB- clearly different, and an additional band Huxley ultramicrotome and observed on a (band IV) was obtained (Fig. 1). The ultrastructure of these two latter bands JEM-T 8 electron microscope. and that of the unsedimented material in RESULTS the fifth band are thus described. The upper part of the third band, just The general outlines of the Hyamine effect on the ultrastructure of bovine above the surface of the 1.57 M sucrose spermatozoa have been described by layer (Fig. 1), consisted of separate vesiO'Donnel et a1.10 and Multamaki and cles of medium (150 to 200 nm diameter) Pelliniemi. S Their observations were also and large (500 to 700 nm) size and of confirmed in this work. The cell mem- acrosomal vesicles in the form of random brane was removed as a separate entity, aggregates or of an acrosomal cap (Figs. generally in the form of whorls or vesicles 2 to 5). One half of the acrosomal vesicle of different sizes, or it was fused with the was composed of the cell membrane and outer acrosomal membrane to form small the other half was composed of the outer vesicles (Figs. 2 and 3). The vesicles acrosomal membrane with acrosomal maappeared in random clusters or retained terial tightly bound to its outer surface the acrosomal cap form (Fig. 3). In both (Fig. 4). The separate large- and mediumcases, acrosomal material was left at- sized vesicles (Figs. 3 and 5) had a typical tached to that part of the vesicles which unit membrane structure; some of them originated from the outer acrosomal mem- were cut in oblique planes and were brane (Fig. 4). When the cell membrane therefore thickwalled. These mediumwas removed as a separate unit, the outer and large-sized vesicles were not coated by acrosomal membrane was detached as a the fuzzy material that was always whole cap and it glided unbroken over attached to that part of the acrosomal the sperm head. Also in this case, acro- vesicle originating from the outer acrosomal material was attached to the inner somal membrane. Separate, nonvesicusurface of the outer acrosomal membrane. lated, outer acrosomal membranes were By the three-step gradient, four dif- never observed in the upper part of the ferent bands within the sucrose and a fifth third band, but were left in the lower part band representing the layered sample of the same band inside the 1.57 M sucrose above the sucrose were obtained and layer. s The fourth band (Figs. 6 and 7) conanalyzed biochemically. 9 These bands are presented schematically in Figure 1. The tained large, medium, and small (30 to pellets obtained after the pooling of the 50 nm) vesicles and some membrane fractions from each band were submitted profiles; each of these structures had a for electron microscopic examination. trilaminar unit membrane. Within this Consistent with earlier results, the first fourth band, there were no membranes or and second band and the lower part of the vesicles with any attached fuzzy material third band showed the same fine structure on one side, which was characteristic as that observed earlier in fractions from a of the acrosomal vesicles in the upper two-step sucrose gradient because they part of the third band and of the separate were all below the upper surface of the 1.57 acrosomal caps in the lower part of the M sucrose, and this part of the gradient third band. The fifth band represented the unsediwas left unchanged (Fig. 1). Therefore, their ultrastructures is not presented in mented or dissolved material of the sam-
934
MULTAMAKI ET AL.
September 1975
Vol. 26, No.9
ULTRASTRUCTURE OF SPERMATOZOAL MEMBRANE FRACTIONS
pIe placed on the sucrose gradient. The sections made from the pellets of the pooled fractions of this band consisted mainly of small vesicles and small membrane fragments (Figs. 8 and 10). A narrow margin of the pellet also contained medium-sized vesicles similar to those found in band IV (Figs. 9 and 11). The main part (Fig. 8) did not contain acrosomal caps or vesicles in cap form. Even though the size of the small vesicles in the main part was equal to that of the acrosomal vesicles, they were clearly different because the whole circle of the vesicle wall had a uniform structure and no acrosomal material was seen bound to the membranes. DISCUSSION
These results confirm the advantageous effect of the third additional sucrose step on the separation of the cell membrane and acrosomal components, also noted biochemically earlier.9 The main part of the smooth membrane vesicles of different sizes was separated as a new additional zone (band IV), and the acrosomal vesicles, either in the form of aggregates or acrosomal caps, were left in the upper part of band III. The formation of these acrosomal vesicles has been reported earlier,S and it is similar to the acrosome reactionY On the other hand, separate outer acrosomal membranes sedimented in the lower part of band III, which was just below the surface of the 1.57 M sucrose step. The main part of the smooth membrane vesicles of different sizes was found in band IV, and those found in the upper part of band III were apparently a marginal contamination.
935
On the basis of these findings it can be concluded that the density of the outer acrosomal membrane, or that of the acrosomal material bound to it, is higher than the density of the cell membrane alone. If the membranous particles were totally composed of the outer acrosomal membrane, they were sedimented in the 1.57 M sucrose; however, if they also contained cell membrane, like the acrosomal vesicles, they were settled just above the surface of the 1.57 M surcrose (upper part of band III). If the vesicles were formed solely from the cell membrane, they were left in the upper part of the 1.0 M sucrose (band IV). The vesicles found in band IV did not contain any material bound to their surface, like the acrosomal vesicles or outer acrosomal membranes in the third band. The trilaminar unit membrane structure was also visible in the vesicles of this band. Therefore, it seems obvious that these vesicles originate from the cell membrane; the material in this band is thus very suitable for studies in which reasonably pure cell membranes are needed. The fifth band, the saline portion with material unsedimented in the sucrose, contained mainly small uniform vesicles and small membrane fragments, both with a trilaminar structure. In one part of the thin section, medium-sized vesicles were noted. They probably represented a contamination from the fourth band, and were sedimented in the bottom of the block during the preparation process. On a morphologic basis, the origin of the small vesicles within the fifth band is not clear. Their size was similar to that of the acrosomal vesicles, but they were without the attached acrosomal material which
FIGs. 2 TO 5. Upper part of band III. FIG. 2. A survey picture showing various vesicles and aggregates of acrosomal vesicles (thin arrow) and one vesiculated acrosomal cap (thick arrow). F, Fibrous sheath (x 7,000). FIG. 3. Vesiculated acrosomal cap (A V) and large (LV)- and medium-sized (MV) vesicles (x 30,000). FIG. 4. AV, A portion of a vesiculated acrosomal cap; AD, outer acrosomal membrane with attached acrosomal material; M, cell membrane (x 70,000). FIG. 5. Large, smooth membrane vesicles (x 70,000).
936
MULTAMAKI ET AL.
September 1975
FIG. 6. A survey picture of band IV containing various vesicles (x 7,000). FIG.7. Medium (MV)- and small-sized (BV) vesicles in band IV (x 70,000).
was always seen in the acrosomal vesicles. On the other hand, they also looked quite similar to the vesicles and bulges formed from the inner acrosomal membrane by a prolonged treatment with Hyamine or by a subsequent treatment with Triton X-IOO.12 The fifth band also demonstrated lactate dehydrogenase and isocitrate dehydrogenase activites. 9 On this basis it was assumed that both the material visualized in the picture of
the pellet from this band and the solubilized components of the same band originated from various parts rather than from a single component of the spermatozoa. The present results indicate that the cell and outer acrosomal membrane of bull spermatozoa can be separated by centrifugation on a sucrose gradient, depending on the density differences of these two structures, but the vesicular acrosomal
Vol. 26, No.9
ULTRASTRUCTURE OF SPERMATOZOAL MEMBRANE FRACTIONS
FIGs. 8 TO 11. Supernatant material (band V). FIG. 8. A survey picture of the main part of the pellet composed ofsmall vesicles (x 7,000). FIG. 9. A survey picture of the marginal part of the pellet showing small vesicles on the left and medium vesicles on the right (x 7,000). FIG. 10. Main part of the pellet containing various sections of small vesicles (BV) and some membrane fragments (arrows) (x 70,000). FIG. 11. Marginal part of the pellet composed of medium vesicles (MV) and some small vesicles (BV) (x 70,000).
937
938
MULTAMAKI ET AL.
fractions always contain some cell membrane as a component of the vesicle wall. SUMMARY
Ejaculated bull spermatozoa were washed and incubated with a cationic detergent, Hyamine 2389, and the material removed was centrifuged on a threestep sucrose gradient. Five different bands were obtained, three of which contained cell and acrosomal membranes and also acrosomal material. The first of these, band III, was composed of two parts. The lower part contained V-shaped membrane profiles originating from the outer acrosomal membrane, acrosomal material attached to them, and vesicles of different sizes. The upper part, in addition to various vesicles (probably formed from cell membrane), contained acrosomal vesicles arranged in the form of aggregates or caps. The latter vesicles were formed by fusion and vesiculation of the cell and outer acrosomal membranes with tightly bound acrosomal material. Band IV was uniform and comprised various round vesicles with a smooth membrane, which had a typical unit-membrane structure. There were obviously formed from the cell membrane. The fifth band, in the saline portion with material unsedimented in the sucrose, included small vesicles and membrane fragments, the origin of which was obscure. The solubilized material was also left in this fraction. Acknowledgments. The authors thank Mrs. Marita Aaltonen, Mrs. Sirpa From, Miss Ulla Miintylii, Mr. Mauno Lehtimiiki, and Mr. Urpo Reunanen for their skillful technical assistance, and Mrs. Airi Ketola for secretarial work.
September 1975 REFERENCES
1. Hartree EF, Srivastava PN: Chemical composition of the acrosomes of ram spermatozoa. J Reprod Fertil 9:47, 1965 2. Zaneveld LJD, Srivastava PN, Williams WL: Relationship of a trypsinlike enzyme in rabbit spermatozoa to capacitation. J Reprod Fertil 20:337, 1969 3. Polakoski KL, Zaneveld LJD, Williams WL: Purification of a proteolytic enzyme from rabbit acrosome. Bioi Reprod 6:23, 1972 4. Stambaugh R, Smith M: An evaluation of several extraction procedures for acrosomal enzymes. Bioi Reprod 7:100, 1972 5. Multamiiki S, Niemi M: Trypsin-like proteolytic activity in an acrosomal extract of bull spermatozoa. Int J Fertil17:43, 1942 6. Stambaugh R, Smith M: A comparison of several extraction procedures for rabbit acrosomal enzymes. J Reprod Fertil 35:127, 1973 7. Stambaugh R, Buckley J: Identification and subcellular localization of the enzymes effecting penetration of the zona pellucida by rabbit spermatozoa. J Reprod Fertil 19:423, 1969 8. Multamiiki S, Pelliniemi LJ: Ultrastructure of subcellular fractions of bull spermatozoa after Hyamine treatment. Z Zellforsch Mikrosk Anat 144:395, 1973 9. Multamiiki S: Isolation of pure acrosomes by subcellular fractionation of bull spermatozoa. Int J Fertil 18:193, 1973 10. O'Donnel JM, Symons DBA, Wooding FBP: Immunofluorescence and electron microscopy of acrosomes of bull spermatozoa. J Physiol (Lond) 210:120P, 1970 11. Bedford JM: Sperm capacitation and fertilization in mammals. BioI Reprod [Suppl] 2:128, 1970 12. Multamiiki S, Suominen J: Distribution and removal of acrosin of bull spermatozoa. Int J Fertil, submitted for publication