SILVER
METHENAMINE
STAINING
AND PHOSPHOTUNGSTIC
OF THE ACROSOME
OF MYTZLUS
ACID
EDULZS
SACHIKO END0 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo, and Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo, Japan
SUMMARY The Mytilus acrosome was investigated by histochemical methods combined with electron microscopy, using silver methenamine (SM) and phosphotungstic acid (PTA) staining, as well as some chemical and enzymatic pretreatments followed by the staining. As one of two major components in the Mytilus acrosome, the egg-membrane lysin was conspicuously stainable with PTA and susceptible to pronase digestion. The other component, that occupies the space between the acrosomal membrane and the axially located strand containing lysin, was stained with SM very specifically. This staining property was not affected by pronase digestion or treatment that blocked aldehyde and SH groups.
Several electron micrographical studies on the acrosome of Mytifus edulis have investigated in considerable detail its fine structure [l, 2, 31, its mode of formation [4] and the changes that occur as it reacts at the egg surface to release an “egg-membrane lysin” and form an acrosomal process [5]. The results of these studies have shown that the Mytifus acrosome contains two major components located in a characteristic and constant pattern. An earlier investigation by Wada et al. [6] indicated that the egg-membrane lysin is released during the acrosomal reaction and it has been identified [5] as the substance which, in the intact acrosome, forms an annular deposit in the “basal ring” [l] and an axially located strand in its distal part. This material is solubilized within a fraction of a second after the apex of the acrosomal vesicle opens at the beginning of the reaction.
The second major component lies immediately inside the outer acrosomal membrane in a thick layer extending from the apex to the basal deposit of lysin. This material is observed to swell sequentially [5], from the the tip toward the base, as sea water enters the acrosomal vesicle through the apical opening. This swelling has been postulated as the agent that causes the reversion of the outer acrosomal and plasma membranes, exposing the inner acrosomal membrane which elongates as the membrane of the acrosomal process [5]. The acrosome of Hydroides hexagonus [7] also includes a layer of material that lines the outer acrosomal membrane and appears to undergo similar hydration and swelling as the acrosome opens [8]. In view of the fact that this second component occupies at least half of the space within the Mytilus acrosomal vesicle and appears to play an important part in the
72
S. Endo microtome, expanded with chloroform vapour, and treated by floating them on the surface of each one or some combination of the various solutions. In the case of serial treatments, the sections were transferred with a platinum loop from one solution to deionized water and then to the next solution.
Silver methenamine staining (SM staining) The sections were floated on a solution of 0.1% silver nitrate containing 0.025 M borate buffer (pH 9.2) and 1% hexamethylenetetramine for 30 min at 60°C. The stained sections were washed with I % thiosulfate solution for about 30 min at room temperature [ 121.
Phosphotungstic acid staining (PTA staining) The sections were exposed to a solution of 1% phosphotungstic acid and 10% chromic acid (pH 0.3) [ 131 for 2 or 4 h at room temperature.
Deionized water treatment (DW treatment) I. Diagrammatic longitudinal section of Myrilus acrosome. AM, acrosomal membrane: CP, central proximal part; D, distal part; La, lysin in axially located strand; Lh, lysin in the basal ring; LM, lumen formed by invagination of acrosomal membrane; N, nucleus; PM, plasma membrane; PP, peripheral proximal part; R. rod.
The sections were floated on deionized water for more than 25 h at 60°C.
Fig.
Blockage of aldehyde and SH groups In order to sequester aldehyde groups, the sections were floated on a 2 % sodium bisulfite solution for 2 h at 60°C [l2]. As for sulfhydryl groups, the sections were floated on 0.1 M iodoacetic acid solution adjusted to pH 8 with ammonium hydroxide for 2 h at 60°C [12], or on 0.1 M N-ethylmaleimide solution containing 10 mM Tris buffer (pH 8) for l-l.5 h at 60°C.
acrosome reaction, its chemical properties are of some interest, particularly since a significant amount of calcium-dependent Pronase digestion ATPase activity has been found in associa- The sections were exposed to a solution of 0.01% tion with it [9]. In this study, the acrosome pronase and 0.025 M borate buffer (pH 9.2) for 15-30 min or 2 h at 37°C. of Mytilus edulis has been investigated by histochemical methods combined with Fig. 2. SM staining of late spermatids in Myrilus testis. electron microscopy, using silver methen- Immature spermatid (short arrow) has a large, irregular nucleus. Mature spermatozoon (long arrow) has a amine and PTA staining, as well as some small spheroidal one. High electron density is conchemical and enzymatic pretreatments fol- spicuous in the acrosome of immature spermatids and the nucleus of mature spermatozoa. A, acrosome; lowed by staining. M, mitochondrion; N, nucleus. X 8 500. Fig. 3. SM staining after DW treatment of spermatids
MATERIALS
AND METHODS
Small pieces of Myths testis were fixed with 2.5% glutaraldehyde in sea water, and dehydrated with acetone for SM staining or ethyleneglycol [IO] for PTA staining, followed by embedding in a Rigolac mixture [I I]. Sections were cut on a Porter-Blum MT-l ultraE.rp Cc/I Rr.5 100 (1976)
as in fig. 2. The stainability of the nucleus is markedly reduced. A, acrosome; F, flagellum; M, mitochondrion; N, nucleus. x8500. Fig. 4. Longitudinal section of an immature spermatid stained with SM after treatment with DW. High electron density is limited to D and CP. Cross section at D region is seen at lower right (arrow). CP, central proximal part; D, distal part; N, nucleus. ~40000.
Cytochemical staining of acrosome
73
74
S. Endo
Fig. 5. SM staining after pronase digestion for 2 h without DW treatment. Pronase digestion does not change the SM staining of the acrosomes. The egg-
membrane lysin in mature acrosome is digested (long arrow), but that of immature acrosome is not (short arrow). x 17 500.
After these treatments the sections were finally mounted directly on grids coated with mesh-cement [l4] and o b served with a Hitachi HU-IIB electron microscope.
SM staining Fig. 2 shows part of a testis containing spermatids in late stages of spermiogenesis. The immature spermatids have large, irregularly shaped nuclei (fig. 2, short arrow), while the nuclei of mature spermatozoa are smaller and more spherical (fig. 2, long arrow). Areas of high electron density caused by the deposition of silver particles are
RESULTS Fig. 1 is a diagram of a longitudinal section of the acrosome of Myrifus edufis. The acrosome consists of a large, conical, acrosomal vesicle. The posterior part of the vesicle is invaginated to form a lumen, in which a slender axial rod is contained. The egg-membrane lysin fills the basal ring (fig. 1, Lb) and the axial part distal to the invagination (fig. 1, La). On the basis of differences in their staining properties, the regions consisting mainly of the other vesicular component are designated as peripheral proximal (PP), central proximal (CP), and distal (D) parts, as shown in fig. 1.
Fig. 6. (a) PTA staining for 4 h. Longitudinal section through apex of Myths acrosome. The egg-membrane lysin (La, Lb) and PP are stained. A material lying just inside the acrosome at its apex is distinguished from La (arrow). La, lysin in axially located strand; Lb, lysin in the basal ring; PP, peripheral proximal part. x68000; (b) PTA staining for 2 h. Longitudinal section through apex of Myrilus acrosome. Arrow indicates layer of substance filling space between plasma and acrosomal membranes; layer is much thinner at apex (*---*). x128000. Fig. 7. PTA staining after pronase digestion for 30 min. Lb and part of La are digested; the staining properties in other parts are unchanged. x25000.
fochemical staining of acrosome
75
76
S. Endo
conspicuous on the acrosomes of the immature spermatids and nuclei of the mature spermatozoa; the acrosomes of the latter showed only faint stain. SM staining after DW treatment DW treatment prior to SM staining (fig. 3) markedly reduced the stainability of the nucleus, and the silver particles scattered on the background were all eliminated. However, the degree and pattern of staining in the acrosome did not change. With higher resolution, it was found that the staining was highly specific for the CP and D parts of the acrosome, and the regions containing lysin (La and Lb) were entirely unstained (fig. 4). SM staining after pronase digestion Pronase treatment for a period of 2 h did not influence the SM staining of the acrosome. In the mature acrosomes, however, pronase digested the lysin and the material contained in the PP part (fig. 5, long arrow). SM staining after blocking aldehyde and SH groups Treatment of the sections with sodium bisulfite, iodoacetic acid or N-ethylmaleimide did not significantly affect the SM staining of the acrosomes. PTA staining The testis used for this staining contained mostly mature spermatozoa. PTA was found to stain La, Lb and PP parts strongly; the electron density of Lb was especially prominent (fig. 6a). A small amount of material lying just inside the acrosome at its apex was distinguished from La by its greater affinity for PTA. It formed a small mass (fig. 6a, arrow) or layer (fig. 6b). E.rp Cdl Rr.\ lo0 119761
A substance that fills the space between the plasma and acrosomal membranes became visible after this staining (fig. 6b, arrow). Since the material was fixed with glutaraldehyde alone, the membranes are not preserved, and this substance looks as if it is situated on the most outer surface of the acrosome. It forms a uniform layer 10 nm thick over all of the acrosome except for a small region at the apex where it is much thinner and is sometimes broken (fig. 66, *---*). The nucleus, mitochondria and the microtubules of the flagellum were also stained by PTA. PTA staining after pronase treatment Pronase treatment for a period of 30 min digested the lysin in Lb and a part of La, without affecting the PTA staining of the other parts of the acrosome (fig. 7). After 2 h treatment, all of the egg-membrane lysin and the material contained in PP was eliminated. The results obtained with pronase digestion and PTA staining suggest that PP consists at least in part of egg-membrane lysin. The material at the apex of the acrosome (fig. 6a, arrow) and the substance between the plasma and acrosomal membranes were found not to be digested by pronase.
DISCUSSION Many investigators have reported that silver-methenamine staining of sections identifies aldehyde groups [12, 15-191 and sulfhydryl groups [12, 15, 19, 203. It has been suggested that when aldehyde fixatives are used, some of the fixative remaining in the tissue might affect the SM staining. It seems probable that this is not so in
Cytochemical staining of acrosome the present case, because SM staining of the acrosome after fixing with osmium tetroxide alone gives the same result. The same is also true when staining is carried out prior to glutaraldehyde fixation (Endo, unpublished). To check the possibility that aldehyde or sulfhydryl, or both groups are primarily responsible for the observed SM staining, treatments for sequestering these groups were applied, but they failed to affect the staining under the experimental conditions used. At present, no definite conclusion can be drawn about the mechanism of SM staining in this material, although the possibility remains that other reducing groups might be responsible for the reaction. With respect to the mechanism and specificity of PTA staining, Rambourg et al [ 131 reported that a substance in rat cells which reacts with PTA should be glycoprotein. They assume that saccharide moieties are stained with PTA. However, under the same experimental conditions except for a staining time of 15 min instead of 2 min, the lysin is strongly stained (Endo, unpublished). Its marked susceptibility to pronase digestion (fig. 7) indicates that it consists mainly of protein. This is in line with the finding of Haino [21], that the eggmembrane lysin carried in the acrosome of another mollusc consists of a simple basic protein. In this connection, PTA in an aqueous solution at pH 4.5 stains collagen fibers, the reactive sites of which are known to be basic amino acids [22]. When collagen fibers were stained at pH 0.3, they showed a pattern quite similar to that obtained at pH 4.5 (Nishigai & Endo, unpublished). This result suggests that basic amino acids may well be responsible for the pattern of PTA staining observed in the Mytilus acrosome. This study has found that major con-
77
stituents of the Mytilus acrosome, occupying the zones D and CP which include about half of the material within the acrosomal vesicle, shows a unique affinity for the SM stain. Although it has not proved possible to determine the chemical nature of this substance, its resistance to digestion by pronase indicates that it is probably not a simple protein. The component D represents the material that has been proposed as the agent for turning back the conjoined plasma and outer acrosomal membranes during the acrosome reaction [5]. To accomplish this in a manner consonant with the successive morphological changes observed in thin sections, the D material should first swell, thereby exerting an outward pressure against the membrane, and then immediately dissolve or disperse in the sea water. On the other hand, the material in CP with the same affinity for SM persists at the base of the acrosomal process, as though to lend physical support to it. The reaction of the Mytilus edulis acrosome is known to begin with a structural change at its apex [5]. Dan et al. [2] observed that the plasma and acrosomal membranes are in especially close contact with each other only in this region. In the present study, moreover, it was found that the layer of substance filling the space between the plasma and acrosomal membranes is much thinner at the apex than in other places. There is a small amount of material lying just inside the acrosome at its apex that is particularly stained by PTA. Recently Dan et al. [3] found an electron-lucent component at the same locus, suggesting that this material is rather labile and may function in the initiation of the acrosomal reaction. Three points should be mentioned in relation to the stage-dependency of differences in the chemical susceptibility to staining E.rp Cc,// Hr.\
100 (1976)
78
S. Endo
and enzyme action of cellular components during late spermiogenesis. (1) Immature acrosomes are stained very heavily by SM, but fully matured acrosomes, only faintly. (2) In later stages of spermiogenesis, the nuclei are more strongly stained. It is generally known in many animals that the spermatid nucleus becomes more basic as spermiogenesis proceeds. Kaye & Kaye [23] found that the amount of acidic protein in the spermatid nucleus of house cricket decreases during spermiogenesis leaving basic protein content constant. According to MacRae & Meets [24], the nucleus of chick erythrocytes in the early maturation stage exhibits little or no ammoniacal silver reaction, but becomes reactive as maturation proceeds, as the result of an alteration of histone species from lysine-rich to argininerich. (3) Pronase treatment for 2 h digested the egg-membrane lysin only when the acrosome was fully matured. These three phenomena suggest that the chemical as well as the ultrastructural properties of each component in the spermatid are modified in the course of spermiogenesis. The author wishes to express her thanks to Dr J. C. Dan for her encouragement throughout the work and assistance with the manuscript. She is also indebted to Dr H. Sakai and Dr M. Nishigai for their interest and valuable discussions.
E.rp Cell
Rr$
100 (1976)
REFERENCES 1. Niijima, L & Dan, J C, J cell biol 25 (1965) 243. 2. Dan, J C, Kakizawa, Y, Kushida, H & Fujita, K, Exp cell res 72 (1972) 60. 3. Dan, J C, Hashimoto, S, Kubo, M & Yonehara, K, The functional anatomy of the spermatozoon (ed B A Afzelius) p. 39. Pergamon Press, Oxford and New York (1974). 4. Suzuki, F & Endo, S. Unpublished. 5. Niiiima, L & Dan, J C. J cell biol 25 (1965) 249. 6. Wada, S K, Collier, J R & Dan, J C. Exp cell res IO (1956) 168. 7. Colwin, A L & Colwin, L H, J biophys biochem cytol 10(1961)211. 8. Colwin, L H & Colwin, A L, J biophys biochem cytol IO (1961) 231. 9. Mabuchi. Y & Mabuchi. I. EXD cell res 82 (1973) 271. IO. Leduc, E H & Bernhard, W, J ultrastruct res 19 (1967) 196. Il. Kushida, H, J electron microsc 9 (1961) 113. 12. Pickett-Heaps, J D, J histochem cytochem I5 (1967) 442. 13. Rambourg, A, Hernandez. W & Leblond, C P, J cell biol 40 (1969) 395. 14. Kushida, H & Fujita, K, J electron microsc I3 (1964) 27. IS. Pickett-Heaps, J D, J cell sci 3 (1968) 55. 16. Swift, J A & Saxton, C A, J ultrastruct res 17 (1967) 23. 17. Rambourg, A & Leblond, C P, J cell biol 32 (l%7) 27. 18. Rambourg, A, J histochem cytochem I5 (1967) 409. 19. Burr, F A, J histochem cytochem 21 (1973) 386. 20. Swift, J A, Histochemistry 19 (1969) 88. Haino, K, Biochim biophys acta 229 (1971) 459. ::: Hodge, A J & Schmitt, F 0, Proc natl acad sci US 46 (1960) 186. 23. Kaye, J S & Kaye, R M, J cell biol31 (1966) 159. 24. MacRae, E K & Meets, G D, J cell biol 45 (1970) 235. Received November 26, 1975 Accepted January 9, 1976
METHENAMINE
STAINING
AND PHOSPHOTUNGSTIC
OF THE ACROSOME
OF MYTZLUS
ACID
EDULZS
SACHIKO END0 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo, and Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo, Japan
SUMMARY The Mytilus acrosome was investigated by histochemical methods combined with electron microscopy, using silver methenamine (SM) and phosphotungstic acid (PTA) staining, as well as some chemical and enzymatic pretreatments followed by the staining. As one of two major components in the Mytilus acrosome, the egg-membrane lysin was conspicuously stainable with PTA and susceptible to pronase digestion. The other component, that occupies the space between the acrosomal membrane and the axially located strand containing lysin, was stained with SM very specifically. This staining property was not affected by pronase digestion or treatment that blocked aldehyde and SH groups.
Several electron micrographical studies on the acrosome of Mytifus edulis have investigated in considerable detail its fine structure [l, 2, 31, its mode of formation [4] and the changes that occur as it reacts at the egg surface to release an “egg-membrane lysin” and form an acrosomal process [5]. The results of these studies have shown that the Mytifus acrosome contains two major components located in a characteristic and constant pattern. An earlier investigation by Wada et al. [6] indicated that the egg-membrane lysin is released during the acrosomal reaction and it has been identified [5] as the substance which, in the intact acrosome, forms an annular deposit in the “basal ring” [l] and an axially located strand in its distal part. This material is solubilized within a fraction of a second after the apex of the acrosomal vesicle opens at the beginning of the reaction.
The second major component lies immediately inside the outer acrosomal membrane in a thick layer extending from the apex to the basal deposit of lysin. This material is observed to swell sequentially [5], from the the tip toward the base, as sea water enters the acrosomal vesicle through the apical opening. This swelling has been postulated as the agent that causes the reversion of the outer acrosomal and plasma membranes, exposing the inner acrosomal membrane which elongates as the membrane of the acrosomal process [5]. The acrosome of Hydroides hexagonus [7] also includes a layer of material that lines the outer acrosomal membrane and appears to undergo similar hydration and swelling as the acrosome opens [8]. In view of the fact that this second component occupies at least half of the space within the Mytilus acrosomal vesicle and appears to play an important part in the
72
S. Endo microtome, expanded with chloroform vapour, and treated by floating them on the surface of each one or some combination of the various solutions. In the case of serial treatments, the sections were transferred with a platinum loop from one solution to deionized water and then to the next solution.
Silver methenamine staining (SM staining) The sections were floated on a solution of 0.1% silver nitrate containing 0.025 M borate buffer (pH 9.2) and 1% hexamethylenetetramine for 30 min at 60°C. The stained sections were washed with I % thiosulfate solution for about 30 min at room temperature [ 121.
Phosphotungstic acid staining (PTA staining) The sections were exposed to a solution of 1% phosphotungstic acid and 10% chromic acid (pH 0.3) [ 131 for 2 or 4 h at room temperature.
Deionized water treatment (DW treatment) I. Diagrammatic longitudinal section of Myrilus acrosome. AM, acrosomal membrane: CP, central proximal part; D, distal part; La, lysin in axially located strand; Lh, lysin in the basal ring; LM, lumen formed by invagination of acrosomal membrane; N, nucleus; PM, plasma membrane; PP, peripheral proximal part; R. rod.
The sections were floated on deionized water for more than 25 h at 60°C.
Fig.
Blockage of aldehyde and SH groups In order to sequester aldehyde groups, the sections were floated on a 2 % sodium bisulfite solution for 2 h at 60°C [l2]. As for sulfhydryl groups, the sections were floated on 0.1 M iodoacetic acid solution adjusted to pH 8 with ammonium hydroxide for 2 h at 60°C [12], or on 0.1 M N-ethylmaleimide solution containing 10 mM Tris buffer (pH 8) for l-l.5 h at 60°C.
acrosome reaction, its chemical properties are of some interest, particularly since a significant amount of calcium-dependent Pronase digestion ATPase activity has been found in associa- The sections were exposed to a solution of 0.01% tion with it [9]. In this study, the acrosome pronase and 0.025 M borate buffer (pH 9.2) for 15-30 min or 2 h at 37°C. of Mytilus edulis has been investigated by histochemical methods combined with Fig. 2. SM staining of late spermatids in Myrilus testis. electron microscopy, using silver methen- Immature spermatid (short arrow) has a large, irregular nucleus. Mature spermatozoon (long arrow) has a amine and PTA staining, as well as some small spheroidal one. High electron density is conchemical and enzymatic pretreatments fol- spicuous in the acrosome of immature spermatids and the nucleus of mature spermatozoa. A, acrosome; lowed by staining. M, mitochondrion; N, nucleus. X 8 500. Fig. 3. SM staining after DW treatment of spermatids
MATERIALS
AND METHODS
Small pieces of Myths testis were fixed with 2.5% glutaraldehyde in sea water, and dehydrated with acetone for SM staining or ethyleneglycol [IO] for PTA staining, followed by embedding in a Rigolac mixture [I I]. Sections were cut on a Porter-Blum MT-l ultraE.rp Cc/I Rr.5 100 (1976)
as in fig. 2. The stainability of the nucleus is markedly reduced. A, acrosome; F, flagellum; M, mitochondrion; N, nucleus. x8500. Fig. 4. Longitudinal section of an immature spermatid stained with SM after treatment with DW. High electron density is limited to D and CP. Cross section at D region is seen at lower right (arrow). CP, central proximal part; D, distal part; N, nucleus. ~40000.
Cytochemical staining of acrosome
73
74
S. Endo
Fig. 5. SM staining after pronase digestion for 2 h without DW treatment. Pronase digestion does not change the SM staining of the acrosomes. The egg-
membrane lysin in mature acrosome is digested (long arrow), but that of immature acrosome is not (short arrow). x 17 500.
After these treatments the sections were finally mounted directly on grids coated with mesh-cement [l4] and o b served with a Hitachi HU-IIB electron microscope.
SM staining Fig. 2 shows part of a testis containing spermatids in late stages of spermiogenesis. The immature spermatids have large, irregularly shaped nuclei (fig. 2, short arrow), while the nuclei of mature spermatozoa are smaller and more spherical (fig. 2, long arrow). Areas of high electron density caused by the deposition of silver particles are
RESULTS Fig. 1 is a diagram of a longitudinal section of the acrosome of Myrifus edufis. The acrosome consists of a large, conical, acrosomal vesicle. The posterior part of the vesicle is invaginated to form a lumen, in which a slender axial rod is contained. The egg-membrane lysin fills the basal ring (fig. 1, Lb) and the axial part distal to the invagination (fig. 1, La). On the basis of differences in their staining properties, the regions consisting mainly of the other vesicular component are designated as peripheral proximal (PP), central proximal (CP), and distal (D) parts, as shown in fig. 1.
Fig. 6. (a) PTA staining for 4 h. Longitudinal section through apex of Myths acrosome. The egg-membrane lysin (La, Lb) and PP are stained. A material lying just inside the acrosome at its apex is distinguished from La (arrow). La, lysin in axially located strand; Lb, lysin in the basal ring; PP, peripheral proximal part. x68000; (b) PTA staining for 2 h. Longitudinal section through apex of Myrilus acrosome. Arrow indicates layer of substance filling space between plasma and acrosomal membranes; layer is much thinner at apex (*---*). x128000. Fig. 7. PTA staining after pronase digestion for 30 min. Lb and part of La are digested; the staining properties in other parts are unchanged. x25000.
fochemical staining of acrosome
75
76
S. Endo
conspicuous on the acrosomes of the immature spermatids and nuclei of the mature spermatozoa; the acrosomes of the latter showed only faint stain. SM staining after DW treatment DW treatment prior to SM staining (fig. 3) markedly reduced the stainability of the nucleus, and the silver particles scattered on the background were all eliminated. However, the degree and pattern of staining in the acrosome did not change. With higher resolution, it was found that the staining was highly specific for the CP and D parts of the acrosome, and the regions containing lysin (La and Lb) were entirely unstained (fig. 4). SM staining after pronase digestion Pronase treatment for a period of 2 h did not influence the SM staining of the acrosome. In the mature acrosomes, however, pronase digested the lysin and the material contained in the PP part (fig. 5, long arrow). SM staining after blocking aldehyde and SH groups Treatment of the sections with sodium bisulfite, iodoacetic acid or N-ethylmaleimide did not significantly affect the SM staining of the acrosomes. PTA staining The testis used for this staining contained mostly mature spermatozoa. PTA was found to stain La, Lb and PP parts strongly; the electron density of Lb was especially prominent (fig. 6a). A small amount of material lying just inside the acrosome at its apex was distinguished from La by its greater affinity for PTA. It formed a small mass (fig. 6a, arrow) or layer (fig. 6b). E.rp Cdl Rr.\ lo0 119761
A substance that fills the space between the plasma and acrosomal membranes became visible after this staining (fig. 6b, arrow). Since the material was fixed with glutaraldehyde alone, the membranes are not preserved, and this substance looks as if it is situated on the most outer surface of the acrosome. It forms a uniform layer 10 nm thick over all of the acrosome except for a small region at the apex where it is much thinner and is sometimes broken (fig. 66, *---*). The nucleus, mitochondria and the microtubules of the flagellum were also stained by PTA. PTA staining after pronase treatment Pronase treatment for a period of 30 min digested the lysin in Lb and a part of La, without affecting the PTA staining of the other parts of the acrosome (fig. 7). After 2 h treatment, all of the egg-membrane lysin and the material contained in PP was eliminated. The results obtained with pronase digestion and PTA staining suggest that PP consists at least in part of egg-membrane lysin. The material at the apex of the acrosome (fig. 6a, arrow) and the substance between the plasma and acrosomal membranes were found not to be digested by pronase.
DISCUSSION Many investigators have reported that silver-methenamine staining of sections identifies aldehyde groups [12, 15-191 and sulfhydryl groups [12, 15, 19, 203. It has been suggested that when aldehyde fixatives are used, some of the fixative remaining in the tissue might affect the SM staining. It seems probable that this is not so in
Cytochemical staining of acrosome the present case, because SM staining of the acrosome after fixing with osmium tetroxide alone gives the same result. The same is also true when staining is carried out prior to glutaraldehyde fixation (Endo, unpublished). To check the possibility that aldehyde or sulfhydryl, or both groups are primarily responsible for the observed SM staining, treatments for sequestering these groups were applied, but they failed to affect the staining under the experimental conditions used. At present, no definite conclusion can be drawn about the mechanism of SM staining in this material, although the possibility remains that other reducing groups might be responsible for the reaction. With respect to the mechanism and specificity of PTA staining, Rambourg et al [ 131 reported that a substance in rat cells which reacts with PTA should be glycoprotein. They assume that saccharide moieties are stained with PTA. However, under the same experimental conditions except for a staining time of 15 min instead of 2 min, the lysin is strongly stained (Endo, unpublished). Its marked susceptibility to pronase digestion (fig. 7) indicates that it consists mainly of protein. This is in line with the finding of Haino [21], that the eggmembrane lysin carried in the acrosome of another mollusc consists of a simple basic protein. In this connection, PTA in an aqueous solution at pH 4.5 stains collagen fibers, the reactive sites of which are known to be basic amino acids [22]. When collagen fibers were stained at pH 0.3, they showed a pattern quite similar to that obtained at pH 4.5 (Nishigai & Endo, unpublished). This result suggests that basic amino acids may well be responsible for the pattern of PTA staining observed in the Mytilus acrosome. This study has found that major con-
77
stituents of the Mytilus acrosome, occupying the zones D and CP which include about half of the material within the acrosomal vesicle, shows a unique affinity for the SM stain. Although it has not proved possible to determine the chemical nature of this substance, its resistance to digestion by pronase indicates that it is probably not a simple protein. The component D represents the material that has been proposed as the agent for turning back the conjoined plasma and outer acrosomal membranes during the acrosome reaction [5]. To accomplish this in a manner consonant with the successive morphological changes observed in thin sections, the D material should first swell, thereby exerting an outward pressure against the membrane, and then immediately dissolve or disperse in the sea water. On the other hand, the material in CP with the same affinity for SM persists at the base of the acrosomal process, as though to lend physical support to it. The reaction of the Mytilus edulis acrosome is known to begin with a structural change at its apex [5]. Dan et al. [2] observed that the plasma and acrosomal membranes are in especially close contact with each other only in this region. In the present study, moreover, it was found that the layer of substance filling the space between the plasma and acrosomal membranes is much thinner at the apex than in other places. There is a small amount of material lying just inside the acrosome at its apex that is particularly stained by PTA. Recently Dan et al. [3] found an electron-lucent component at the same locus, suggesting that this material is rather labile and may function in the initiation of the acrosomal reaction. Three points should be mentioned in relation to the stage-dependency of differences in the chemical susceptibility to staining E.rp Cc,// Hr.\
100 (1976)
78
S. Endo
and enzyme action of cellular components during late spermiogenesis. (1) Immature acrosomes are stained very heavily by SM, but fully matured acrosomes, only faintly. (2) In later stages of spermiogenesis, the nuclei are more strongly stained. It is generally known in many animals that the spermatid nucleus becomes more basic as spermiogenesis proceeds. Kaye & Kaye [23] found that the amount of acidic protein in the spermatid nucleus of house cricket decreases during spermiogenesis leaving basic protein content constant. According to MacRae & Meets [24], the nucleus of chick erythrocytes in the early maturation stage exhibits little or no ammoniacal silver reaction, but becomes reactive as maturation proceeds, as the result of an alteration of histone species from lysine-rich to argininerich. (3) Pronase treatment for 2 h digested the egg-membrane lysin only when the acrosome was fully matured. These three phenomena suggest that the chemical as well as the ultrastructural properties of each component in the spermatid are modified in the course of spermiogenesis. The author wishes to express her thanks to Dr J. C. Dan for her encouragement throughout the work and assistance with the manuscript. She is also indebted to Dr H. Sakai and Dr M. Nishigai for their interest and valuable discussions.
E.rp Cell
Rr$
100 (1976)
REFERENCES 1. Niijima, L & Dan, J C, J cell biol 25 (1965) 243. 2. Dan, J C, Kakizawa, Y, Kushida, H & Fujita, K, Exp cell res 72 (1972) 60. 3. Dan, J C, Hashimoto, S, Kubo, M & Yonehara, K, The functional anatomy of the spermatozoon (ed B A Afzelius) p. 39. Pergamon Press, Oxford and New York (1974). 4. Suzuki, F & Endo, S. Unpublished. 5. Niiiima, L & Dan, J C. J cell biol 25 (1965) 249. 6. Wada, S K, Collier, J R & Dan, J C. Exp cell res IO (1956) 168. 7. Colwin, A L & Colwin, L H, J biophys biochem cytol 10(1961)211. 8. Colwin, L H & Colwin, A L, J biophys biochem cytol IO (1961) 231. 9. Mabuchi. Y & Mabuchi. I. EXD cell res 82 (1973) 271. IO. Leduc, E H & Bernhard, W, J ultrastruct res 19 (1967) 196. Il. Kushida, H, J electron microsc 9 (1961) 113. 12. Pickett-Heaps, J D, J histochem cytochem I5 (1967) 442. 13. Rambourg, A, Hernandez. W & Leblond, C P, J cell biol 40 (1969) 395. 14. Kushida, H & Fujita, K, J electron microsc I3 (1964) 27. IS. Pickett-Heaps, J D, J cell sci 3 (1968) 55. 16. Swift, J A & Saxton, C A, J ultrastruct res 17 (1967) 23. 17. Rambourg, A & Leblond, C P, J cell biol 32 (l%7) 27. 18. Rambourg, A, J histochem cytochem I5 (1967) 409. 19. Burr, F A, J histochem cytochem 21 (1973) 386. 20. Swift, J A, Histochemistry 19 (1969) 88. Haino, K, Biochim biophys acta 229 (1971) 459. ::: Hodge, A J & Schmitt, F 0, Proc natl acad sci US 46 (1960) 186. 23. Kaye, J S & Kaye, R M, J cell biol31 (1966) 159. 24. MacRae, E K & Meets, G D, J cell biol 45 (1970) 235. Received November 26, 1975 Accepted January 9, 1976
