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Electron microscopic evidence for the presence of an acrosomal reaction in Ascaris lumbricoides var. Suum
Acrosomd
reaction
in A. lumbricoides
vnr. suum
643
RNA which was trapped in clumped metaphase figures. The nuclei showed a very heterogeneous labeling pattern. In any region of any of the stages there were nuclei which showed all variations between light and heavy labeling. Incubation of the slides of Rana with ribonuclease (0.02 per cent RNase in 0.2 n/l acetate buffer, pH 5.5 for 2 hr) before coating with the liquid emulsion virtually abolished the subsequent appearance of silver grains in nuclei. This indicates that the 3H-5-uridine was not incorporated into DNA. In conclusion, results of our autoradiographic study show that labeled RNA is not retained by metaphase chromosomes, but the data do not indicate at what time before metaphase the recently synthesized RNA is lost from the nuclei. REFERENCES 1. MAIO, J. Jr,, and SCHILDRANT, C., L., J. Mol. Biol. 24, 29 (1967). 2. NW, M C. and TWITTY, V. C., Proc. Nafl Acad. Sci. Wash. 34, 985 (1953). 3. PRESCOTT, D. M. and BENDER, M. A., Expff Cell Res. 26, 260 (1962). 4. RUGH, R., Experimental Embryology, p. 90. Burgess Publishing Co., Minneapolis, 1962. 5. SALZMAK, N. P., MOORE, D. E. and R~ESDELSOHN, J., Proc. I\‘afl Acad. Sci. r.S. 56, 1449 (1966). 6. SHUMWAY, W., Anat. Rec. 78, 139 (1940).
ELECTRON THE PRESENCE ASCARIS
MICROSCOPIC
EVIDENCE
OF AN ACROSOMAL LUMBRICOIDES
W. H. CLARK, JR., R. L. MORETTI
FOR
REACTION
IN
VAR. SUUM. and W. W. THOMSON
Department of Life Sciences and the Air Pollution Research Center, University of California, Riverside, Calif., U.S.A.
Received February 14, 1967l
A LTHOUGH an acrosome has been found in most sperm, its presence in the nonflagellated sperm of nematodes has been a controversial subject for many years. According to light microscope studies [5-81, the mature nematode sperm is slightly elongated with one end narrower than the other. There is a centrally located nucleus surrounded by mitochondria. Occupying most of the narrow end is a refractile, gram positive cone which is composed of a protein, ascaridine [3]. Bowen [l] suggested that the refringent cone might correspond to the acrosome. Sturdivant [8], using Ascaris megalocephala, also considered the refringent cone to be acrosomal in nature. Recently Favard [4], in an electron microscope study of A. megalocephala, showed that the refringent cone did not originate from the Golgi complex as is true of acrosome formation described to date. It has also been shown that the refringent cone does not exhibit a positive histochemical test for mucopolysaccharides which is a generally accepted criterion for acrosomal identification [2]. 1 Revised
version
received
April
24, 196i.
Experimental
Cell Research 47
644
W. H. Clark, Jr., R. L. Moretti
and W. W. Thomson
Favard [4] described a layer of proacrosomal vesicles between the refringent cone and the plasma membrane of the mature sperm. These vesicles arose from the Golgi bodies as does the acrosome, but no particular function was ascribed to them. Our work indicates that these proacrosomal bodies may function in a manner analogous to the typical acrosome. Materials and Methods.-Live adult dscaris lumbricoides var. suum were collected from intestines of freshly killed pigs. Male and female reproductive systems were removed from the worms and immediately placed in 2.5 per cent gluteraldehyde fixative buffered with 0.1 M phosphate at pH 7. While in the fixative, pieces were removed from the distal portions of the oviduct and spermatic duct. These sections remained in the fixative for 1 hr and were then washed with buffer for 1 min and post fixed with I per cent phosphate buffered osmium for 30 min. The pieces were dehydrated in acetone and embedded in Maraglas. Thin sections were cut on a PorterBlum MT-2 ultramicrotome, picked up on 400 mesh uncoated grids, stained on the grids for 1 hr with uranyl acetate followed by 30 min with lead citrate, and studied with a Hitachi Hu 11 electron microscope. Observations and Discussion.-Fig. 1 shows a spermatid from the spermatic duct of a male A. Zumbricoides var. suum. A small, extremely electron-dense nucleus is present in the center of the cell (Figs 1 and 2). A trilaminar nuclear membrane is not apparent; however, the nucleus is bordered by an irregular electron-dense layer which often contains spheres of varying density (Figs 1 and 2). Several large, relatively electron-translucent bodies are present in the cytoplasm (Fig. 1). Many of these bodies have dense bands across them which are probably artifacts caused by localized folding in the section. Large spherical mitochondria (Fig. 1) containing few cristae are distributed throughout the cytoplasm except in regions containing fibrillar networks (Fig. 1). These fibrillar regions are thought to represent the pseudopodia of the late spermatid. Also present in the cytoplasm are membrane-bound vesicles which contain whorls of fibrillar strands (Figs 1 and 2). The tripartite bounding membrane is asymmetric with the thicker, dense layer bordering the cytoplasm (Figs 3 and 4). X’umerous finger-like invaginations of the membrane protrude into the matrix of the vesicles. The vesicles, which are generally aligned around the periphery of the spermatid, are apparently analogous to the proacrosomal bodies described by Favard [4]. Fig. 5 shows a mature sperm from a section of the distal portion of the oviduct approximately 3 mm anterior to the beginning of the uterus. It is approximately Fig. l.--An electron micrograph showing a spermatid from the spermatic duct of a male A. Zumbricoides var. suum. A small dense nucleus (A’) is present in the center of the cell. Several refringent bodies (R), and numerous mitochondria (M) are present throughout the cytoplasm except in fibrillar regions which are thought to represent pseudopodia (Es). Proacrosomal bodies (P) are generally situated along the periphery of the cell. x 6700. Fig. 2.-A high magnification micrograph illustrating the irregular electron-dense ing the nucleus which often contains spheres of varying density. x 24,200. Fig. 3.-A high magnification micrograph and their fibrillar matrix. x 43,100. Fig. 4.-A high magnification micrograph of a proacrosomal body. x 135,500. Experimental
Cell Research 47
illustrating
the proacrosomal
showing the asymmetric
layer surround-
bodies of the spermatid
tripartite
bounding
membrane
Acrosomal reaction
in A. lumbricoides
var. suum
645
Experimental
Cell Research 47
616
Experimental
W. H. Clark, Jr., R. I,. Moretti
Cell Research 47
and 1%‘.W. Thomson
ilcrosomal
reaction
in A. lumbricoides
var. smm~
645
10.6 ,U long and 5.9 p in width and corresponds in morphology with work provided by light microscopists [5-S]. However, no refringent cone is found in this spermatozoan. In A. megnlocephala Favard [4] noted that the spermatozoa not participating in fertilization undergo degeneration, and that this degeneration first manifests itself by the disappearance of the refringent cone. Several poorly defined areas of varying electron density, situated throughout the cytoplasm, are believed to correspond with degeneration products of the cone (Fig. 5). Several differences are found in the spermatozoa as compared to the spermatids. The nuclei are similar except for the absence of the dense spheres in the layer which surrounds the nucleus (Fig. 5). The mitochondria (Fig. 5) contain few cristae and The vesicles are found along the the matrix now appears more electron-dense. periphery of the conical portion of the sperm and in many instances the bounding membranes of the vesicles are fused with the plasma membrane (Figs 5 and 6). The vesicles fused with the plasma membrane are devoid of fibrillar content. This would suggest that the vesicles have a secretory function and may be analogous in this sense to the acrosome of the flagellate sperm. If these vesicles represent the acrosome it is an unusual acrosome in that it is composed of many individual membrane-bound bodies instead of being a single structure. This work was supported in part by a National Science Foundation Grant, GB 4513, and a grant AP 00356-OlAl from the Division of Air Pollution, Bureau of State Services of Public Health Service. We wish to thank Dr Thomas D. Pitts for his encouragement and advice. REFERENCES
BOWEN, R. H., Anat. Rec. 31, 201 (1925). CLERYONT, Y. and LEBLOSD, C. P., Am. .I. And 96, 229 (1955). FAURO-FROMIET, E., Arch. Anat. Micr. 15, 435 (1913). FAVARD, P., Ann. Sci. Xuf. Zool., Ser. 12, 53 (1961). HIRSCHLER, .J., Arch. Zellf. 9, 351 (1913). 6. SATH, V., GUPTA, B. L. and KOCHHAR, D. &I., Qunrt. J. Micro. Sci. 102, 39 (1961). 7. PASTEELS, J., Arch. Biol. 59, 405 (1948). 8. STURDlV.4ST, H. P., .I. ,~~orl-‘h. 55, 435 (1934).
1. 2. 3. 4. 5.
Fig. 5.-An electron micrograph showing a mature sperm from distal portion of the oviduct. Arrows indicate material believed to correspond with degeneration products of the refringent cone. A centrally located nucleus (N) and numerous mitochondria (M) are found in the cytoplasm. Proacrosomal bodies (I’) are located around the periphery of the conical portion of the sperm. In some instances the bounding membranes of these vesicles are fused with the plasma membrane of the sperm. x 16,500. Fig. 6.-A high magnification micrograph illustrating the fusion of the proacrosomal bounding membrane with the plasma membrane of the sperm. x 22,300.
Experimenfal
Cell Research 47
reaction
in A. lumbricoides
vnr. suum
643
RNA which was trapped in clumped metaphase figures. The nuclei showed a very heterogeneous labeling pattern. In any region of any of the stages there were nuclei which showed all variations between light and heavy labeling. Incubation of the slides of Rana with ribonuclease (0.02 per cent RNase in 0.2 n/l acetate buffer, pH 5.5 for 2 hr) before coating with the liquid emulsion virtually abolished the subsequent appearance of silver grains in nuclei. This indicates that the 3H-5-uridine was not incorporated into DNA. In conclusion, results of our autoradiographic study show that labeled RNA is not retained by metaphase chromosomes, but the data do not indicate at what time before metaphase the recently synthesized RNA is lost from the nuclei. REFERENCES 1. MAIO, J. Jr,, and SCHILDRANT, C., L., J. Mol. Biol. 24, 29 (1967). 2. NW, M C. and TWITTY, V. C., Proc. Nafl Acad. Sci. Wash. 34, 985 (1953). 3. PRESCOTT, D. M. and BENDER, M. A., Expff Cell Res. 26, 260 (1962). 4. RUGH, R., Experimental Embryology, p. 90. Burgess Publishing Co., Minneapolis, 1962. 5. SALZMAK, N. P., MOORE, D. E. and R~ESDELSOHN, J., Proc. I\‘afl Acad. Sci. r.S. 56, 1449 (1966). 6. SHUMWAY, W., Anat. Rec. 78, 139 (1940).
ELECTRON THE PRESENCE ASCARIS
MICROSCOPIC
EVIDENCE
OF AN ACROSOMAL LUMBRICOIDES
W. H. CLARK, JR., R. L. MORETTI
FOR
REACTION
IN
VAR. SUUM. and W. W. THOMSON
Department of Life Sciences and the Air Pollution Research Center, University of California, Riverside, Calif., U.S.A.
Received February 14, 1967l
A LTHOUGH an acrosome has been found in most sperm, its presence in the nonflagellated sperm of nematodes has been a controversial subject for many years. According to light microscope studies [5-81, the mature nematode sperm is slightly elongated with one end narrower than the other. There is a centrally located nucleus surrounded by mitochondria. Occupying most of the narrow end is a refractile, gram positive cone which is composed of a protein, ascaridine [3]. Bowen [l] suggested that the refringent cone might correspond to the acrosome. Sturdivant [8], using Ascaris megalocephala, also considered the refringent cone to be acrosomal in nature. Recently Favard [4], in an electron microscope study of A. megalocephala, showed that the refringent cone did not originate from the Golgi complex as is true of acrosome formation described to date. It has also been shown that the refringent cone does not exhibit a positive histochemical test for mucopolysaccharides which is a generally accepted criterion for acrosomal identification [2]. 1 Revised
version
received
April
24, 196i.
Experimental
Cell Research 47
644
W. H. Clark, Jr., R. L. Moretti
and W. W. Thomson
Favard [4] described a layer of proacrosomal vesicles between the refringent cone and the plasma membrane of the mature sperm. These vesicles arose from the Golgi bodies as does the acrosome, but no particular function was ascribed to them. Our work indicates that these proacrosomal bodies may function in a manner analogous to the typical acrosome. Materials and Methods.-Live adult dscaris lumbricoides var. suum were collected from intestines of freshly killed pigs. Male and female reproductive systems were removed from the worms and immediately placed in 2.5 per cent gluteraldehyde fixative buffered with 0.1 M phosphate at pH 7. While in the fixative, pieces were removed from the distal portions of the oviduct and spermatic duct. These sections remained in the fixative for 1 hr and were then washed with buffer for 1 min and post fixed with I per cent phosphate buffered osmium for 30 min. The pieces were dehydrated in acetone and embedded in Maraglas. Thin sections were cut on a PorterBlum MT-2 ultramicrotome, picked up on 400 mesh uncoated grids, stained on the grids for 1 hr with uranyl acetate followed by 30 min with lead citrate, and studied with a Hitachi Hu 11 electron microscope. Observations and Discussion.-Fig. 1 shows a spermatid from the spermatic duct of a male A. Zumbricoides var. suum. A small, extremely electron-dense nucleus is present in the center of the cell (Figs 1 and 2). A trilaminar nuclear membrane is not apparent; however, the nucleus is bordered by an irregular electron-dense layer which often contains spheres of varying density (Figs 1 and 2). Several large, relatively electron-translucent bodies are present in the cytoplasm (Fig. 1). Many of these bodies have dense bands across them which are probably artifacts caused by localized folding in the section. Large spherical mitochondria (Fig. 1) containing few cristae are distributed throughout the cytoplasm except in regions containing fibrillar networks (Fig. 1). These fibrillar regions are thought to represent the pseudopodia of the late spermatid. Also present in the cytoplasm are membrane-bound vesicles which contain whorls of fibrillar strands (Figs 1 and 2). The tripartite bounding membrane is asymmetric with the thicker, dense layer bordering the cytoplasm (Figs 3 and 4). X’umerous finger-like invaginations of the membrane protrude into the matrix of the vesicles. The vesicles, which are generally aligned around the periphery of the spermatid, are apparently analogous to the proacrosomal bodies described by Favard [4]. Fig. 5 shows a mature sperm from a section of the distal portion of the oviduct approximately 3 mm anterior to the beginning of the uterus. It is approximately Fig. l.--An electron micrograph showing a spermatid from the spermatic duct of a male A. Zumbricoides var. suum. A small dense nucleus (A’) is present in the center of the cell. Several refringent bodies (R), and numerous mitochondria (M) are present throughout the cytoplasm except in fibrillar regions which are thought to represent pseudopodia (Es). Proacrosomal bodies (P) are generally situated along the periphery of the cell. x 6700. Fig. 2.-A high magnification micrograph illustrating the irregular electron-dense ing the nucleus which often contains spheres of varying density. x 24,200. Fig. 3.-A high magnification micrograph and their fibrillar matrix. x 43,100. Fig. 4.-A high magnification micrograph of a proacrosomal body. x 135,500. Experimental
Cell Research 47
illustrating
the proacrosomal
showing the asymmetric
layer surround-
bodies of the spermatid
tripartite
bounding
membrane
Acrosomal reaction
in A. lumbricoides
var. suum
645
Experimental
Cell Research 47
616
Experimental
W. H. Clark, Jr., R. I,. Moretti
Cell Research 47
and 1%‘.W. Thomson
ilcrosomal
reaction
in A. lumbricoides
var. smm~
645
10.6 ,U long and 5.9 p in width and corresponds in morphology with work provided by light microscopists [5-S]. However, no refringent cone is found in this spermatozoan. In A. megnlocephala Favard [4] noted that the spermatozoa not participating in fertilization undergo degeneration, and that this degeneration first manifests itself by the disappearance of the refringent cone. Several poorly defined areas of varying electron density, situated throughout the cytoplasm, are believed to correspond with degeneration products of the cone (Fig. 5). Several differences are found in the spermatozoa as compared to the spermatids. The nuclei are similar except for the absence of the dense spheres in the layer which surrounds the nucleus (Fig. 5). The mitochondria (Fig. 5) contain few cristae and The vesicles are found along the the matrix now appears more electron-dense. periphery of the conical portion of the sperm and in many instances the bounding membranes of the vesicles are fused with the plasma membrane (Figs 5 and 6). The vesicles fused with the plasma membrane are devoid of fibrillar content. This would suggest that the vesicles have a secretory function and may be analogous in this sense to the acrosome of the flagellate sperm. If these vesicles represent the acrosome it is an unusual acrosome in that it is composed of many individual membrane-bound bodies instead of being a single structure. This work was supported in part by a National Science Foundation Grant, GB 4513, and a grant AP 00356-OlAl from the Division of Air Pollution, Bureau of State Services of Public Health Service. We wish to thank Dr Thomas D. Pitts for his encouragement and advice. REFERENCES
BOWEN, R. H., Anat. Rec. 31, 201 (1925). CLERYONT, Y. and LEBLOSD, C. P., Am. .I. And 96, 229 (1955). FAURO-FROMIET, E., Arch. Anat. Micr. 15, 435 (1913). FAVARD, P., Ann. Sci. Xuf. Zool., Ser. 12, 53 (1961). HIRSCHLER, .J., Arch. Zellf. 9, 351 (1913). 6. SATH, V., GUPTA, B. L. and KOCHHAR, D. &I., Qunrt. J. Micro. Sci. 102, 39 (1961). 7. PASTEELS, J., Arch. Biol. 59, 405 (1948). 8. STURDlV.4ST, H. P., .I. ,~~orl-‘h. 55, 435 (1934).
1. 2. 3. 4. 5.
Fig. 5.-An electron micrograph showing a mature sperm from distal portion of the oviduct. Arrows indicate material believed to correspond with degeneration products of the refringent cone. A centrally located nucleus (N) and numerous mitochondria (M) are found in the cytoplasm. Proacrosomal bodies (I’) are located around the periphery of the conical portion of the sperm. In some instances the bounding membranes of these vesicles are fused with the plasma membrane of the sperm. x 16,500. Fig. 6.-A high magnification micrograph illustrating the fusion of the proacrosomal bounding membrane with the plasma membrane of the sperm. x 22,300.
Experimenfal
Cell Research 47