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The fine structure of nuclei in mature sperm
© 1971 by Academic Press, Inc.
J. ULTRASTRUCTURERESEARCI~36, 237-248 (1971)
237
The Fine Structure of Nuclei in M a t u r e Sperm I. Application of the Langmuir Trough-Critical Point Method to Histone-Containing Sperm Nuclei z BARRY R. ZIRKIN
Department of Zoology, University of California, Davis, California 95616 Received October 21, 1970, and in revised form December 3, 1970 The fine structure of histone-containing sperm nuclei of the leopard frog, sea urchin, and goldfish, were studied by the Langmuir trough-critical point method. Striking similarities in nuclear fine structure were observed in these sperm. In each case, well separated, randomly distributed fibers of similar appearance and dimensions were the only structures seen. Measurements of nuclear fibers revealed mean diameters of 164 A for the sea urchin, 171 A for the goldfish, and 179 A for the frog. The relationship of these diameters to previously reported fiber diameters in isolated nuclei and chromosomes is discussed. Previous work has established that the basic protein composition of sperm nuclei may differ in different organisms. Histones similar to those in somatic cell nuclei are present in the sperm nuclei of some organisms; in others, histones unusually rich in arginine or protamines are present (6, 7, 11, 2l). The structure of nuclear elements probably is determined in large part by the basic proteins present. Sperm nuclei whose basic proteins differ might thus be expected to show differences in fine structure. Conversely, sperm nuclei whose basic proteins are similar might be expected to show similarities in fine structure. The extent to which basic protein composition and fine structure are related in sperm nuclei, however, is still uncertain. One source of difficulty in examining this problem is that, in most cases, the fine structure of sperm nuclei cannot be studied by conventional electron microscope techniques. In sections, most sperm nuclei appear uniformly electron dense, suggesting that nuclear elements are too closely packed to be resolved (17). One exception has been a previous study of leopard frog sperm, in which closely packed fibers were resolved in very thin sections of sperm nuclei (35). The Langmuir trough method, introduced by Gall (12), does not require fixation z This study was supported by N I H Postdoctoral Fellowships 1 F02 HD31695-01 and 5 F02 HD31695-02 from the National Institute of Child Health and Human Development.
238
ZIRKIN
and sectioning, and has been used extensively in studies of nuclear and chromosomal structure (24, 32). By this technique, cells are burst at an air-water interface on the surface of a Langmuir trough. Material is then picked up on grids from the surface of the trough, dried, and examined under an electron microscope. The fibers seen in somatic nuclei and chromosomes prepared this way are considerably thicker than comparable fibers viewed in sections (17, 32), and usually are separated from each other to a greater extent than in sections. The Langmuir trough technique also has been applied to studies of spermatid (13) and sperm (20, 23, 26) nuclei. These studies have shown that, under some conditions, the sperm nuclei of at least some organisms spread sufficiently for their elements to be resolved by electron microscopy. It seemed possible that further application of this technique would reveal substructure in a variety of sperm nuclei. If this were the case, correlated fine structural and chemical analyses might reveal a relationship between structure and basic protein composition in sperm nuclei, and thus might provide information on the role of various basic proteins in the packing of DNA. In the present work, the fine structure of nuclei in the histone-containing sperm of the frog, Rana pipiens, sea urchin, Strongylocentrotus purpuratus, and goldfish, Carassius auratus, has been studied by the Langmuir trough-critical point method.
MATERIALS AND METHODS
Electron microscopy. The frogs (Rana pipiens) used in this study were obtained from a supplier in Vermont. During the winter months, the testes of frogs from the northeastern United States contain mature sperm, spermatogonia, and Sertoli cells within the seminiferous tubules, and connective cells between the tubules (25, 34). The mature sperm can easily be distinguished from other cells in surface-spread preparations. For studies of frog sperm, the testes of a winter frog were dissected out and macerated in a few drops of distilled water. Small droplets of the suspension were transferred on the end of a glass rod to the distilled water surface of a Langmuir trough. The resultant surface film was compressed and picked up by touching Formvar-carbon coated grids to the surface. Grids were stained for 15 minute3 in 2 % aqueous uranyl acetate, washed in distilled water, loaded into a grid carrier , and dehydrated in ethanol. The grids were then transferred to amyl acetate and dried by the critical point method (4). Shed sperm from the sea urchin, Strongylocentrotus purpuratus, were obtained by 0.5 M KC1 treatment. Sperm from the goldfish, Carassius auratus, were obtained by stripping. Droplets of the sperm suspensions were transferred with a glass rod to the distilled water surface of a Langmuir trough, and subsequently treated as above. All preparations were examined under an Hitachi 11A electron microscope. Light microscopy. Sperm suspensions were fixed in 10% neutral buffered formalin (l hour, room temperature). Fixed sperm were smeared on glass slides and permitted to air dry. Slides were stained for histones according to the Alfert and Geschwind (3) procedure; DNA was removed with hot (87-90°C) trichloroacetic acid prior to staining with alkaline fast green
ULTRASTRUCTUREOF HISTONE-CONTAININGSPERM NUCLEI
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FIG. 1. Sea urchin sperm stained for histones. Nuclei stain intensely, x 2 100. FIG. 2. Sea urchin sperm stained for arginine-rich histones. Considerably less stain is bound by the nuclei after deamination, x 2 100. FIG. 3. Goldfish sperm stained for histones. Nuclei stain intensely, x 2 100. FIG. 4. Goldfish sperm stained for arginine-rich histones. Considerably less stain is bound by the nuclei, x 2 100.
(pH 8.1-8.3). To test for arginine-rich histones, slides were treated with hot TCA, then with nitrous acid (2 changes, 15 minutes each), and stained with alkaline fast green. The nitrous acid was prepared just before use by adding together equal volumes of 10 % TCA and 10 % sodium nitrite (9). Measurement of nuclear fibers. The thickness of fibers in sperm nuclei was measured with a reticle, on prints enlarged to a final magnification of 100 000 times. The scale was divided into tenths of a millimeter and viewed at 7 x magnification. Nuclear elements were measured in the following way: A transparent sheet ruled at 0.5 cm intervals was placed over the print, and the fibers crossed by ruled lines were measured. The print was rotated 90 ° and measurements again were made (31). Calibration of the electron microscope was done at intervals during the course of this study, by means of a diffraction grating replica of 28 800 lines/inch. Variation in magnification was less than 5 %.
240
ZIRKIN
TABLE I MEASUREMENTS OF DIAMETERS OF NUCLEAR FIBERS
Source
Mean Diameter (~)
Standard Deviation (A)
R~ge
Frog sperm Sea urchin sperm Goldfish sperm Bovine kidney chromosomes
179 164 171 264
41.7 38.7 42.5 37.5
90-300 80-280 60-300 200-350
OBSERVATIONS
Cytochemistry The nuclei of sea urchin and goldfish sperm stain intensely with alkaline fast green after hot TCA treatment (Figs. 1 and 3). After deamination with nitrous acid, considerably less alkaline fast green is bound by the nuclei (Figs. 2 and 4). A previous cytochemical study has shown similar staining in sperm nuclei of the leopard frog (34).
Electron microscopy Frog sea urchin, and goldfish sperm spread readily on distilled water in a Langmuir trough (Figs. 5, 7, and 9). Nuclear spreading was achieved without pretreatment of the sperm in thioglycolate or EDTA, used previously to spread bull (20) and sea urchin (26) sperm nuclei, respectively. Most of the sperm nuclei observed on each grid showed substantial degrees of spreading. At high magnification, frog, sea urchin, and goldfish sperm nuclei show striking similarities in fine structure (Figs. 6, 8 and 10). In each case, nuclei are composed entirely of fibers. The fibers appear well separated, randomly distributed, lumpy in outline, and without visible substructure. Measurements of the diameter of the fibers indicate similarity in dimensions as well (Table 1). The fibers in frog, sea urchin, and goldfish sperm nuclei show mean diameters of 179 A, 164 A, and 171 A, respectively. For comparison, the diameter of fibers in surface spread bovine kidney cell chromosomes (Fig. 11) was measured. The methods used for the kidney cells were identical to the methods used for sperm preparations. A mean diameter of 264 A was found (Table 1). This diameter is consistent with previously reported measurements of fibers in these chromosomes (29, 30). The frequency distributions shown in Fig. 12 are approximately normal for each sample of measurements, indicating that the sample of fibers which was measured in each case constitutes a single class. FiG. 5. Frog sperm isolated on distilled water by the Langmuir trough-critical point method. × 7 800.
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Fie. 6. A portion of an isolated frog sperm nucleus. The nucleus is composed entirely of fibers whose average diameter is 179/~. × 100 000.
FIG. 7. Isolated sea urchin sperm. × 26 000. FIG. 8. A portion of an isolated sea urchin sperm nucleus. The nucleus is composed of fibers whose average diameter is 164/~. × 100 000.
ULTRASTRUCTURE OF HISTONE-CONTAININGSPERM NUCLEI
245
FIG. 11. A portion of an isolated bovine kideny chromosome. The fibers average 264/~ in diameter. × 100 000. DISCUSSION Chemical analyses have indicated that the basic proteins in frog (5, 28), sea urchin and carp (18) sperm nuclei resemble those in somatic cell nuclei. These findings are supported by cytochemical analyses of sea urchin and goldfish sperm reported in this study, and of frog sperm reported previously (34). Intense alkaline fast green staining occurs in the sperm nuclei after hot T C A treatment, indicating that protamines are not present (2). Staining intensities are considerably reduced following deamination, indicating that histones with substantially higher arginine contents than those in somatic nuclei are not present (9). Application of the Langmuir trough-critical point method has revealed striking similarities in nuclear fine structure in the sperm nuclei of these organisms. In each
(15),
FIG. 9. Two isolated goldfish sperm, x 13 500. FIG. 10. A portion of an isolated goldfish sperm nucleus. The nucleus is composed of fibers whose average diameter is 171 A. x 100 000.
246
ZIRKIN
SEA URCHIN SPERM
FROG SPERM 40
40
20
>-
,ZL I00
200
20
I O0
500
200
500
hi LU LI-
BOVINE KIDNEY CHROMOSOMES
GOLDFISH SPERM 40
40
20
2O
I
P I00
200
300
r
r I00
200
500
FIBER DIAMETER (A) FIG. 12. Frequency distributions of fiber diameters in surface-spread frog, sea urchin, and goldfish sperm nuclei, and bovine kidney chromosomes. case, randomly distributed fibers of similar appearance and dimensions were the only structures seen. Measurements of sperm nuclear fibers revealed mean diameters of 164 A for the sea urchin, 171 A for the goldfish, and 179 A for the frog. These small differences in diameter are not regarded as meaningful in view of the errors introduced in the measurements. Microscope magnification changes (up to 5 %), contamination of fibers in the electron beam, and stretching of fibers, might account for the 15 A difference. The fiber diameters obtained in this study probably are somewhat lower than the actual diameters of the isolated fibers, since stretched fibers were included in the measurements. The occurrence of fibers in the surface-spread preparations is consistent with previous electron microscope studies, in which fibers have been demonstrated in sectioned nuclei of frog sperm (35) and sea urchin spermatids (19), and in surface-spread nuclei of sea urchin sperm (26). Highly oriented fibers, lamellae, and sheets occur in middle and late spermatid nuclei of some organisms (17). These unusual structures are strikingly different from the fibrous elements which occur in early spermatids, interphase chromatin, and
ULTRASTRUCTURE OF HISTONE-CONTAININGSPERM NUCLEI
247
chromosomes (17). Unusual structures have not been seen in the sperm nuclei of the frog, sea urchin, and goldfish. Sheets have been demonstrated both in surface spread (13) and sectioned (14) nuclei of grasshopper spermatids, indicating that if sheets were present in the sperm nuclei under study, they could have been demonstrated by the techniques used. It has been suggested that the occurrence of unusual structures might be correlated with the replacement of histones by arginine-rich histones and protamines (9, 16, 22). The sperm nuclei of the frog, sea urchin, and goldfish contain somatic-type histones. The absence of unusual structures in these nuclei might thus reflect the absence of unusual basic proteins. The diameter of fibers in surface spread interphase nuclei and mitotic and meiotic chromosomes generally has been reported to average about 250 A (1, 10, 13, 24, 27, 29-33). This diameter is consistent with the diameter of fibers in surface spread bovine kidney chromosomes reported in this study (264 A). The fiber diameters measured in surface spread sperm nuclei (164-179 A), however, are substantially lower than the common 250 A diameter. The difference appears to be too great to be attributed solely to errors in the measurements. One possible explanation is that surface spread sperm chromatin, even from sperm nuclei that contain somatic-type histones, is different in at least some respects from surface spread interphase or metaphase chromatin. However, Wolfe (31) has reported that unfixed, surface spread barley leaflet and root tip chromosomes contain fibers that average 187 /k and 170 /~, respectively. These diameters are very similar to the sperm diameters reported in the present study. On the other hand, barley endosperm chromosome fibers were reported by Wolfe (31) to have a mean diameter of 244 A, similar to the commonly reported 250 ,&_diameter, and substantially greater than the diameters found in barley leaflet and root tip chromosomes. The magnitude of the differences in fiber diameters in the various barley chromosomes is similar to the differences in diameters seen in the sperm and kidney nuclear fibers reported in the present study. The explanation for these differences is not yet known. I thank Drs. Stephen L. Wolfe and Ronald J. Baskin for their suggestions and critical reading of the manuscript, and Mr John Mais for his excellent technical assistance. I also thank Dr. Dennis Barrett for supplying sea urchins.
REFERENCES l. 2. 3. 4. 5.
ABUELLO,J. G. and MOORE, D. E., J. Cell Biol. 41, 73 0969). ALFERT,M., J. Biophys. Biochem. Cytol. 2, 109 (1956). ALFERT, M. and GESCHWIND,I. I., Proc. Nat. Acad. Sci. U.S. 39, 992 (1953). ANDERSON,T. F., Trans. N. Y. Acad. Sci. 13, 130 (1951). BLOCH,D. P., J. Histochem. Cytochem. 10, 137 (1962).
248
6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24. 25. 26. 27. 28.
29. 30. 31. 32. 33. 34. 35.
ZIRKIN
-Protoplasmatologia 5: 3d, 1 0966). -Genetics 61: 1, Suppl., 93 (1969). BLOCH, D. P. and BRACK, S. D., J. Cell Biol. 22, 327 (1964). BLOCH, D. P. and HEW, H. Y. C., J. Biophys. Biochem. Cytol. "7, 515 (1960). DuPRAw, E. J., Proe. Nat. Acad. Sci. U.S. 53, 161 (1965). FELIX, K., FISCHER, H., and KREKELS,A., Progr. Biophys. Biophys. Chem. 6, 2 (1956). GALL,J. G., Science 139, 120 (1963). -Chromosoma 20, 221 (1966). GIBBONS,I. R., and BRADFIELD,J. R. G., J. Biophys. Biochem. Cytol. 3, 133 (1957). HAMER, D., Biol. Bull. 108, 35 (1955). KAYE, J. S., in LEVINE,L. (Ed.), The Cell in Mitosis, p. 221. Academic Press, New York, 1963. KAYE, J. S., in L1MA-DE-FARIA,A. (Ed.), Handbook of Molecular Cytology, p. 361. North-Holland Publ. Co., Amsterdam, 1969. KOSSEL,A., in The Protamines and Histones. Longmans, Green, New York, 1928. LONGO, F. J., and ANDERSON,E., J. Ultrastruct. Res. 27, 486 (1969). LUNG, B., J. Ultrastruct. Res. 22, 485 (1968). PHILLIPS,D. M. P., Progr. Biophys. Biophys. Chem. 12, 213 (1962). RIs, H., Can. J. Genet. Cytol. 3, 95 (1961). -Proc. Roy. Soc. Set. B 164, 279 (1966). -in LIMA-DE-FARIA,A. (Ed.), Handbook of Molecular Cytology, p. 221. NorthHolland Publ. Co., Amsterdam, 1969. RUGH, R., J. Exp. Zool. 80, 81 (1939). SOLARI,A. J., Exp. Cell Res. 53, 553 (1968). TOKUYASU,K., MADDEN, S. C., and ZELDIS, L. J., Jr. Cell Biol. 39, 630 (1968). VENDRELY,R., Arch. Klaus-Stift. Vererb.-Forsch. 32, 538 (1957). WOLFE,S. L., J. Ultrastruct. Res. 12, 104 (1965). -Exp. Cell Res. 37, 45 (1965). -J. Cell Biol. 37, 610 (1968). -in BITTAR, E. E. (Ed.), The Biological Basis of Medicine, p. 3. Academic Press, New York, 1969. WOLFE, S. L., and GRIM, J. N., J. Ultrastruct. Res. 19, 382 (1967). ZmK~N, B. R., Chromosoma 31, 231 (1970). -J. Ultrastruct. Res. 34, 159 (1971).
J. ULTRASTRUCTURERESEARCI~36, 237-248 (1971)
237
The Fine Structure of Nuclei in M a t u r e Sperm I. Application of the Langmuir Trough-Critical Point Method to Histone-Containing Sperm Nuclei z BARRY R. ZIRKIN
Department of Zoology, University of California, Davis, California 95616 Received October 21, 1970, and in revised form December 3, 1970 The fine structure of histone-containing sperm nuclei of the leopard frog, sea urchin, and goldfish, were studied by the Langmuir trough-critical point method. Striking similarities in nuclear fine structure were observed in these sperm. In each case, well separated, randomly distributed fibers of similar appearance and dimensions were the only structures seen. Measurements of nuclear fibers revealed mean diameters of 164 A for the sea urchin, 171 A for the goldfish, and 179 A for the frog. The relationship of these diameters to previously reported fiber diameters in isolated nuclei and chromosomes is discussed. Previous work has established that the basic protein composition of sperm nuclei may differ in different organisms. Histones similar to those in somatic cell nuclei are present in the sperm nuclei of some organisms; in others, histones unusually rich in arginine or protamines are present (6, 7, 11, 2l). The structure of nuclear elements probably is determined in large part by the basic proteins present. Sperm nuclei whose basic proteins differ might thus be expected to show differences in fine structure. Conversely, sperm nuclei whose basic proteins are similar might be expected to show similarities in fine structure. The extent to which basic protein composition and fine structure are related in sperm nuclei, however, is still uncertain. One source of difficulty in examining this problem is that, in most cases, the fine structure of sperm nuclei cannot be studied by conventional electron microscope techniques. In sections, most sperm nuclei appear uniformly electron dense, suggesting that nuclear elements are too closely packed to be resolved (17). One exception has been a previous study of leopard frog sperm, in which closely packed fibers were resolved in very thin sections of sperm nuclei (35). The Langmuir trough method, introduced by Gall (12), does not require fixation z This study was supported by N I H Postdoctoral Fellowships 1 F02 HD31695-01 and 5 F02 HD31695-02 from the National Institute of Child Health and Human Development.
238
ZIRKIN
and sectioning, and has been used extensively in studies of nuclear and chromosomal structure (24, 32). By this technique, cells are burst at an air-water interface on the surface of a Langmuir trough. Material is then picked up on grids from the surface of the trough, dried, and examined under an electron microscope. The fibers seen in somatic nuclei and chromosomes prepared this way are considerably thicker than comparable fibers viewed in sections (17, 32), and usually are separated from each other to a greater extent than in sections. The Langmuir trough technique also has been applied to studies of spermatid (13) and sperm (20, 23, 26) nuclei. These studies have shown that, under some conditions, the sperm nuclei of at least some organisms spread sufficiently for their elements to be resolved by electron microscopy. It seemed possible that further application of this technique would reveal substructure in a variety of sperm nuclei. If this were the case, correlated fine structural and chemical analyses might reveal a relationship between structure and basic protein composition in sperm nuclei, and thus might provide information on the role of various basic proteins in the packing of DNA. In the present work, the fine structure of nuclei in the histone-containing sperm of the frog, Rana pipiens, sea urchin, Strongylocentrotus purpuratus, and goldfish, Carassius auratus, has been studied by the Langmuir trough-critical point method.
MATERIALS AND METHODS
Electron microscopy. The frogs (Rana pipiens) used in this study were obtained from a supplier in Vermont. During the winter months, the testes of frogs from the northeastern United States contain mature sperm, spermatogonia, and Sertoli cells within the seminiferous tubules, and connective cells between the tubules (25, 34). The mature sperm can easily be distinguished from other cells in surface-spread preparations. For studies of frog sperm, the testes of a winter frog were dissected out and macerated in a few drops of distilled water. Small droplets of the suspension were transferred on the end of a glass rod to the distilled water surface of a Langmuir trough. The resultant surface film was compressed and picked up by touching Formvar-carbon coated grids to the surface. Grids were stained for 15 minute3 in 2 % aqueous uranyl acetate, washed in distilled water, loaded into a grid carrier , and dehydrated in ethanol. The grids were then transferred to amyl acetate and dried by the critical point method (4). Shed sperm from the sea urchin, Strongylocentrotus purpuratus, were obtained by 0.5 M KC1 treatment. Sperm from the goldfish, Carassius auratus, were obtained by stripping. Droplets of the sperm suspensions were transferred with a glass rod to the distilled water surface of a Langmuir trough, and subsequently treated as above. All preparations were examined under an Hitachi 11A electron microscope. Light microscopy. Sperm suspensions were fixed in 10% neutral buffered formalin (l hour, room temperature). Fixed sperm were smeared on glass slides and permitted to air dry. Slides were stained for histones according to the Alfert and Geschwind (3) procedure; DNA was removed with hot (87-90°C) trichloroacetic acid prior to staining with alkaline fast green
ULTRASTRUCTUREOF HISTONE-CONTAININGSPERM NUCLEI
239
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FIG. 1. Sea urchin sperm stained for histones. Nuclei stain intensely, x 2 100. FIG. 2. Sea urchin sperm stained for arginine-rich histones. Considerably less stain is bound by the nuclei after deamination, x 2 100. FIG. 3. Goldfish sperm stained for histones. Nuclei stain intensely, x 2 100. FIG. 4. Goldfish sperm stained for arginine-rich histones. Considerably less stain is bound by the nuclei, x 2 100.
(pH 8.1-8.3). To test for arginine-rich histones, slides were treated with hot TCA, then with nitrous acid (2 changes, 15 minutes each), and stained with alkaline fast green. The nitrous acid was prepared just before use by adding together equal volumes of 10 % TCA and 10 % sodium nitrite (9). Measurement of nuclear fibers. The thickness of fibers in sperm nuclei was measured with a reticle, on prints enlarged to a final magnification of 100 000 times. The scale was divided into tenths of a millimeter and viewed at 7 x magnification. Nuclear elements were measured in the following way: A transparent sheet ruled at 0.5 cm intervals was placed over the print, and the fibers crossed by ruled lines were measured. The print was rotated 90 ° and measurements again were made (31). Calibration of the electron microscope was done at intervals during the course of this study, by means of a diffraction grating replica of 28 800 lines/inch. Variation in magnification was less than 5 %.
240
ZIRKIN
TABLE I MEASUREMENTS OF DIAMETERS OF NUCLEAR FIBERS
Source
Mean Diameter (~)
Standard Deviation (A)
R~ge
Frog sperm Sea urchin sperm Goldfish sperm Bovine kidney chromosomes
179 164 171 264
41.7 38.7 42.5 37.5
90-300 80-280 60-300 200-350
OBSERVATIONS
Cytochemistry The nuclei of sea urchin and goldfish sperm stain intensely with alkaline fast green after hot TCA treatment (Figs. 1 and 3). After deamination with nitrous acid, considerably less alkaline fast green is bound by the nuclei (Figs. 2 and 4). A previous cytochemical study has shown similar staining in sperm nuclei of the leopard frog (34).
Electron microscopy Frog sea urchin, and goldfish sperm spread readily on distilled water in a Langmuir trough (Figs. 5, 7, and 9). Nuclear spreading was achieved without pretreatment of the sperm in thioglycolate or EDTA, used previously to spread bull (20) and sea urchin (26) sperm nuclei, respectively. Most of the sperm nuclei observed on each grid showed substantial degrees of spreading. At high magnification, frog, sea urchin, and goldfish sperm nuclei show striking similarities in fine structure (Figs. 6, 8 and 10). In each case, nuclei are composed entirely of fibers. The fibers appear well separated, randomly distributed, lumpy in outline, and without visible substructure. Measurements of the diameter of the fibers indicate similarity in dimensions as well (Table 1). The fibers in frog, sea urchin, and goldfish sperm nuclei show mean diameters of 179 A, 164 A, and 171 A, respectively. For comparison, the diameter of fibers in surface spread bovine kidney cell chromosomes (Fig. 11) was measured. The methods used for the kidney cells were identical to the methods used for sperm preparations. A mean diameter of 264 A was found (Table 1). This diameter is consistent with previously reported measurements of fibers in these chromosomes (29, 30). The frequency distributions shown in Fig. 12 are approximately normal for each sample of measurements, indicating that the sample of fibers which was measured in each case constitutes a single class. FiG. 5. Frog sperm isolated on distilled water by the Langmuir trough-critical point method. × 7 800.
U L T R A S T R U C T U R E O1~ H I S T O N E - C O N T A I N 1 N G SPERM N U C L E I
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J . Ultrastructure Research
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242
ZIRKIN
Fie. 6. A portion of an isolated frog sperm nucleus. The nucleus is composed entirely of fibers whose average diameter is 179/~. × 100 000.
FIG. 7. Isolated sea urchin sperm. × 26 000. FIG. 8. A portion of an isolated sea urchin sperm nucleus. The nucleus is composed of fibers whose average diameter is 164/~. × 100 000.
ULTRASTRUCTURE OF HISTONE-CONTAININGSPERM NUCLEI
245
FIG. 11. A portion of an isolated bovine kideny chromosome. The fibers average 264/~ in diameter. × 100 000. DISCUSSION Chemical analyses have indicated that the basic proteins in frog (5, 28), sea urchin and carp (18) sperm nuclei resemble those in somatic cell nuclei. These findings are supported by cytochemical analyses of sea urchin and goldfish sperm reported in this study, and of frog sperm reported previously (34). Intense alkaline fast green staining occurs in the sperm nuclei after hot T C A treatment, indicating that protamines are not present (2). Staining intensities are considerably reduced following deamination, indicating that histones with substantially higher arginine contents than those in somatic nuclei are not present (9). Application of the Langmuir trough-critical point method has revealed striking similarities in nuclear fine structure in the sperm nuclei of these organisms. In each
(15),
FIG. 9. Two isolated goldfish sperm, x 13 500. FIG. 10. A portion of an isolated goldfish sperm nucleus. The nucleus is composed of fibers whose average diameter is 171 A. x 100 000.
246
ZIRKIN
SEA URCHIN SPERM
FROG SPERM 40
40
20
>-
,ZL I00
200
20
I O0
500
200
500
hi LU LI-
BOVINE KIDNEY CHROMOSOMES
GOLDFISH SPERM 40
40
20
2O
I
P I00
200
300
r
r I00
200
500
FIBER DIAMETER (A) FIG. 12. Frequency distributions of fiber diameters in surface-spread frog, sea urchin, and goldfish sperm nuclei, and bovine kidney chromosomes. case, randomly distributed fibers of similar appearance and dimensions were the only structures seen. Measurements of sperm nuclear fibers revealed mean diameters of 164 A for the sea urchin, 171 A for the goldfish, and 179 A for the frog. These small differences in diameter are not regarded as meaningful in view of the errors introduced in the measurements. Microscope magnification changes (up to 5 %), contamination of fibers in the electron beam, and stretching of fibers, might account for the 15 A difference. The fiber diameters obtained in this study probably are somewhat lower than the actual diameters of the isolated fibers, since stretched fibers were included in the measurements. The occurrence of fibers in the surface-spread preparations is consistent with previous electron microscope studies, in which fibers have been demonstrated in sectioned nuclei of frog sperm (35) and sea urchin spermatids (19), and in surface-spread nuclei of sea urchin sperm (26). Highly oriented fibers, lamellae, and sheets occur in middle and late spermatid nuclei of some organisms (17). These unusual structures are strikingly different from the fibrous elements which occur in early spermatids, interphase chromatin, and
ULTRASTRUCTURE OF HISTONE-CONTAININGSPERM NUCLEI
247
chromosomes (17). Unusual structures have not been seen in the sperm nuclei of the frog, sea urchin, and goldfish. Sheets have been demonstrated both in surface spread (13) and sectioned (14) nuclei of grasshopper spermatids, indicating that if sheets were present in the sperm nuclei under study, they could have been demonstrated by the techniques used. It has been suggested that the occurrence of unusual structures might be correlated with the replacement of histones by arginine-rich histones and protamines (9, 16, 22). The sperm nuclei of the frog, sea urchin, and goldfish contain somatic-type histones. The absence of unusual structures in these nuclei might thus reflect the absence of unusual basic proteins. The diameter of fibers in surface spread interphase nuclei and mitotic and meiotic chromosomes generally has been reported to average about 250 A (1, 10, 13, 24, 27, 29-33). This diameter is consistent with the diameter of fibers in surface spread bovine kidney chromosomes reported in this study (264 A). The fiber diameters measured in surface spread sperm nuclei (164-179 A), however, are substantially lower than the common 250 A diameter. The difference appears to be too great to be attributed solely to errors in the measurements. One possible explanation is that surface spread sperm chromatin, even from sperm nuclei that contain somatic-type histones, is different in at least some respects from surface spread interphase or metaphase chromatin. However, Wolfe (31) has reported that unfixed, surface spread barley leaflet and root tip chromosomes contain fibers that average 187 /k and 170 /~, respectively. These diameters are very similar to the sperm diameters reported in the present study. On the other hand, barley endosperm chromosome fibers were reported by Wolfe (31) to have a mean diameter of 244 A, similar to the commonly reported 250 ,&_diameter, and substantially greater than the diameters found in barley leaflet and root tip chromosomes. The magnitude of the differences in fiber diameters in the various barley chromosomes is similar to the differences in diameters seen in the sperm and kidney nuclear fibers reported in the present study. The explanation for these differences is not yet known. I thank Drs. Stephen L. Wolfe and Ronald J. Baskin for their suggestions and critical reading of the manuscript, and Mr John Mais for his excellent technical assistance. I also thank Dr. Dennis Barrett for supplying sea urchins.
REFERENCES l. 2. 3. 4. 5.
ABUELLO,J. G. and MOORE, D. E., J. Cell Biol. 41, 73 0969). ALFERT,M., J. Biophys. Biochem. Cytol. 2, 109 (1956). ALFERT, M. and GESCHWIND,I. I., Proc. Nat. Acad. Sci. U.S. 39, 992 (1953). ANDERSON,T. F., Trans. N. Y. Acad. Sci. 13, 130 (1951). BLOCH,D. P., J. Histochem. Cytochem. 10, 137 (1962).
248
6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24. 25. 26. 27. 28.
29. 30. 31. 32. 33. 34. 35.
ZIRKIN
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