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Effect of Cryopreservation on the Fine Structure of Spermatozoa of Rainbow Trout (Oncorhynchus mykiss) and Brown Trout (Salmo trutta F. fario)
37, 263–270 (1998) CY982123
CRYOBIOLOGY ARTICLE NO.
BRIEF COMMUNICATION Effect of Cryopreservation on the Fine Structure of Spermatozoa of Rainbow Trout (Oncorhynchus mykiss) and Brown Trout (Salmo trutta F. fario) S. Drokin,1 H. Stein,* and H. Bartscherer* Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of the Ukraine, 23 Pereyaslavskeya Street, Kharkov 310015, Ukraine; and *TU Munchen, Freising, Germany Freeze-fracture electron microscopy has revealed that changes are induced in the organization of the plasma membranes of spermatozoa of rainbow and brown trout when they are cryopreserved. Electron-micrographic images of spermatozoa that had not been exposed to cryopreservation showed a protoplasmic surface with particles homogeneously distributed. The concentration of the particles was low. In the median portion of the tail the authors observed longitudinal strips, consisting of particles located along the bands. In the neck of the spermatozoon the particles were aggregated in a chaotic manner. Electron micrographs of trout spermatozoa that had been cryopreserved showed particles grouped in rounded clusters on the protoplasmic surfaces of both the head and the tail. In some spermatozoa, folding of the protoplasmic membrane, with the particle-free sites, were found. The dimensions of the clusters and the spaces that were free of particles suggest that, after thawing, the spermatozoa of the brown trout is likely to experience greater difficulty in restoring their physiological integrity than those of the rainbow trout. These results suggest that the membrane proteins of spermatozoa of the species of trout that were studied possess high motility and diffuse, with the formation of clusters, in very short periods of time (about 30 s). The changes in membrane structure of the trout spermatozoa following cryopreservation appear to indicate high lability. © 1998 Academic Press
Due to their heterogeneity, fish spermatozoa occupy a special place in the history of cryobiology. Evolutionary diversity and great differences in nutrition and the physical–chemical nature of their habitat have resulted in considerable differences between the physiological, biochemical, and morphological properties of spermatozoa of different species. This has produced major challenges to cryobiologists attempting to design effective methods of cryopreservation, and, as reported by numerous researchers, it has sometimes been difficult to achieve this goal. It is common knowledge that the sperm of saltwater fish survive cryopreservation better than those of freshwater fish (5). In this respect rainbow trout and brown trout occupy an intermediate position in addition to their differing evolutionary status. The resistance of sperm to freezing is
Received July 8, 1997; accepted July 27, 1998. 1 To whom correspondence should be addressed.
known to be dependent on the biochemical characteristics of their membranes; the higher the molar ratio of cholesterol to phospholipids and the lower the proportion of polyunsaturated fatty acids, the better the membrane survives osmotic and thermal stress and the mechanical injury caused by ice nucleation (3, 5). The technique of freeze fracture permits the visual evaluation of alterations induced in spermatozoal membranes by cooling and cryopreservation. The clusters that are observed develop as a result of the lateral diffusion of proteins and are indicative of the phase heterogeneity of the phospholipid bilayer; this may be one of the parameters that control the cryoresistance of spermatozoa. The aim of this study was to investigate the morphological structure of the sperm of rainbow trout and brown trout both prior to and following cryopreservation. The authors were unable to find any report on the normal morphology of these fish spermatozoa. Mature milt was obtained during the period 263 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Freeze-fracture electron micrograph of the head of a brown trout spermatozoon prior to cryopreservation (bar 5 0.2 mm).
of natural spawning. The sperm that were to be cryopreserved were diluted by a cryoprotective medium (10), containing 750 mg NaCl, 200 mg NaHCO3, 40 mg KCl, 100 mg glucose, 100 ml distilled water, 20 ml egg yolk, and 12 ml dimethyl sulfoxide (Me2SO) in the ratio of 1:3. The sperm were frozen in solid carbon dioxide (dry ice) at 279°C until completely frozen. The average rate of cooling, measured with a copper-constantan ther-
mocouple, was 40°C/min. Thawing was done in a 40°C water bath. Control spermatozoa were not subjected to cooling but were diluted with the same medium without egg yolk, and the amount of Me2SO was increased to 12%. After cooling to 14°C, sufficient glycerol was added to the frozen–thawed and control sperm to produce a concentration of 10%. The sperm samples were equilibrated at 4–6°C for 1 h. After that, the samples were centrifuged,
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265
FIG. 2. Freeze-fracture electron micrograph of tails of rainbow trout spermatozoa prior to cryopreservation (bar 5 0.1 mm).
and the sperm were placed on gold holders and frozen in subcooled liquid nitrogen. They were then placed in a copper holder and transferred into a Balzers BAF 400 freeze-etch apparatus where fracturing was performed. The samples were washed in distilled water, H2SO4, and sodium hypochlorate and then again in distilled water. After this the replicas were mounted on copper grids and examined with a Philips CM-10 or a Zeiss EM 10 microscope.
Examination of electron micrographs of the spermatozoa (bodies and tails) prior to cryopreservation failed to reveal any significant differences between rainbow and brown trout. The protoplasmic surface demonstrated particles, homogeneously distributed over the surface (Figs. 1 and 2). In some cases the authors observed a chaotic aggregation of particles in the region of the neck and longitudinal stripes consisting of particles in the tail. In both species,
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FIG. 3. Freeze-fracture electron micrograph of the head and tails of brown trout spermatozoa after cryopreservation (bar 5 0.2 mm).
the region of the neck is poorly visualized. The diameter of the tail is very small compared with that of the body. In no cases were belt-like or spiral structures, which are often found in mammalian spermatozoa, identified on the protoplasmic surface of the spermatozoa of brown or rainbow trout (4, 9). The electron micrographs of the spermatozoa of brown and rainbow trout, following cryo-
preservation (Figs. 3–5), showed particles that were grouped into clusters on the protoplasmic surface. This was observed in both the head and the tail. The dimensions of these clusters vary greatly within a single species and were much bigger in the brown trout than the rainbow trout (Figs. 4 and 5). Most often the particles formed rounded or longitudinally shaped clusters in the region of the tail. The protoplasmic membrane
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267
FIG. 4. Freeze-fracture electron micrograph of the midpiece of the head of a brown trout spermatozoon after cryopreservation (bar 5 0.1 mm).
was folded in some spermatozoa of both species of fish, and the particles were distributed inhomogeneously, while the clusters were missing on the protoplasmic surface (Fig. 6). It is common knowledge that cryopreservation induces phase transitions in the lipids of the cytoplasmic membranes of the cells, and this results in a spatial redistribution of their components. This process is caused by the lateral
separation of lipids and the aggregation of membrane proteins which result in changes in cell membrane permeability and impairment of the membrane-transporting enzymes (1, 8). The method of freeze fracturing and electron microscopy permits the identification of clusters on the protoplasmic surface of the cells, which is due to the aggregation of the membrane protein.
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BRIEF COMMUNICATION
FIG. 5. Freeze-fracture electron micrograph of the midpiece of the head of a rainbow trout spermatozoon after cryopreservation (bar 5 0.1 mm).
Trout spermatozoa are of particular interest in this context because the normal temperature of their habitat is very low (11 to 13°C). Previous work by the authors (6, 7) investigated the fatty-acid composition of phospholipids and the molar ratio of cholesterol to phospholipids in some salmon species, the spawning temperature of which is close to the spawning temperature of the trout species un-
der study. Judging by those results, it may be assumed that the phase transition temperature of the bulk of the phospholipids is lower than 220°C. With a cooling rate of 40°C/min, only about 30 s would be available for the formation of protein clusters in the trout spermatozoa. Hence, the production of clusters on the protoplasmic face of the trout spermatozoa during cryopreservation indicates a very high
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269
FIG. 6. Freeze-fracture electron micrograph of the head of a rainbow trout spermatozoon after cryopreservation (bar 5 0.25 mm).
rate of lateral diffusion of membrane proteins. The changes that occur in the membranes of the trout spermatozoa will probably negatively affect its physiological integrity after thawing, although they may prove to be reversible, as is the case with bull and boar spermatozoa (4). The extent of membrane injury, and the probability of its recovery after thawing, may depend on the distance that is traveled by the particles en route from their normal physiological site to form a cluster.
The authors believe the rate of cluster formation in the brown trout is greater than that in the rainbow trout, and it appears that the spermatozoa of the brown trout demonstrate a lower tolerance to the method of cryopreservation used in this study. The changes in the structure of the cell membrane shown in Fig. 6 are probably more significant. The protoplasmic surface is folded, and it has sites that are free of the particles that have not formed clusters. Apparently, in this case the
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spermatozoa have poorly differentiated membranes in which the process of lateral diffusion of molecules at the low temperatures used is limited, and this has resulted in the observed deformation of the membranes and greater injury to the cells, compared with highly differentiated membranes that permit the rapid formation of clusters. Probably the rate of cooling used by the authors (1) was too high for these spermatozoa. Their membranes may have been exposed to vesiculation and the loss of proteins, as well as lipids (2). Such injuries to the cells appear to be irreversible, and the spermatozoa lose their functional integrity. REFERENCES 1. Belous, A. M., and Bondarenko, V. A. Structural changes in biological membranes during cooling. Kiev. Naukova dumka publ. 255 (1982). [in Russian] 2. Bong, Y. Y., Ryan, M. A., and Wiggs, A. J. Loss of protein from spermatozoa of Atlantic salmon (Salmo salar L.) because of cryopreservation. Can. J. Zool. 65, 9 –13 (1987). 3. Darin-Bennet, A., and White, I. G. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold shock. Cryobiology 24, 466 – 470 (1977).
4. De Leeuw, F. E., Hsiao-Ching Chen, Colenbrander, B., and Verkleij, A. J. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 27, 171–183 (1990). 5. Drokin, S. I., Kopeika, E. F., and Grischenko, V. I. Differences in the resistance to cryopreservation and specificity of lipid content of spermatozoa of marine and freshwater fish species. Rep. USSR Acad. Sci. 304, 1493–1496 (1989). [in Russian] 6. Drokin, S. I., and Kopeika, E. F. Peculiarities of lipid composition of spermatozoa of chum salmon and pink salmon. II. Symp. Ecol. Biochem., Rostov the Great, Russia 74 (1990). [in Russian]. 7. Drokin, S. I., and Cherepanov, V. V. Peculiarities of the fatty acid compositions of sperm, phospholipids of the autumn-spawning Oncorhynchus keta and Oncorhynchus gorbusha. VIII Sci. Conf. Fish Ecol. Physiol. Biochem. Abstr. Rep.-Petrozavodsk. (1991). [in Russian]. 8. Holt, W. Y., and North, R. D. Partially irreversible cold-induced lipid phase transition in mammalian sperm plasma membrane domains: Freeze-fracture study. J. Exp. Zool. 230, 473– 483 (1984). 9. Koehler, J. K. Fine structure observations in frozenetched bovine spermatozoa. J. Ultrastruct. Res. 16, 359 –375 (1966). 10. Stein, H. Einfluss verschiedener Gefrier-und Auftaugeschwindigkeiten auf die Befruchtungsf higkeit von tiefgefrorenem Forellensperma. Berl. Muench. Tieraerzfl. Wochenschr. 97, 138 –139 (1984).
CRYOBIOLOGY ARTICLE NO.
BRIEF COMMUNICATION Effect of Cryopreservation on the Fine Structure of Spermatozoa of Rainbow Trout (Oncorhynchus mykiss) and Brown Trout (Salmo trutta F. fario) S. Drokin,1 H. Stein,* and H. Bartscherer* Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of the Ukraine, 23 Pereyaslavskeya Street, Kharkov 310015, Ukraine; and *TU Munchen, Freising, Germany Freeze-fracture electron microscopy has revealed that changes are induced in the organization of the plasma membranes of spermatozoa of rainbow and brown trout when they are cryopreserved. Electron-micrographic images of spermatozoa that had not been exposed to cryopreservation showed a protoplasmic surface with particles homogeneously distributed. The concentration of the particles was low. In the median portion of the tail the authors observed longitudinal strips, consisting of particles located along the bands. In the neck of the spermatozoon the particles were aggregated in a chaotic manner. Electron micrographs of trout spermatozoa that had been cryopreserved showed particles grouped in rounded clusters on the protoplasmic surfaces of both the head and the tail. In some spermatozoa, folding of the protoplasmic membrane, with the particle-free sites, were found. The dimensions of the clusters and the spaces that were free of particles suggest that, after thawing, the spermatozoa of the brown trout is likely to experience greater difficulty in restoring their physiological integrity than those of the rainbow trout. These results suggest that the membrane proteins of spermatozoa of the species of trout that were studied possess high motility and diffuse, with the formation of clusters, in very short periods of time (about 30 s). The changes in membrane structure of the trout spermatozoa following cryopreservation appear to indicate high lability. © 1998 Academic Press
Due to their heterogeneity, fish spermatozoa occupy a special place in the history of cryobiology. Evolutionary diversity and great differences in nutrition and the physical–chemical nature of their habitat have resulted in considerable differences between the physiological, biochemical, and morphological properties of spermatozoa of different species. This has produced major challenges to cryobiologists attempting to design effective methods of cryopreservation, and, as reported by numerous researchers, it has sometimes been difficult to achieve this goal. It is common knowledge that the sperm of saltwater fish survive cryopreservation better than those of freshwater fish (5). In this respect rainbow trout and brown trout occupy an intermediate position in addition to their differing evolutionary status. The resistance of sperm to freezing is
Received July 8, 1997; accepted July 27, 1998. 1 To whom correspondence should be addressed.
known to be dependent on the biochemical characteristics of their membranes; the higher the molar ratio of cholesterol to phospholipids and the lower the proportion of polyunsaturated fatty acids, the better the membrane survives osmotic and thermal stress and the mechanical injury caused by ice nucleation (3, 5). The technique of freeze fracture permits the visual evaluation of alterations induced in spermatozoal membranes by cooling and cryopreservation. The clusters that are observed develop as a result of the lateral diffusion of proteins and are indicative of the phase heterogeneity of the phospholipid bilayer; this may be one of the parameters that control the cryoresistance of spermatozoa. The aim of this study was to investigate the morphological structure of the sperm of rainbow trout and brown trout both prior to and following cryopreservation. The authors were unable to find any report on the normal morphology of these fish spermatozoa. Mature milt was obtained during the period 263 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
264
BRIEF COMMUNICATION
FIG. 1. Freeze-fracture electron micrograph of the head of a brown trout spermatozoon prior to cryopreservation (bar 5 0.2 mm).
of natural spawning. The sperm that were to be cryopreserved were diluted by a cryoprotective medium (10), containing 750 mg NaCl, 200 mg NaHCO3, 40 mg KCl, 100 mg glucose, 100 ml distilled water, 20 ml egg yolk, and 12 ml dimethyl sulfoxide (Me2SO) in the ratio of 1:3. The sperm were frozen in solid carbon dioxide (dry ice) at 279°C until completely frozen. The average rate of cooling, measured with a copper-constantan ther-
mocouple, was 40°C/min. Thawing was done in a 40°C water bath. Control spermatozoa were not subjected to cooling but were diluted with the same medium without egg yolk, and the amount of Me2SO was increased to 12%. After cooling to 14°C, sufficient glycerol was added to the frozen–thawed and control sperm to produce a concentration of 10%. The sperm samples were equilibrated at 4–6°C for 1 h. After that, the samples were centrifuged,
BRIEF COMMUNICATION
265
FIG. 2. Freeze-fracture electron micrograph of tails of rainbow trout spermatozoa prior to cryopreservation (bar 5 0.1 mm).
and the sperm were placed on gold holders and frozen in subcooled liquid nitrogen. They were then placed in a copper holder and transferred into a Balzers BAF 400 freeze-etch apparatus where fracturing was performed. The samples were washed in distilled water, H2SO4, and sodium hypochlorate and then again in distilled water. After this the replicas were mounted on copper grids and examined with a Philips CM-10 or a Zeiss EM 10 microscope.
Examination of electron micrographs of the spermatozoa (bodies and tails) prior to cryopreservation failed to reveal any significant differences between rainbow and brown trout. The protoplasmic surface demonstrated particles, homogeneously distributed over the surface (Figs. 1 and 2). In some cases the authors observed a chaotic aggregation of particles in the region of the neck and longitudinal stripes consisting of particles in the tail. In both species,
266
BRIEF COMMUNICATION
FIG. 3. Freeze-fracture electron micrograph of the head and tails of brown trout spermatozoa after cryopreservation (bar 5 0.2 mm).
the region of the neck is poorly visualized. The diameter of the tail is very small compared with that of the body. In no cases were belt-like or spiral structures, which are often found in mammalian spermatozoa, identified on the protoplasmic surface of the spermatozoa of brown or rainbow trout (4, 9). The electron micrographs of the spermatozoa of brown and rainbow trout, following cryo-
preservation (Figs. 3–5), showed particles that were grouped into clusters on the protoplasmic surface. This was observed in both the head and the tail. The dimensions of these clusters vary greatly within a single species and were much bigger in the brown trout than the rainbow trout (Figs. 4 and 5). Most often the particles formed rounded or longitudinally shaped clusters in the region of the tail. The protoplasmic membrane
BRIEF COMMUNICATION
267
FIG. 4. Freeze-fracture electron micrograph of the midpiece of the head of a brown trout spermatozoon after cryopreservation (bar 5 0.1 mm).
was folded in some spermatozoa of both species of fish, and the particles were distributed inhomogeneously, while the clusters were missing on the protoplasmic surface (Fig. 6). It is common knowledge that cryopreservation induces phase transitions in the lipids of the cytoplasmic membranes of the cells, and this results in a spatial redistribution of their components. This process is caused by the lateral
separation of lipids and the aggregation of membrane proteins which result in changes in cell membrane permeability and impairment of the membrane-transporting enzymes (1, 8). The method of freeze fracturing and electron microscopy permits the identification of clusters on the protoplasmic surface of the cells, which is due to the aggregation of the membrane protein.
268
BRIEF COMMUNICATION
FIG. 5. Freeze-fracture electron micrograph of the midpiece of the head of a rainbow trout spermatozoon after cryopreservation (bar 5 0.1 mm).
Trout spermatozoa are of particular interest in this context because the normal temperature of their habitat is very low (11 to 13°C). Previous work by the authors (6, 7) investigated the fatty-acid composition of phospholipids and the molar ratio of cholesterol to phospholipids in some salmon species, the spawning temperature of which is close to the spawning temperature of the trout species un-
der study. Judging by those results, it may be assumed that the phase transition temperature of the bulk of the phospholipids is lower than 220°C. With a cooling rate of 40°C/min, only about 30 s would be available for the formation of protein clusters in the trout spermatozoa. Hence, the production of clusters on the protoplasmic face of the trout spermatozoa during cryopreservation indicates a very high
BRIEF COMMUNICATION
269
FIG. 6. Freeze-fracture electron micrograph of the head of a rainbow trout spermatozoon after cryopreservation (bar 5 0.25 mm).
rate of lateral diffusion of membrane proteins. The changes that occur in the membranes of the trout spermatozoa will probably negatively affect its physiological integrity after thawing, although they may prove to be reversible, as is the case with bull and boar spermatozoa (4). The extent of membrane injury, and the probability of its recovery after thawing, may depend on the distance that is traveled by the particles en route from their normal physiological site to form a cluster.
The authors believe the rate of cluster formation in the brown trout is greater than that in the rainbow trout, and it appears that the spermatozoa of the brown trout demonstrate a lower tolerance to the method of cryopreservation used in this study. The changes in the structure of the cell membrane shown in Fig. 6 are probably more significant. The protoplasmic surface is folded, and it has sites that are free of the particles that have not formed clusters. Apparently, in this case the
270
BRIEF COMMUNICATION
spermatozoa have poorly differentiated membranes in which the process of lateral diffusion of molecules at the low temperatures used is limited, and this has resulted in the observed deformation of the membranes and greater injury to the cells, compared with highly differentiated membranes that permit the rapid formation of clusters. Probably the rate of cooling used by the authors (1) was too high for these spermatozoa. Their membranes may have been exposed to vesiculation and the loss of proteins, as well as lipids (2). Such injuries to the cells appear to be irreversible, and the spermatozoa lose their functional integrity. REFERENCES 1. Belous, A. M., and Bondarenko, V. A. Structural changes in biological membranes during cooling. Kiev. Naukova dumka publ. 255 (1982). [in Russian] 2. Bong, Y. Y., Ryan, M. A., and Wiggs, A. J. Loss of protein from spermatozoa of Atlantic salmon (Salmo salar L.) because of cryopreservation. Can. J. Zool. 65, 9 –13 (1987). 3. Darin-Bennet, A., and White, I. G. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold shock. Cryobiology 24, 466 – 470 (1977).
4. De Leeuw, F. E., Hsiao-Ching Chen, Colenbrander, B., and Verkleij, A. J. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 27, 171–183 (1990). 5. Drokin, S. I., Kopeika, E. F., and Grischenko, V. I. Differences in the resistance to cryopreservation and specificity of lipid content of spermatozoa of marine and freshwater fish species. Rep. USSR Acad. Sci. 304, 1493–1496 (1989). [in Russian] 6. Drokin, S. I., and Kopeika, E. F. Peculiarities of lipid composition of spermatozoa of chum salmon and pink salmon. II. Symp. Ecol. Biochem., Rostov the Great, Russia 74 (1990). [in Russian]. 7. Drokin, S. I., and Cherepanov, V. V. Peculiarities of the fatty acid compositions of sperm, phospholipids of the autumn-spawning Oncorhynchus keta and Oncorhynchus gorbusha. VIII Sci. Conf. Fish Ecol. Physiol. Biochem. Abstr. Rep.-Petrozavodsk. (1991). [in Russian]. 8. Holt, W. Y., and North, R. D. Partially irreversible cold-induced lipid phase transition in mammalian sperm plasma membrane domains: Freeze-fracture study. J. Exp. Zool. 230, 473– 483 (1984). 9. Koehler, J. K. Fine structure observations in frozenetched bovine spermatozoa. J. Ultrastruct. Res. 16, 359 –375 (1966). 10. Stein, H. Einfluss verschiedener Gefrier-und Auftaugeschwindigkeiten auf die Befruchtungsf higkeit von tiefgefrorenem Forellensperma. Berl. Muench. Tieraerzfl. Wochenschr. 97, 138 –139 (1984).