- Email: [email protected]
Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membranes after cryopreservation
CRYOBIOLOGY
26,
70-75 (1989)
Changes in the Phospholipid Composition and Phospholipid Asymmetry of Ram Sperm Plasma Membranes after Cryopreservation V. HINKOVSKA-GALCHEVA,
D. PETKOVA, AND K. KOUMANOV Central Laboratory of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the transIocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane. 0 1989 Academic
Press, Inc.
Comparative studies on the different methods for sperm conservation through freezing in the presence of cryoprotectors have shown that the success of such methods depends on the species. Spermatozoa of bulls and stallions tolerate deep freezing relatively well. Ram and boar spermatozoa show a lower fertilizing capacity under similar conditions (9). The biochemical nature of the spermatozoan is quite complex and of unstable structure. Its membrane can be easily disturbed even in the presence of cryoprotectors . The mechanisms of cryodamage affecting the separate components of biomembranes are of great interest in the study of its structural and functional peculiarities. The lipid components prove to be particularly instable. The lipid/protein interactions play an important role in keeping the membrane intact and functionally active. Many membrane-bound enzymes require a special conformation in order to display their optimal activity. This state is maintained largely by the membrane lipids (2, 8, 10,
20). It is well known that many important enzymes involved in the acrosome reaction of the sperm that ensure its fertilizing capacity are located on the cell surface. The membrane fusion in the acrosome reaction and the release of the acrosomal content are due to changes in the existing organization of the membrane macromolecules (28). The extent to which the structures of sperm plasma membranes follow a definite pattern is very important for the proper performance of the fusion processes. In earlier investigations we established that the distribution of the lipids in the spermatozoa plasma membranes is asymmetric (13). Temperature is one of the important factors affecting the diffusion of the membrane components. Therefore, we investigated the changes in the lipid distribution following a decrease in temperature to - 196°C. MATERIALS
Semen was obtained from Romney Marsh rams. Immediately after collection of semen with an artificial vagina, semen
Received March 1, 1988; accepted June 29, 1988.
70 OOll-2240/89 $3.00 Copyright All rights
Q 1989 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
SPERMAL
PLASMA
MEMBRANES
PHOSPHOLIPIDS
volume and spermatozoa motility were recorded. Spermatozoa motility, assessed as the percentage of motile spermatozoa (O-100%) was determined microscopically. The semen was cooled to 5°C at 0.3”C/min without diluent. After equilibration for 4 hr at 5°C the semen was cooled immediately to -78°C in pellets, and then stored in liquid nitrogen (- 196°C). The frozen semen was thawed at 37°C for 3 min. Only ejaculates having a high motility were used for plasma membrane isolation. As control we used fresh nondiluted semen. Plasma membranes were isolated by the procedure of Lunstra et al. (19). The purity of the membrane fraction thus obtained was estimated by the specific activity of marker enzymes and by electron microscopy. We determined the activities of 5’-nucleotidase (21), succinate dehydrogenase (17)) DNA (4)) and acrosin (29) in the plasma membrane preparations. The spermatozoa1 and the plasma membrane lipids were extracted (11) and cholesterol (24) and phospholipid phosphorus (15) were determined. The different phospholipid fractions were separated by thin-layer chromatography (TLC)’ on silica gel 60 precoated plates (Merck) in a solvent system containing chloroform/methanol/isopropano1/0.25% KCUtriethylamine (30/9/25/ 6/18 (v/v)). The distribution of the membrane phospholipids was investigated by enzymatic degradation of the membrane phospholipids with phospholipase A, (Naja naja; Sigma), phospholipase C (Clostridium welchii; Sigma), and sphingomyelinase (Staphylococcus aureus). The enzymatic treatment was performed at a ratio of l:O. 1 (membrane protein:enzyme protein). The Abbreviations used: TLC, Thin-layer chromatography; TNBS, Trinitrobenzene sulfonate; PG, Phosphatidylglycerol; PS, Phosphatidylserine; PE, Phosphatidylethanolamine; DPG, Diphosphatidylglycerol; SM, Sphingomyelin; SMIPC, Sphingomyelinlphosphatidylcholine ratio; PI, Phosphatidylinositol; CHIPL, CholesteroYphospholipid ratio.
AND
CRYOPRESERVATION
71
mixture was shaken constantly for 15 min at 37°C in a 10 mA4Tris buffer (pH 7.4). The buffer contained 5 mM Ca2’ for phospholipases or 40 miU Mg2+ for sphingomyelinase. After the corresponding treatment the plasma membranes were isolated and washed twice with the same 10 mM Tris buffer, and the phospholipids were extracted and quantitated as described above. Trinitrobenzene sulphonate (TNBS) was also used for assessing the aminophospholipid distribution in membranes (23). The protein was determined by the procedure of Lowry et al. (18). Statistical analysis was carried out using Student’s t test. All the results were obtained from analysis of 10 semen samples. RESULTS
The freezing of ram spermatozoa to - 196°Cwithout the addition of cryoprotectors and the subsequent thawing led to certain alterations in their lipid composition. Table 1 shows that the amount of the total phospholipids of isolated plasma membranes obtained from sperm cells frozen without a cryoprotector to - 196°C remained almost unchanged as compared to the controls. Some individual phospholipid fractions underwent greater or smaller alterations. Phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine (PG, PS, PE) were diminished, while DPG was increased after freezing to - 196°C and subsequently thawed. The results presented in Table 1 show that the total amounts of phospholipids of intact sperm cells remained almost unchanged but certain rearrangements of the separate phospholipid fractions were observed. The increase of SM was followed by an augmentation of the SM/PC ratio. Phosphatidylinositol (PI) was augmented, whereas PE and DPG were reduced. As a result of the cholesterol diminution the CH/PL ratio was lowered in whole cells and plasma membranes compared to controls
72
HINKOVSKA-GALCHEVA,
PETKOVA, AND KOUMANOV
TABLE 1 Phospholipid Composition of Ram Spermatozoa Plasma Membranes before and after Freezing Spermatozoa (pg PWl.8 x 10’ cells)
Plasma membranes (pg PL/mg protein) Phospholipids SM PC PS PI PE PC DPG TPL SMPC CHiPL
Control 20.37 k 94.76 + 5.47 * 2.69 f 19.70 T 9.68 + 13.90 f
2.8 8.1 0.9 0.1 2.7 2.2 1.7
167.80 + 3.5 0.236 0.280
- 196°C
Control
20.36 + 2.4 88.22 2 1.2 4.32 -c 2.6* 2.25 2 0.6* 15.89 ” 0.9* 6.46 2 0.5*** 16.42 f 0.5***
58.00 + 5.1 159.00 + 5.8 8.80 2 2.9 6.60 -+ 1.3 49.09 k 2.9 15.50 + 2.1 26.80 rf: 4.8
62.45 + 153.73 f 9.13 + 8.31 + 41.18 + 14.05 + 20.85 f
323.79 + 4.7 0.364 0.769
314.10 + 1.2 0.406 0.630
153.92 * 1.2 0.230 0.250
- 196°C 1.9*** 1.4* 0.7 1.8* 2.7** 1.9* 3.5***
Note. Values are means k SD (n = 10). TPL, total phospholipids; SM, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; CH, cholesterol; PL[ phospholipids. * P < 0.05. ** P < 0.01. *** P < 0.001.
(CH/PL, plasma membranes, 0.360-0.250 and cells, 0.769-0.630). Deep freezing of the spermatozoa led to considerable alterations in the distribution of the separate phospholipids between the inner and outer monolayers of the membrane (Fig. 1). In the control membranes 99% of the diphosphatidylglycerol (DPG) was found in the inner monolayer; in the plasma membranes, isolated from cells subjected to low temperatures, this percentage was only 58%. In the control membranes 47% of PS was found in the inner monolayer. After freezing to - 196°C this percentage increased to 61% in the inner monolayer of the plasma membranes.
IOO-
50I-
DISCUSSION
The phospholipid composition of spermatozoal plasma membranes is of great interest since the phospholipids play an important role in the acrosome reaction (7,25, 27, 28). Moreover this composition is of particular importance for the successful survival of the spermatozoa subjected to cryopreservation.
FIG. 1. Phospholipid distribution in the inner and outer monolayer of ram spermatozoa plasma membranes before (open column) and after (hatched columns) freezing to - 196°C.
SPERMAL
PLASMA
MEMBRANES
PHOSPHOLIPIDS
Freezing of ram sperm to - 196°C with no cryoprotectors and subsequent thawing induced certain changes in the lipid composition. These changes affected both the spermatozoa and their plasma membranes. The total phospholipids of the plasma membranes remained almost unchanged compared to that of the control and only individual phospholipid fractions underwent certain changes. PG, PS, and PE showed a tendency to decrease, whereas DPG was increased in the course of freezing to - 196°C. An interesting feature of the structure of ram sperm plasma membranes was the characteristic asymmetric distribution of the phospholipids (13). Lipid asymmetry is a property of many biological membranes (3, 12, 30). An important condition for the proper functioning of the membranes is the firm association between their lipid and protein components. Therefore the changes in the metabolic processes are probably related to alterations in the lipid distribution. The structural and functional properties of many membrane-bound enzymes may undergo both primary and secondary alterations due to the effect of the disturbed molecular organization of their lipid microenvironment (5). Spermatozoa freezing leads also to alterations in the phospholipids’ distribution between the two monolayers of the membrane lipid bilayer. As our results indicated the most significant changes occurred in the DPG distribution. DPG is localized mainly in the inner monolayer of the native membranes, whereas as a result of freezing the DPG molecules underwent translocation and became almost evenly distributed in the outer and inner lipid bilayers. This may be an important change since DPG is necessary for the normal acrosome reaction (1). The transfer of DPG from the inner to the outer monolayer may inhibit the acrosome reaction which starts with the fusion of the inner monolayer of the plasma membrane and the outer monolayer of the acrosomal
AND
CRYOPRESERVATION
73
membrane. The fertilizing capacity of the cells subjected to low temperatures may be lowered. Some hypothesis, explaining the biochemical mechanism of membrane fusion in the course of the acrosome reaction, includes the participation of phospholipase A,. This enzyme is known to induce accumulation of lysophospholipids and free fatty acids, causing changes in the membrane bilayer (6). As we mentioned above, many membrane-bound enzyme activities depend strongly upon membrane physicochemical properties. The changes in the phospholipid distribution between the inner and outer membrane leaflets may provoke local changes in the membrane fluidity (16). In previous works of ours we demonstrated that the activity of the rat liver plasma membrane-bound phospholipase A, was correlated to the membrane fluidity (22). We established the same behavior for the ram spermatozoa membrane phospholipase A, too (14) and we suggest mechanisms including regulation of phospholipase A, activity by the changes in membrane fluidity and thus on membrane fusion and on the acrosome reaction. It is very likely that the preservation of the normal phospholipid distribution between the two membrane monolayers is one way of ensuring a comparatively high fertilizing capacity of ram spermatozoa. This distribution maintains an optimal lipid environment for the membrane enzymes which participate in the acrosome reaction and may be necessary for the normal occurrence of the acrosome reaction. ACKNOWLEDGMENT
We thank Dr. Roelofsen (Utrecht) for being so kind as to give us a highly purified sphingomyelinase from s. aureus. REFERENCES
1. Bearer, E. L., and Friend, D. S. Modifications of anionic phospholipid distribution preceding
74
2.
3.
4.
5.
6. 7.
8.
9.
10.
HINKOVSKA-GALCHEVA,
PETKOVA,
membrane fusion. J. Cell Biol. 81, 534-546 (1980). Benga, Cl., Hodamau, A., and Bohm, B. Human liver mitochondria relation of a particular lipid composition to the mobility of spin-labelled lipids. Eur. J. Biochem. 84, 625-628 (1978). Bishop, D. G., Op Den Kamp, A. F., and Van Deenen, L. L. M. The distribution of lipids in the protoplast membranes of Bacillus subtilis. Study with phospholipase C and TNBS. Eur. J. Biochem. 80, 381-398 (1977). Burton K. In “Methods in Enzymology” (E. Grossman and K. Moldave, Eds.), Vol. 7, pp. 163-166. Academic Press, New York/London, 1968. Chapman, D., Peel, W. E., Kingston, B., and Lilley, T. H. Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers. Biochim. Biophys. Acta, 464, 260-275 (1977). Clegg, E. D., and Foote, R. H. Mechanism of mammalian sperm capacitation. J. Reprod. Fertil. 34, 379-383 (1973). Clegg, E. D., Morre, D. J., and Lunstra, D. D. In “The Biology of the Male Gamete” (J. G. Duckett and P. A. Racey, Eds.), Vol. 7, pp. 321-335. Academic Press, New York, 1975. Comte, J., Maisterrena, B., and Gautheron, D. C. Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria comparison with microsomes. Biochim. Biophys. Acta 419, 271-284 (1976). Darin-Bennett, A., and White, I. G. Influence of the cholesterol content of mammalian sperm on susceptibility to cold shock. Cryobiology 14, 466-470 (1977). DePierre, J. W., and Dallner, G. Structural aspects of the membrane of the endoplasmic reticulum. Biochim. Biophys. Acta 415, 411-172
(1975). 11. Folch, J., Lees, M., and Sloane-Staney. A simple
method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-507 (1957). 12. Fong, B. S., and Brown, J. C. Asymmetric distribution of phosphatidylethanolamine fatty acid chains in the membrane of vesicular stomatitis virus. Biochim. Biophys. Acta 510, 230-241 (1978). 13. Hinkovska, V., Dimitrov, G., and Koumanov, K. Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes. Znt. J. Biochem. 18, 1115-1121(1986). 14. Hinkovska-Galcheva, V., Peeva, D., Momchilova-Pankova, A., Pekova, D., and Koumanov, K. Phosphatidylcholine and phosphatidyletha-
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
AND
KOUMANOV
nolamine derivatives, membrane fluidity and changes in the lipolytic activity of ram spermatozoa plasma membranes during cryoconservation. Znt. J. Biochem., in press. Kahovkova, J., and Odavic, R. A simple method for analysis of phospholipids separated on thin layer chromatography. J. Chromatogr. 40, 9095 (1969). Kimelberg, H. K. Alterations in phospholipid dependent Na+-K+ ATPase activity due to lipid fluidity. Biochim. Biophys. Acta 413, 143-152 (1975). King, T. E. Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. In “Methods in Enzymology” (E. Grossman and K. Moldave, Eds.), Vol. 10, pp. 322-331. Academic Press, New York, 1967. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). Lunstra, D. D., Clegg, E. D., and Morre, D. J. Isolation of plasma membrane from porcine spermatozoa. Prep. Biochem. 4, 341-352 (1974). Masotti, L., Lenaz, G., Spisni, A., and Urry, D. W. Effect of phospholipids on the protein conformation in the inner mitochondrial membranes. Biochem. Biophys. Res. Commun. 56, 892-897 (1974). Michel, R. H., and Hawthorne, J. N. The site of diphosphoinositide synthesis in rat liver. Biothem. Biophys. Res. Commun. 21, 333-338 (1965). Momchilova, A. B., Pekova, D. H., Koumanov, K. S. Phospholipid composition modifications influence phospholipase A, acitivity in rat liver plasma membranes. Znt. I. Biochem. 18, 945952 (1986). Nordlund, J. R., Schmidt, C. F., and Dicken, S. N., and Thompson, T. E. Transbilayer distribution of phosphatidylethanolamine in large and small unilamellar vesicles. Biochemistry 20, 3237-3241 (1981). Sperry, W. M., and Webb, M. A revision of Shoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187, 97-106 (1950). Yanagimachi, R. The movement of golden hamster spermatozoa before, and after capacitation. J. Reprod. Fertil. 23, 193-l% (1970). Yanagimachi, R. Specificity of sperm egg interaction. In “Immunology of Gametes” (M. Eddin and M. H. Johnson, Eds.), pp. 255-295. Cambridge Univ. Press., London, 1977. Yanagimachi, R. Sperm-egg association in mammals. In “Current Topics in Developmental
SPERMAL PLASMA MEMBRANES PHOSPHOLIPIDS AND CRYOPRESERVATION Biology” (H. A. Moskova and A. Monray, Eds.), pp. 83-105. Academic Press, New York, 1978. 28. Yanagimachi, R. Mechanisms of fertilization in mammals. In “Fertilization and Embryonic Development in Vitro” (L. Mastrianm and J. D. Biggers, Eds.), pp. 81-155. Plenum, New York/London, 1981. 29. Zaneveld, L. J. D., Polakowski, K. L., and
75
Williams, W. L. A proteinase inhibitor of mammalian sperm acrosomes. Biol. Reprod. 9, 219225 (1973). 30. Zwaal, R. F. A., Roelofsen, B., Comfurius, P., and Van Deenen, L. L. L. Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases. Biochim. Biophys. Acta 406, 83-96 (1975).
26,
70-75 (1989)
Changes in the Phospholipid Composition and Phospholipid Asymmetry of Ram Sperm Plasma Membranes after Cryopreservation V. HINKOVSKA-GALCHEVA,
D. PETKOVA, AND K. KOUMANOV Central Laboratory of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the transIocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane. 0 1989 Academic
Press, Inc.
Comparative studies on the different methods for sperm conservation through freezing in the presence of cryoprotectors have shown that the success of such methods depends on the species. Spermatozoa of bulls and stallions tolerate deep freezing relatively well. Ram and boar spermatozoa show a lower fertilizing capacity under similar conditions (9). The biochemical nature of the spermatozoan is quite complex and of unstable structure. Its membrane can be easily disturbed even in the presence of cryoprotectors . The mechanisms of cryodamage affecting the separate components of biomembranes are of great interest in the study of its structural and functional peculiarities. The lipid components prove to be particularly instable. The lipid/protein interactions play an important role in keeping the membrane intact and functionally active. Many membrane-bound enzymes require a special conformation in order to display their optimal activity. This state is maintained largely by the membrane lipids (2, 8, 10,
20). It is well known that many important enzymes involved in the acrosome reaction of the sperm that ensure its fertilizing capacity are located on the cell surface. The membrane fusion in the acrosome reaction and the release of the acrosomal content are due to changes in the existing organization of the membrane macromolecules (28). The extent to which the structures of sperm plasma membranes follow a definite pattern is very important for the proper performance of the fusion processes. In earlier investigations we established that the distribution of the lipids in the spermatozoa plasma membranes is asymmetric (13). Temperature is one of the important factors affecting the diffusion of the membrane components. Therefore, we investigated the changes in the lipid distribution following a decrease in temperature to - 196°C. MATERIALS
Semen was obtained from Romney Marsh rams. Immediately after collection of semen with an artificial vagina, semen
Received March 1, 1988; accepted June 29, 1988.
70 OOll-2240/89 $3.00 Copyright All rights
Q 1989 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
SPERMAL
PLASMA
MEMBRANES
PHOSPHOLIPIDS
volume and spermatozoa motility were recorded. Spermatozoa motility, assessed as the percentage of motile spermatozoa (O-100%) was determined microscopically. The semen was cooled to 5°C at 0.3”C/min without diluent. After equilibration for 4 hr at 5°C the semen was cooled immediately to -78°C in pellets, and then stored in liquid nitrogen (- 196°C). The frozen semen was thawed at 37°C for 3 min. Only ejaculates having a high motility were used for plasma membrane isolation. As control we used fresh nondiluted semen. Plasma membranes were isolated by the procedure of Lunstra et al. (19). The purity of the membrane fraction thus obtained was estimated by the specific activity of marker enzymes and by electron microscopy. We determined the activities of 5’-nucleotidase (21), succinate dehydrogenase (17)) DNA (4)) and acrosin (29) in the plasma membrane preparations. The spermatozoa1 and the plasma membrane lipids were extracted (11) and cholesterol (24) and phospholipid phosphorus (15) were determined. The different phospholipid fractions were separated by thin-layer chromatography (TLC)’ on silica gel 60 precoated plates (Merck) in a solvent system containing chloroform/methanol/isopropano1/0.25% KCUtriethylamine (30/9/25/ 6/18 (v/v)). The distribution of the membrane phospholipids was investigated by enzymatic degradation of the membrane phospholipids with phospholipase A, (Naja naja; Sigma), phospholipase C (Clostridium welchii; Sigma), and sphingomyelinase (Staphylococcus aureus). The enzymatic treatment was performed at a ratio of l:O. 1 (membrane protein:enzyme protein). The Abbreviations used: TLC, Thin-layer chromatography; TNBS, Trinitrobenzene sulfonate; PG, Phosphatidylglycerol; PS, Phosphatidylserine; PE, Phosphatidylethanolamine; DPG, Diphosphatidylglycerol; SM, Sphingomyelin; SMIPC, Sphingomyelinlphosphatidylcholine ratio; PI, Phosphatidylinositol; CHIPL, CholesteroYphospholipid ratio.
AND
CRYOPRESERVATION
71
mixture was shaken constantly for 15 min at 37°C in a 10 mA4Tris buffer (pH 7.4). The buffer contained 5 mM Ca2’ for phospholipases or 40 miU Mg2+ for sphingomyelinase. After the corresponding treatment the plasma membranes were isolated and washed twice with the same 10 mM Tris buffer, and the phospholipids were extracted and quantitated as described above. Trinitrobenzene sulphonate (TNBS) was also used for assessing the aminophospholipid distribution in membranes (23). The protein was determined by the procedure of Lowry et al. (18). Statistical analysis was carried out using Student’s t test. All the results were obtained from analysis of 10 semen samples. RESULTS
The freezing of ram spermatozoa to - 196°Cwithout the addition of cryoprotectors and the subsequent thawing led to certain alterations in their lipid composition. Table 1 shows that the amount of the total phospholipids of isolated plasma membranes obtained from sperm cells frozen without a cryoprotector to - 196°C remained almost unchanged as compared to the controls. Some individual phospholipid fractions underwent greater or smaller alterations. Phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine (PG, PS, PE) were diminished, while DPG was increased after freezing to - 196°C and subsequently thawed. The results presented in Table 1 show that the total amounts of phospholipids of intact sperm cells remained almost unchanged but certain rearrangements of the separate phospholipid fractions were observed. The increase of SM was followed by an augmentation of the SM/PC ratio. Phosphatidylinositol (PI) was augmented, whereas PE and DPG were reduced. As a result of the cholesterol diminution the CH/PL ratio was lowered in whole cells and plasma membranes compared to controls
72
HINKOVSKA-GALCHEVA,
PETKOVA, AND KOUMANOV
TABLE 1 Phospholipid Composition of Ram Spermatozoa Plasma Membranes before and after Freezing Spermatozoa (pg PWl.8 x 10’ cells)
Plasma membranes (pg PL/mg protein) Phospholipids SM PC PS PI PE PC DPG TPL SMPC CHiPL
Control 20.37 k 94.76 + 5.47 * 2.69 f 19.70 T 9.68 + 13.90 f
2.8 8.1 0.9 0.1 2.7 2.2 1.7
167.80 + 3.5 0.236 0.280
- 196°C
Control
20.36 + 2.4 88.22 2 1.2 4.32 -c 2.6* 2.25 2 0.6* 15.89 ” 0.9* 6.46 2 0.5*** 16.42 f 0.5***
58.00 + 5.1 159.00 + 5.8 8.80 2 2.9 6.60 -+ 1.3 49.09 k 2.9 15.50 + 2.1 26.80 rf: 4.8
62.45 + 153.73 f 9.13 + 8.31 + 41.18 + 14.05 + 20.85 f
323.79 + 4.7 0.364 0.769
314.10 + 1.2 0.406 0.630
153.92 * 1.2 0.230 0.250
- 196°C 1.9*** 1.4* 0.7 1.8* 2.7** 1.9* 3.5***
Note. Values are means k SD (n = 10). TPL, total phospholipids; SM, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; CH, cholesterol; PL[ phospholipids. * P < 0.05. ** P < 0.01. *** P < 0.001.
(CH/PL, plasma membranes, 0.360-0.250 and cells, 0.769-0.630). Deep freezing of the spermatozoa led to considerable alterations in the distribution of the separate phospholipids between the inner and outer monolayers of the membrane (Fig. 1). In the control membranes 99% of the diphosphatidylglycerol (DPG) was found in the inner monolayer; in the plasma membranes, isolated from cells subjected to low temperatures, this percentage was only 58%. In the control membranes 47% of PS was found in the inner monolayer. After freezing to - 196°C this percentage increased to 61% in the inner monolayer of the plasma membranes.
IOO-
50I-
DISCUSSION
The phospholipid composition of spermatozoal plasma membranes is of great interest since the phospholipids play an important role in the acrosome reaction (7,25, 27, 28). Moreover this composition is of particular importance for the successful survival of the spermatozoa subjected to cryopreservation.
FIG. 1. Phospholipid distribution in the inner and outer monolayer of ram spermatozoa plasma membranes before (open column) and after (hatched columns) freezing to - 196°C.
SPERMAL
PLASMA
MEMBRANES
PHOSPHOLIPIDS
Freezing of ram sperm to - 196°C with no cryoprotectors and subsequent thawing induced certain changes in the lipid composition. These changes affected both the spermatozoa and their plasma membranes. The total phospholipids of the plasma membranes remained almost unchanged compared to that of the control and only individual phospholipid fractions underwent certain changes. PG, PS, and PE showed a tendency to decrease, whereas DPG was increased in the course of freezing to - 196°C. An interesting feature of the structure of ram sperm plasma membranes was the characteristic asymmetric distribution of the phospholipids (13). Lipid asymmetry is a property of many biological membranes (3, 12, 30). An important condition for the proper functioning of the membranes is the firm association between their lipid and protein components. Therefore the changes in the metabolic processes are probably related to alterations in the lipid distribution. The structural and functional properties of many membrane-bound enzymes may undergo both primary and secondary alterations due to the effect of the disturbed molecular organization of their lipid microenvironment (5). Spermatozoa freezing leads also to alterations in the phospholipids’ distribution between the two monolayers of the membrane lipid bilayer. As our results indicated the most significant changes occurred in the DPG distribution. DPG is localized mainly in the inner monolayer of the native membranes, whereas as a result of freezing the DPG molecules underwent translocation and became almost evenly distributed in the outer and inner lipid bilayers. This may be an important change since DPG is necessary for the normal acrosome reaction (1). The transfer of DPG from the inner to the outer monolayer may inhibit the acrosome reaction which starts with the fusion of the inner monolayer of the plasma membrane and the outer monolayer of the acrosomal
AND
CRYOPRESERVATION
73
membrane. The fertilizing capacity of the cells subjected to low temperatures may be lowered. Some hypothesis, explaining the biochemical mechanism of membrane fusion in the course of the acrosome reaction, includes the participation of phospholipase A,. This enzyme is known to induce accumulation of lysophospholipids and free fatty acids, causing changes in the membrane bilayer (6). As we mentioned above, many membrane-bound enzyme activities depend strongly upon membrane physicochemical properties. The changes in the phospholipid distribution between the inner and outer membrane leaflets may provoke local changes in the membrane fluidity (16). In previous works of ours we demonstrated that the activity of the rat liver plasma membrane-bound phospholipase A, was correlated to the membrane fluidity (22). We established the same behavior for the ram spermatozoa membrane phospholipase A, too (14) and we suggest mechanisms including regulation of phospholipase A, activity by the changes in membrane fluidity and thus on membrane fusion and on the acrosome reaction. It is very likely that the preservation of the normal phospholipid distribution between the two membrane monolayers is one way of ensuring a comparatively high fertilizing capacity of ram spermatozoa. This distribution maintains an optimal lipid environment for the membrane enzymes which participate in the acrosome reaction and may be necessary for the normal occurrence of the acrosome reaction. ACKNOWLEDGMENT
We thank Dr. Roelofsen (Utrecht) for being so kind as to give us a highly purified sphingomyelinase from s. aureus. REFERENCES
1. Bearer, E. L., and Friend, D. S. Modifications of anionic phospholipid distribution preceding
74
2.
3.
4.
5.
6. 7.
8.
9.
10.
HINKOVSKA-GALCHEVA,
PETKOVA,
membrane fusion. J. Cell Biol. 81, 534-546 (1980). Benga, Cl., Hodamau, A., and Bohm, B. Human liver mitochondria relation of a particular lipid composition to the mobility of spin-labelled lipids. Eur. J. Biochem. 84, 625-628 (1978). Bishop, D. G., Op Den Kamp, A. F., and Van Deenen, L. L. M. The distribution of lipids in the protoplast membranes of Bacillus subtilis. Study with phospholipase C and TNBS. Eur. J. Biochem. 80, 381-398 (1977). Burton K. In “Methods in Enzymology” (E. Grossman and K. Moldave, Eds.), Vol. 7, pp. 163-166. Academic Press, New York/London, 1968. Chapman, D., Peel, W. E., Kingston, B., and Lilley, T. H. Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers. Biochim. Biophys. Acta, 464, 260-275 (1977). Clegg, E. D., and Foote, R. H. Mechanism of mammalian sperm capacitation. J. Reprod. Fertil. 34, 379-383 (1973). Clegg, E. D., Morre, D. J., and Lunstra, D. D. In “The Biology of the Male Gamete” (J. G. Duckett and P. A. Racey, Eds.), Vol. 7, pp. 321-335. Academic Press, New York, 1975. Comte, J., Maisterrena, B., and Gautheron, D. C. Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria comparison with microsomes. Biochim. Biophys. Acta 419, 271-284 (1976). Darin-Bennett, A., and White, I. G. Influence of the cholesterol content of mammalian sperm on susceptibility to cold shock. Cryobiology 14, 466-470 (1977). DePierre, J. W., and Dallner, G. Structural aspects of the membrane of the endoplasmic reticulum. Biochim. Biophys. Acta 415, 411-172
(1975). 11. Folch, J., Lees, M., and Sloane-Staney. A simple
method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-507 (1957). 12. Fong, B. S., and Brown, J. C. Asymmetric distribution of phosphatidylethanolamine fatty acid chains in the membrane of vesicular stomatitis virus. Biochim. Biophys. Acta 510, 230-241 (1978). 13. Hinkovska, V., Dimitrov, G., and Koumanov, K. Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes. Znt. J. Biochem. 18, 1115-1121(1986). 14. Hinkovska-Galcheva, V., Peeva, D., Momchilova-Pankova, A., Pekova, D., and Koumanov, K. Phosphatidylcholine and phosphatidyletha-
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
AND
KOUMANOV
nolamine derivatives, membrane fluidity and changes in the lipolytic activity of ram spermatozoa plasma membranes during cryoconservation. Znt. J. Biochem., in press. Kahovkova, J., and Odavic, R. A simple method for analysis of phospholipids separated on thin layer chromatography. J. Chromatogr. 40, 9095 (1969). Kimelberg, H. K. Alterations in phospholipid dependent Na+-K+ ATPase activity due to lipid fluidity. Biochim. Biophys. Acta 413, 143-152 (1975). King, T. E. Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. In “Methods in Enzymology” (E. Grossman and K. Moldave, Eds.), Vol. 10, pp. 322-331. Academic Press, New York, 1967. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). Lunstra, D. D., Clegg, E. D., and Morre, D. J. Isolation of plasma membrane from porcine spermatozoa. Prep. Biochem. 4, 341-352 (1974). Masotti, L., Lenaz, G., Spisni, A., and Urry, D. W. Effect of phospholipids on the protein conformation in the inner mitochondrial membranes. Biochem. Biophys. Res. Commun. 56, 892-897 (1974). Michel, R. H., and Hawthorne, J. N. The site of diphosphoinositide synthesis in rat liver. Biothem. Biophys. Res. Commun. 21, 333-338 (1965). Momchilova, A. B., Pekova, D. H., Koumanov, K. S. Phospholipid composition modifications influence phospholipase A, acitivity in rat liver plasma membranes. Znt. I. Biochem. 18, 945952 (1986). Nordlund, J. R., Schmidt, C. F., and Dicken, S. N., and Thompson, T. E. Transbilayer distribution of phosphatidylethanolamine in large and small unilamellar vesicles. Biochemistry 20, 3237-3241 (1981). Sperry, W. M., and Webb, M. A revision of Shoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187, 97-106 (1950). Yanagimachi, R. The movement of golden hamster spermatozoa before, and after capacitation. J. Reprod. Fertil. 23, 193-l% (1970). Yanagimachi, R. Specificity of sperm egg interaction. In “Immunology of Gametes” (M. Eddin and M. H. Johnson, Eds.), pp. 255-295. Cambridge Univ. Press., London, 1977. Yanagimachi, R. Sperm-egg association in mammals. In “Current Topics in Developmental
SPERMAL PLASMA MEMBRANES PHOSPHOLIPIDS AND CRYOPRESERVATION Biology” (H. A. Moskova and A. Monray, Eds.), pp. 83-105. Academic Press, New York, 1978. 28. Yanagimachi, R. Mechanisms of fertilization in mammals. In “Fertilization and Embryonic Development in Vitro” (L. Mastrianm and J. D. Biggers, Eds.), pp. 81-155. Plenum, New York/London, 1981. 29. Zaneveld, L. J. D., Polakowski, K. L., and
75
Williams, W. L. A proteinase inhibitor of mammalian sperm acrosomes. Biol. Reprod. 9, 219225 (1973). 30. Zwaal, R. F. A., Roelofsen, B., Comfurius, P., and Van Deenen, L. L. L. Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases. Biochim. Biophys. Acta 406, 83-96 (1975).