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Loss of plasma membrane proteins of bull spermatozoa through the freezing -thawing process
ELSEVIER
LOSS OF PLASMA MEMBRANE PROTEINS OF BULL SPERMATOZOA THROUGH THE FREEZING -THAWING PROCESS M. Ollero, O. Besc0s, J. A. Cebri~n-Perez and T. MuiSo-Blanco Departamento de Bioquimica y Biologia Molecular y Celular Facultad de Veterinaria, Zaragoza, Spain Received for publication: July 24, 1996 Accepted: June 10, 1997 ABSTRACT The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 108 frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 °(3 with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozenthawed semen treated with Tween 20 showed that the higher amount of- extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process. © 1998 by ElsevierScience Inc.
Key words: spermatozoa, plasma membrane proteins, electrophoresis Acknowledgments This work was supported by grant DGICYT AGF-94-0614 and DGA 96 P/03. The authors thank C. Rinc6n and F. Quinfin (CENSYRA, Zaragoza) for providing semen samples, and J. Medina for his technical assistance. Theriogenology 49:547-555, 1998 © 1998 by Elsevier Science Inc.
0093-691)(/98/$19.00 P(I S0093-691X(98)00006-5
Theriogenology
548 INTRODUCTION
The biological and biophysical properties of sperm cell membranes, as well as other biological membranes, are ultimately determined by their molecular composition. Sperm membrane composition is of great interest because of its influence on spermoocyte membrane fusion events required for fertilization (1, 15, 31). The main characteristics of the ejaculated sperm surface are acquired in the epididymis (maturation process) and at ejaculation by the adsorption of specific components from the seminal plasma (2, 6, 18). One of the modifications that occurs in the epididymis is the acquisition, by sperm surface specific regions, of elements from the epididymal plasma (5, 12, 28). These elements are mainly glycoproteins, hence the increase of saccharide residues on the sperm surface during maturation (17, 27). Some of these glycoproteins will be masked by other elements adsorbed onto the surface from the seminal plasma. It is well known that adsorption of elements by the cell surface is a temperaturedependent process. In the case of sperm cells, adsorption would probably be more affected at freezing temperatures, since some rearrangement of the sperm plasma membrane takes place during freezing and thawing (16, 30). Thus, frozen spermatozoa would present a different qualitative adsorption of proteins on their surface compared to those maintained at room temperature. On the other hand, the strength and nature of the adsorption of all these elements to the cell varies, with some components being removed by simple washing techniques, others by treatment with hypertonic saline, and still others requiring detergent treatment (24, 26). The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility from artificial insemination is poorer than that from fresh semen in most species. Even using the best preservation techniques, post-thaw survival of the sperm population is approximately 50%. Most surviving spermatozoa have characteristics which distinguish them from spermatozoa before cryopreservation (29). Further developments in our understanding need more detailed studies of the sperm membrane functional organization. The aim of this work was to compare the protein composition of fresh and frozenthawed bull sperm plasma membrane surface. Experiments were also conducted to determine the effect of the removal of sperm surface protein on sperm motility and viability. MATERIALS AND METHODS Preparation of Cell Samples The experiments with frozen semen were carried out with spermatozoa from 4 proven fertile bulls. Semen was frozen in 0.25-ml straws. The suspension medium, commercially used for routine cryopreservation of bovine sperm cells, was composed of egg yolk, Triladyl (a commercial solution containing Tris, citric acid, fructose, streptomycin sulphate and glycerol -Stethaimer-Apotheke, D8300 Landshut/Bayern), and water (1:1:3).
Theriogenology
549
For each experiment, either fresh or frozen semen, approximately 2 x 108 cells, was used. For the frozen semen assays, 7 straws were thawed at 35°C for 21 sec. The samples were washed by sedimentation through a Percoll gradient (10). The resultant pellet was resuspended with 5 ml saline medium containing 140 mM NaCI, 10 mM glucose, 2.5 mM KCI and 20 mM Hepes (Sigma Chemical Co, St. Louis, MO, USA; 10) and centrifuged for 5 min at 700 x g at 20°C. The supernatant was removed and the pellet was resuspended up to 1 ml with saline medium and then incubated with different concentrations of Tween 20 for 15 min at 20°C. Then, the samples were centrifuged for 20 sec at 700 xg at 200(3. The supernatant was collected, and protein concentration and SDS-PAGE analyses were performed. The pellet containing the spermatozoa was resuspended with 1 ml of saline medium, after which motility and cell viability were determined. Evaluation of Semen Samples Progressive individual sperm cell motility of the semen was subjectively assessed by visual estimation using a television microscopy system (Nikon Alphaphot 2), which was maintained at 37°C (7). Two magnifications were used (x 100 and x 400), and the percentage of progressively motile spermatozoa was estimated at 10% increments. Semen motility evaluations were performed by the same person throughout the study. Cell viability was assessed (11) by fluorescent staining with carboxifluorescein diacetate and propidium iodide (Sigma). The cells were then examined under a Nikon fluorescence microscope, and the numbers of fluorescein-positive (membrane-intact) spermatozoa and propidium iodide-positive (membrane-damaged) spermatozoa per 100 cells were stimated and recorded. At least 200 cells were counted in duplicate for each sample. The results are expressed as the percentage of viable cells in each sample. Sperm concentration was calculated in duplicate using a Neubauer chamber (Marienfeld, Germany). Protein Concentration and SDS-PAGE Analysis Protein concentration was assessed using the Bradford method (3). The SDSPAGE analysis was carried out in a polyacrilamide gel gradient (5 to15%) according to the method described by Laemmli (14), using a protean II vertical slab gel electrophoresis apparatus (Bio Rad). The samples were diluted 4:1 with the sample buffer (20% glycerol, 5% SDS, 0.125 M Tris-HCI, pH 6.8, 5% 2-mercaptoethanol, 10 mM EDTA and 0.1% bromophenol blue; Serva) and heated for 5 rain at 95°C. Electrophoresis was carried out for 20 h at 70 V at room temperature. A mixture of prestained molecular weights ranging from 14 to 116 kDa (Sigma) was used as a standard. The gels were silver-stained for total protein content (19). Densitometric analysis of the stained gel was carried out using a laser light source LKB Ultro Scan XL with Gel Scan software.
Theriogenology
550 RESULTS AND DISCUSSION
Cryopreserved sperm fertility is poorer than that of fresh spermatozoa. The decrease in motility is not the only reason for this, since when equal numbers of motile spermatozoa are inseminated intracervically, the results with cryopreserved samples are still lower than those with fresh semen (29). Several observations support the view that the sperm membrane is altered by cryopreservation (9) and by the freezingthawing process (4, 13, 16, 23, 25). Furthermore, our previous studies using centrifugal counter current distribution in an aqueous two-phase system has shown that the partition behavior of frozen or coldshocked spermatozoa could be mimicked by the removal of sperm membrane proteins (8, 20, 21,22). Thus, the frozen-thawed sperm membrane surface appears to respond as if its proteins were removed. The findings of the current study indicate that the removal of sperm membrane proteins results in a decrease in sperm motility and viability. Table 1 shows the effect of incubation with the mild detergent Tween 20 on frozen-thawed bull spermatozoa. The results are shown as means ± SD of 4 samples. Correlation analysis of means was done using the StatView+Graphics Mac computer program. Sperm cell viability decreased as detergent concentration was increased (correlation coefficient 0.91). Cell motility was completely abolished at the 3 assayed detergent concentrations (0.01, 0.03 and 0.05%). The amount of extracted proteins increased from 0.72 mg to 2 mg with the detergent concentration, reaching 2 mg with 0.05% Tween 20 (correlation coefficient 0.887). However, SDS-PAGE analysis revealed the same bands in all cases (Figure 1). The same 12 bands were identifiable in the 0.01,0.03 and 0.05% Tween 20 extracted sample profiles. The migration patterns displayed by these polypeptides were indistinguishable in semen from selected bulls of different breeds (data not shown). Table 1. Effect of Tween 20 on frozen-thawed bull spermatozoa. Amount of extracted proteins and percentage of viability after 15 min incubation at 20°C. Results are expressed as means ± SD (n=4).
Tween 20 (%)
Extracted proteins (mg)
0 0.01 0.03 0.05
0.72+0.11 1.54±0.2 1.68±O.25 2.01±0.14
Viability (%) 52±6 26='-4.3 10±2 5±1.3
Theriogenology
551 1
2
3
kDa
11_6 66
36
2o
Figure 1.
Separation by SDS-PAGE of extracted polypeptides from frozen bull spermatozoa by incubation with 0.01% (Line 1), 0.03% (Line 2), 0.05% (Line 3) Tween 20. Each line was loaded with 20 pg of proteins.
To compare fresh and frozen semen, fresh bull spermatozoa from 4 different sires were incubated with 0.05% Tween 20. The obtained results from the 4 ejaculates, analyzed under identical conditions, are shown in Table 2. None of the samples showed motility after incubation with 0.05% Tween 20 for 15 min at 20°C. Likewise, cell viability decreased significantly (P<0.000t); a sample with an initial viability value of 59% dropped to only 8% after treatment with 0.05% Tween 20. The amount of extracted proteins was similar in the 4 fresh semen samples studied and was significantly different (P<0.0001) from the amount in the frozen semen samples. This difference was probably due to the composition of the cryoprotectant rather than to the amount in the sperm membrane. The presence of egg yolk proteins in the diluent, which bind to the sperm surface, must also be taken into account. These polypeptides are removed from the sperm surface together with those from the cell membrane during the incubation with the detergent. Table 2. Effect of 0.05% Tween 20 on 4 fresh bull sperm samples from 4 different sires. Amount of extracted proteins and percentage of viability after incubating for 15 minutes at 20°C Concen'cal~ Extracted Progressive Post-treatment Initial sperm (sperm/pl) proteins sperm motility motility viability x t 06 (mg) (%) (%) (%) 1.5 0.25 80 0 67 1.1 0.23 65 0 63 1.4 0.29 35 0 61 0.88 0.35 60 0 59
Post-treatment viability (%) 13 12 10 8
Theriogenology
552
Comparative SDS-PAGE analyses confirm the suggestion above, and show changes in the patterns of sperm membrane polypeptides from fresh and frozen bull spermatozoa (Figure 2). There was a detectable loss of 4 bands in samples from frozen semen. The prominent 3, 6, 7 and 8 polypeptides of the fresh sample profile (Line 1), of 35, 68, 100 and 245 kDa, respectively, were not detected in the frozen sample pattern (Line 2). The appearance of the other different bands in the frozen sample profile came from the egg yolk polypeptides adsorbed onto the sperm surface (Line 3). However, some other bands seen in frozen spermatozoa (approximately 55 and 95 kDa) do not come from the egg yolk lipoproteins. Although a modification of some proteins due to interaction with egg yolk is possible, this is not likely to be the reason for the appearance of different bands. The higher amount of extracted proteins from frozen samples would indicate that other proteins are removed by the detergent incubation. Moreover, the band around 50 kDa became much darker in frozen than in fresh sperm samples. This increase in extracted proteins from frozen-thawed spermatozoa could be interpreted as resulting from cryopreservation injury, which would make the sperm membrane more sensitive to the detergent. 1
2
3
kDa
35
Figure 2.
Polypeptide patterns in 0.05% Tween 20-extracted samples. Separation by SDS-PAGE of fresh (Line 1) and frozen (Line 2) semen sample polypeptides, and egg yolk polypeptides (Line 3).Each line was loaded with 20 pg of proteins.
Densitometric analysis of the stained gel patterns allows for automatic comparison among the separated bands. The electrophoretic densitometric tracing of fresh and frozen sperm-extracted proteins, and egg yolk proteins is shown in Figure 3. The 4 undetectable bands in the frozen sperm sample profile can easily be identified as major components of the fresh sperm membrane. These polypeptides must have been lost as a result of the damage induced by the freezing-thawing process. Other bands
Theriogenology
553
which were only shown in the frozen sperm-extracted sample profile are clearly identified as egg yolk components.
I 35 Figure 3.
I I 68 100
I 245
I kDa
Densitometric tracing of SDS-PAGE of fresh (1) and frozen (2) semen sample polypeptides, and of egg yolk polypeptides (3). kDa = kilodaltons
The difference between the fresh and frozen sperm membrane composition shown in this study is, among other factors (oxidation, temperature, capacitation, acrosome status) a molecular basis for the differences in fertility. The identification of sperm plasma membrane proteins which are involved in the fertilization process might aid in the development of subsequent inexpensive biochemical tests designed to assess the fertility of semen donors in commercial AI operations. This identification could also help in the formulation of better protocols for the cryopreservation of semen.
Theriogenology
554 REFERENCES 1. 2. 3. 4. 5.
6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Ahuja K K. Fertilization: changes in sperm and zona carbohydrates. In: Alexander N J, Griffin D, Spieler J M, Waites G M H (eds), Gamete interaction: Prospects for inmunocontraception. New York: Wiley, 1990; 239-258. Amann R P, Hammerstedt R H, Veeramachaneni D N R. The epididymis and sperm maturation - a perspective. Reprod Fertil Dev 1993; 5: 361-381. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Analyt Biochem 1976; 72: 248-254. Buhr M M, Curtis E F, Kakuda N S. Composition and behaviour of head membrane lipids of fresh and cryopreserved boar sperm. Cryobiology 1994; 31 : 224-238. Dacheux J L, Chevrier C, Dacheux F, Jeulin C, Gatti J L, Pariset C, Paquignon M. Sperm biochemical changes during epididymal maturation. In: Alexander N J, Griffin D, Spieler J M, Waites G M H (eds), Gamete Interaction. New York: Wiley and Sons, 1990; 111-128. Desnoyers L, Manjunath P. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J Biol Chem 1992; 267:10149-10155. Evans G, Maxwell W M C. Manejo y valoracibn del semen. Inseminacibn artificial de ovejas y cabras. Zaragoza: Acribia, 1989; 95-107. Garcia-Lbpez N, Ollero M, Cebri&n-P~rez M, MuiSo-Blanco T. Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems. J Chrom 1996; 680: 137-143. Hammerstedt R H, Graham J K. Cryopreservation of poultry sperm: the enigma of glycerol. Cryobiology 1992; 29: 26-38. Harrison R A P, Dott H M, Foster G C. Bovine serum albumin, sperm motility, and the "dilution effect". J exp Zoo11982; 222: 81-88. Harrison R A P, Vickers S E. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J Reprod Ferti11990; 88: 343-352. Hegde U C, Khole V, Premachandran S. Immunochemical localization and characterization of proteins from mouse cauda epididymis using human sperm-specific monoclonal antibody. Hum Reprod 1991;6: 259-262. Holt W V, Head M F, North R D. Freeze-induced membrane damage in ram spermatozoa is manifested after thawing -Observations with experimental cryomicroscopy. Biol Reprod 1992; 46: 1086-1094. Laemmli, U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685. Langlais J, Roberts K D A. A molecular model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 1985; 16: 183-224. Leeuw de F E, Chen H-C, Colenbrander B, Verkleij A J. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 1990; 27: 171-183. Magargee S F, Kunze E, Hammersted R H. Changes in lectin-binding features of ram sperm surfaces associated with epididymal maturation and ejaculation. Biol Reprod 1988; 38: 667-685. Metz K W, Berger T, Clegg E D. Adsorption of seminal plasma proteins by boar spermatozoa. Theriogenology 1990; 34: 691-700.
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19. Morrissey J H. Silver stain for proteins in polyacrilamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981 ; 117: 307-310. 20. Ollero M, MuiSo-Blanco T, Lbpez-P~rez M J, Cebri~.n-P6rez J A. Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system. Effect of different cryoprotectants. J Chrom 1996; 157-164. 21. Pascual M L, Mui5o-Blanco T, Cebri~.n-P~rez J A, L6pez-P~rez M J. Sperm cell heterogeneity revealed by centrifugal counter current distribution in an aqueous two-phase system. J Chrom 1993; 617: 51-57. 22. Pascual M L, Mui~o-Blanco T, Cebri~.n-P~rez J A, Lbpez-P~rez M. Acquisition of viable-like surface properties of sperm cells by adsorption of seminal plasma proteins revealed by centrifugal countercurrent distribution. Biol Cell 1994; 82: 75-78. 23. Robertson L, Watson P F. The effect of egg yolk on the control of intracellular calcium in ram spermatozoa cooled and stored at 5°C. Anim Reprod Sci 1987; 15: 177-187. 24. Russell L D, Montag B, Hunt W, Peterson R N. Properties of boar sperm plasma membranes (PM): Proteins released by washing and differential solubility in salts, detergents, and sensitivity to surface radiolabelling. Gamete Res 1985; 11 : 237-252. 25. Simpson A M, White I G. Effect of cold shock and coling rate on calcium uptake of ram spermatozoa. Anim Reprod Sci 1986; 12: 131-143. 26. Tanphaichitr N, Zheng Y S, Kates M, Abdullah N. Cholesterol and phospholipid levels of washed and Percoll gradient centrifuged mouse sperm: presence of lipids possessing inhibitory effects on sperm motility. Mol Reprod Dev 1996; 43: 187-195. 27. Tulsiani D R P, Skudlarek M D, Holland M K, Orgebincrist M C. Glycosylation of rat sperm plasma membrane during epididymal maturation. Biol Reprod 1993; 48: 417-428. 28. Vreeburg J T M, Holland M, Orgebin-Crist M C. Binding of epididymal proteins to rat spermatozoa in vivo. Biol Reprod 1992; 47: 588-597. 29. Watson P F. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev 1995; 7: 871-891. 30. Watson P F, Morris G J. Cold shock injury in animal cells. Proc Soc Exp Biol 1987; 41 : 311-340. 31. Yanagimachi R. Mammalian fertilization. In Knobil E, Neill JD (eds),The Physiology of Reproduction. New York: Raven Press, 1994; 245-254.
LOSS OF PLASMA MEMBRANE PROTEINS OF BULL SPERMATOZOA THROUGH THE FREEZING -THAWING PROCESS M. Ollero, O. Besc0s, J. A. Cebri~n-Perez and T. MuiSo-Blanco Departamento de Bioquimica y Biologia Molecular y Celular Facultad de Veterinaria, Zaragoza, Spain Received for publication: July 24, 1996 Accepted: June 10, 1997 ABSTRACT The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 108 frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 °(3 with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozenthawed semen treated with Tween 20 showed that the higher amount of- extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process. © 1998 by ElsevierScience Inc.
Key words: spermatozoa, plasma membrane proteins, electrophoresis Acknowledgments This work was supported by grant DGICYT AGF-94-0614 and DGA 96 P/03. The authors thank C. Rinc6n and F. Quinfin (CENSYRA, Zaragoza) for providing semen samples, and J. Medina for his technical assistance. Theriogenology 49:547-555, 1998 © 1998 by Elsevier Science Inc.
0093-691)(/98/$19.00 P(I S0093-691X(98)00006-5
Theriogenology
548 INTRODUCTION
The biological and biophysical properties of sperm cell membranes, as well as other biological membranes, are ultimately determined by their molecular composition. Sperm membrane composition is of great interest because of its influence on spermoocyte membrane fusion events required for fertilization (1, 15, 31). The main characteristics of the ejaculated sperm surface are acquired in the epididymis (maturation process) and at ejaculation by the adsorption of specific components from the seminal plasma (2, 6, 18). One of the modifications that occurs in the epididymis is the acquisition, by sperm surface specific regions, of elements from the epididymal plasma (5, 12, 28). These elements are mainly glycoproteins, hence the increase of saccharide residues on the sperm surface during maturation (17, 27). Some of these glycoproteins will be masked by other elements adsorbed onto the surface from the seminal plasma. It is well known that adsorption of elements by the cell surface is a temperaturedependent process. In the case of sperm cells, adsorption would probably be more affected at freezing temperatures, since some rearrangement of the sperm plasma membrane takes place during freezing and thawing (16, 30). Thus, frozen spermatozoa would present a different qualitative adsorption of proteins on their surface compared to those maintained at room temperature. On the other hand, the strength and nature of the adsorption of all these elements to the cell varies, with some components being removed by simple washing techniques, others by treatment with hypertonic saline, and still others requiring detergent treatment (24, 26). The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility from artificial insemination is poorer than that from fresh semen in most species. Even using the best preservation techniques, post-thaw survival of the sperm population is approximately 50%. Most surviving spermatozoa have characteristics which distinguish them from spermatozoa before cryopreservation (29). Further developments in our understanding need more detailed studies of the sperm membrane functional organization. The aim of this work was to compare the protein composition of fresh and frozenthawed bull sperm plasma membrane surface. Experiments were also conducted to determine the effect of the removal of sperm surface protein on sperm motility and viability. MATERIALS AND METHODS Preparation of Cell Samples The experiments with frozen semen were carried out with spermatozoa from 4 proven fertile bulls. Semen was frozen in 0.25-ml straws. The suspension medium, commercially used for routine cryopreservation of bovine sperm cells, was composed of egg yolk, Triladyl (a commercial solution containing Tris, citric acid, fructose, streptomycin sulphate and glycerol -Stethaimer-Apotheke, D8300 Landshut/Bayern), and water (1:1:3).
Theriogenology
549
For each experiment, either fresh or frozen semen, approximately 2 x 108 cells, was used. For the frozen semen assays, 7 straws were thawed at 35°C for 21 sec. The samples were washed by sedimentation through a Percoll gradient (10). The resultant pellet was resuspended with 5 ml saline medium containing 140 mM NaCI, 10 mM glucose, 2.5 mM KCI and 20 mM Hepes (Sigma Chemical Co, St. Louis, MO, USA; 10) and centrifuged for 5 min at 700 x g at 20°C. The supernatant was removed and the pellet was resuspended up to 1 ml with saline medium and then incubated with different concentrations of Tween 20 for 15 min at 20°C. Then, the samples were centrifuged for 20 sec at 700 xg at 200(3. The supernatant was collected, and protein concentration and SDS-PAGE analyses were performed. The pellet containing the spermatozoa was resuspended with 1 ml of saline medium, after which motility and cell viability were determined. Evaluation of Semen Samples Progressive individual sperm cell motility of the semen was subjectively assessed by visual estimation using a television microscopy system (Nikon Alphaphot 2), which was maintained at 37°C (7). Two magnifications were used (x 100 and x 400), and the percentage of progressively motile spermatozoa was estimated at 10% increments. Semen motility evaluations were performed by the same person throughout the study. Cell viability was assessed (11) by fluorescent staining with carboxifluorescein diacetate and propidium iodide (Sigma). The cells were then examined under a Nikon fluorescence microscope, and the numbers of fluorescein-positive (membrane-intact) spermatozoa and propidium iodide-positive (membrane-damaged) spermatozoa per 100 cells were stimated and recorded. At least 200 cells were counted in duplicate for each sample. The results are expressed as the percentage of viable cells in each sample. Sperm concentration was calculated in duplicate using a Neubauer chamber (Marienfeld, Germany). Protein Concentration and SDS-PAGE Analysis Protein concentration was assessed using the Bradford method (3). The SDSPAGE analysis was carried out in a polyacrilamide gel gradient (5 to15%) according to the method described by Laemmli (14), using a protean II vertical slab gel electrophoresis apparatus (Bio Rad). The samples were diluted 4:1 with the sample buffer (20% glycerol, 5% SDS, 0.125 M Tris-HCI, pH 6.8, 5% 2-mercaptoethanol, 10 mM EDTA and 0.1% bromophenol blue; Serva) and heated for 5 rain at 95°C. Electrophoresis was carried out for 20 h at 70 V at room temperature. A mixture of prestained molecular weights ranging from 14 to 116 kDa (Sigma) was used as a standard. The gels were silver-stained for total protein content (19). Densitometric analysis of the stained gel was carried out using a laser light source LKB Ultro Scan XL with Gel Scan software.
Theriogenology
550 RESULTS AND DISCUSSION
Cryopreserved sperm fertility is poorer than that of fresh spermatozoa. The decrease in motility is not the only reason for this, since when equal numbers of motile spermatozoa are inseminated intracervically, the results with cryopreserved samples are still lower than those with fresh semen (29). Several observations support the view that the sperm membrane is altered by cryopreservation (9) and by the freezingthawing process (4, 13, 16, 23, 25). Furthermore, our previous studies using centrifugal counter current distribution in an aqueous two-phase system has shown that the partition behavior of frozen or coldshocked spermatozoa could be mimicked by the removal of sperm membrane proteins (8, 20, 21,22). Thus, the frozen-thawed sperm membrane surface appears to respond as if its proteins were removed. The findings of the current study indicate that the removal of sperm membrane proteins results in a decrease in sperm motility and viability. Table 1 shows the effect of incubation with the mild detergent Tween 20 on frozen-thawed bull spermatozoa. The results are shown as means ± SD of 4 samples. Correlation analysis of means was done using the StatView+Graphics Mac computer program. Sperm cell viability decreased as detergent concentration was increased (correlation coefficient 0.91). Cell motility was completely abolished at the 3 assayed detergent concentrations (0.01, 0.03 and 0.05%). The amount of extracted proteins increased from 0.72 mg to 2 mg with the detergent concentration, reaching 2 mg with 0.05% Tween 20 (correlation coefficient 0.887). However, SDS-PAGE analysis revealed the same bands in all cases (Figure 1). The same 12 bands were identifiable in the 0.01,0.03 and 0.05% Tween 20 extracted sample profiles. The migration patterns displayed by these polypeptides were indistinguishable in semen from selected bulls of different breeds (data not shown). Table 1. Effect of Tween 20 on frozen-thawed bull spermatozoa. Amount of extracted proteins and percentage of viability after 15 min incubation at 20°C. Results are expressed as means ± SD (n=4).
Tween 20 (%)
Extracted proteins (mg)
0 0.01 0.03 0.05
0.72+0.11 1.54±0.2 1.68±O.25 2.01±0.14
Viability (%) 52±6 26='-4.3 10±2 5±1.3
Theriogenology
551 1
2
3
kDa
11_6 66
36
2o
Figure 1.
Separation by SDS-PAGE of extracted polypeptides from frozen bull spermatozoa by incubation with 0.01% (Line 1), 0.03% (Line 2), 0.05% (Line 3) Tween 20. Each line was loaded with 20 pg of proteins.
To compare fresh and frozen semen, fresh bull spermatozoa from 4 different sires were incubated with 0.05% Tween 20. The obtained results from the 4 ejaculates, analyzed under identical conditions, are shown in Table 2. None of the samples showed motility after incubation with 0.05% Tween 20 for 15 min at 20°C. Likewise, cell viability decreased significantly (P<0.000t); a sample with an initial viability value of 59% dropped to only 8% after treatment with 0.05% Tween 20. The amount of extracted proteins was similar in the 4 fresh semen samples studied and was significantly different (P<0.0001) from the amount in the frozen semen samples. This difference was probably due to the composition of the cryoprotectant rather than to the amount in the sperm membrane. The presence of egg yolk proteins in the diluent, which bind to the sperm surface, must also be taken into account. These polypeptides are removed from the sperm surface together with those from the cell membrane during the incubation with the detergent. Table 2. Effect of 0.05% Tween 20 on 4 fresh bull sperm samples from 4 different sires. Amount of extracted proteins and percentage of viability after incubating for 15 minutes at 20°C Concen'cal~ Extracted Progressive Post-treatment Initial sperm (sperm/pl) proteins sperm motility motility viability x t 06 (mg) (%) (%) (%) 1.5 0.25 80 0 67 1.1 0.23 65 0 63 1.4 0.29 35 0 61 0.88 0.35 60 0 59
Post-treatment viability (%) 13 12 10 8
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Comparative SDS-PAGE analyses confirm the suggestion above, and show changes in the patterns of sperm membrane polypeptides from fresh and frozen bull spermatozoa (Figure 2). There was a detectable loss of 4 bands in samples from frozen semen. The prominent 3, 6, 7 and 8 polypeptides of the fresh sample profile (Line 1), of 35, 68, 100 and 245 kDa, respectively, were not detected in the frozen sample pattern (Line 2). The appearance of the other different bands in the frozen sample profile came from the egg yolk polypeptides adsorbed onto the sperm surface (Line 3). However, some other bands seen in frozen spermatozoa (approximately 55 and 95 kDa) do not come from the egg yolk lipoproteins. Although a modification of some proteins due to interaction with egg yolk is possible, this is not likely to be the reason for the appearance of different bands. The higher amount of extracted proteins from frozen samples would indicate that other proteins are removed by the detergent incubation. Moreover, the band around 50 kDa became much darker in frozen than in fresh sperm samples. This increase in extracted proteins from frozen-thawed spermatozoa could be interpreted as resulting from cryopreservation injury, which would make the sperm membrane more sensitive to the detergent. 1
2
3
kDa
35
Figure 2.
Polypeptide patterns in 0.05% Tween 20-extracted samples. Separation by SDS-PAGE of fresh (Line 1) and frozen (Line 2) semen sample polypeptides, and egg yolk polypeptides (Line 3).Each line was loaded with 20 pg of proteins.
Densitometric analysis of the stained gel patterns allows for automatic comparison among the separated bands. The electrophoretic densitometric tracing of fresh and frozen sperm-extracted proteins, and egg yolk proteins is shown in Figure 3. The 4 undetectable bands in the frozen sperm sample profile can easily be identified as major components of the fresh sperm membrane. These polypeptides must have been lost as a result of the damage induced by the freezing-thawing process. Other bands
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which were only shown in the frozen sperm-extracted sample profile are clearly identified as egg yolk components.
I 35 Figure 3.
I I 68 100
I 245
I kDa
Densitometric tracing of SDS-PAGE of fresh (1) and frozen (2) semen sample polypeptides, and of egg yolk polypeptides (3). kDa = kilodaltons
The difference between the fresh and frozen sperm membrane composition shown in this study is, among other factors (oxidation, temperature, capacitation, acrosome status) a molecular basis for the differences in fertility. The identification of sperm plasma membrane proteins which are involved in the fertilization process might aid in the development of subsequent inexpensive biochemical tests designed to assess the fertility of semen donors in commercial AI operations. This identification could also help in the formulation of better protocols for the cryopreservation of semen.
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554 REFERENCES 1. 2. 3. 4. 5.
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