ALBUMIN IS REQUIRED FOR THE GUINEA PIG SPERM CAPACITATION BUT IS NOT ESSENTIAL FOR ACROSOME REACTION AND FUSION WITH EGGS
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A. BHATTACHARYYA
The importance of serum albumin in supporting guinea pig sperm capacitation, acrosome reaction, and fusion with eggs in vitro was studied by incubating the spermatozoa in albumin-free medium containing different synthetic polymers. Serum albumin was found to be an obligatory component in the incubating medium for the capacitation of guinea pig spermatozoa. Albumin in the medium is not essential for the acrosome reaction and fusion with the eggs, but these phenomena take place most efficiently in presence of albumin. Key Words: Sperm; Capacitation; Guinea pig; Macromolecules; Factors.
INTRODUCTION In acquiring the ability to fuse with egg plasma membrane, mammalian spermatozoa must undergo the process of capacitation and acrosome reaction, in vivo, in the presence of follicular fluid [ 19, 201. Biochemical and immunochemical studies have revealed that the follicular fluid of most mammals contains the blood plasma proteins and that serum albumin is the major protein in this fluid [ 6 ] . Under in vitro conditions, mammalian sperm fertilization appears to be a complex process [16, 201 and the components of the medium definitely influence the process. The importance of protein, specifically serum albumin, in the fertilization process of hamsters [17], rabbits [15], rats [4], dogs [13], guinea pigs [lo], mice [8], and humans [22] is well documented. However, its exact site of action in the different components of fertilization is unclear and appears to vary, depending on the species studied. The guinea pig is an ideal model for evaluating the effects of albumin on the capacitation, acrosome reaction, and fusion with the eggs because the processes can be differentiated easily [21].
MATERIALS AND METHODS Media. The standard (regular) mT medium used in the present study had the following composition: 108.76 mM NaCl, 2.7 mM KCI, 2.0 mM CaCI,, 0.49 mM MgCl,, 6H,O, 25.07 mM NaHCO,, 5.56
From the Department of Anatomy and Reproductive Biology, University of Hawaii, School of Medicine, Honolulu, HI 96822. Address correspondence to Dr. Amitabha Bhattacharyya, Reproductive Biology Research Section, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India. ARCHIVES OF ANDROLOGY 28:235-239 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
235
236
A. Bhattacharyya
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mM D-glucose, 10 mM Na-lactate; 1 mM Na-pyruvate, and 3 mg/mL bovine serum albumin (Fr.V fatty acid poor). The osmolality and pH of the medium were 308 mosm and 7.8 (gas phase, pure air), respectively. The Ca2’ -free mT was prepared without CaCl, from the regular mT medium. The osrnolality of the Ca2’-free mT was kept constant by adjusting the concentration of NaCl. Albumin-free test mT medium were prepared by replacing bovine serum albumin in the regular or Ca2+-free medium with 3 mg/mL of one of the macromolecules purchased from Sigma Chemical Co. (St. Louis, MO): polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), dextran, Ficoll, and methocel. Sperm Cupacifation. Spermatozoa were collected from one cauda epididymis and vas deferens of mature male guinea pig (body weight 600 g) and were suspended in the 12 mL saline (0.9% NaCl solution). The sperm suspension was centrifuged at 280g for 3-4 min and the pellet was suspended in 2 mL saline. The sperm concentration at this stage was 4-5 x lo7 cells/mL. An aliquot (0.5 mL) of this suspension was mixed with 3 mL Ca2’-free test mT media which had been previously placed under mineral oil (Squibb and Sons, Princeton, NJ) in a plastic petri dish (10 X 35 mm; Falcon Plastics, Oxnard, CA). The concentration of spermatozoa at this stage was 4-6 x 106 cells/mL. After incubation for 16-18 h at 37°C (gas phase, pure air), the sperm suspensions were diluted to 12 mL in a centrifuge tube and centrifuged at 280g for 3-4 min. After discardation of the supernatent, the sperm pellet was diluted at 250 pL saline. An aliquot of this sperm suspension was mixed with 0.3 mL regular mT medium for acrosome reaction. The acrosome reaction of spermatozoon occurs only if they capacitate properly. The percentage of acrosome-related spermatozoa in the entire population was calculated as described by Murphy and Yanagimachi [ 141. Sperm Acrosome Reaction. For the acrosome reaction, capacitated guinea pig spermatozoa were prepared by incubating them at Ca2’ -free mT medium containing a low concentration (85 pg/mL) of lysolecithin (Avanti Biochemical, Birmingham, AL) at 37°C for 1-1.5 h [7]. Following incubation, the sperm suspension containing capacitated spermatozoa was diluted to 12 mL in a centrifuged tube and was centrifuged at 280g for 3-4 min. The pellet was resuspended in 0.25 mL saline. An aliquot (10 pL) of this sperm suspension was mixed with 0.3 mL of regular and albumin-free test mT medium that had been previously placed under mineral oil at 37°C in gas phase of pure air. The sperm concentration at this stage was 1.5-2 x lo6 cells/mL. After incubation for 15-20 min the overall percentage of motile spermatozoa and quality of motility were visually estimated and the acrosome-reacted spermatozoa were examined by phase contrast microscopy ( x 450) [ 141. Sperm-Egg Fusion. To determine the ability of fusion of spermatozoa with the zona-free eggs, fully acrosome-reacted guinea pig spermatozoa were prepared by incubating the same in regular mT medium following capacitation in Ca” -free lysolecithin containing capacitation medium (see above). Insemination was performed by introducing washed acrosome-reacted sperm suspension in 0.3 mL of regular or test mT medium containing zona-free guinea pig eggs. The zona-free guinea pig eggs were prepared following the method of Bhattacharyya and Yanagimachi [3]. After incubation of the eggs with spermatozoa (sperm concentration was 1 X lo5 cells/mL) for 1-1.5 h at 37°C under gas phase, pure air, the eggs were removed from the medium, cleaned, and mounted in between a slide and cover slip. The fusion was evaluated by phase contrast microscopy [ 181. An egg was considered fertilized when at least one decondensed sperm head was visible within its cytoplasm [3].
RESULTS When incubated continuously in Ca2‘-free mT medium (which contained 0.3% albumin) for 16-18 h, about 65-75 % spermatozoa underwent “synthronous” acrosome reaction after transfer into Ca2’ -containing medium (Table 1). Among the acrosome-reacted spermatozoa, many were vigorously motile (hyperactivated type). As shown in Table 1, guinea pig sper-
Capacitation of Guinea Pig Sperm
237
TABLE 1 Effect of Different Polymers on the Capacitation of Guinea Pig Spermatozoa ~
~
~
~
~
Sperm 15-20 After in Regular mT
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Media Contents (0.3%) Albumin PVP PVA Methocel Dextran Ficol None
% Motile Sperm After Incubation
85 51 65 36 60 78 21
5.6 12.8 10.4 18.5 12.7 16.8 f 19.7
f f f f 2 f
Quality
% Acrosome Reacted
f 5.2
3
f 15.9 f 15.9 17.7 f 1.5 k 9.9 f 30.1
1 1 1 1 1
69.7 f 3.8 0 0 0 0 0
1
0
% Motile
82 43 66 54 52 52 37
*
Nore. Values are means f SD of six experiments using spermatozoa from different males. Washed spermatozoa were incubated in 3 mL Ca2+-free different test mT medium for 16-18 h. After washing with saline, 10 pl of sperm suspension (in saline) was added to 0.3 mL regular mT medium.
matozoa are unable to undergo acrosome reaction when the capacitating medium contains synthetic macromolecules instead of albumin. This is due to inhibition of capacitation, since acrosome reaction occurs only in presence of Ca2+,after a successful completion of capacitation. Guinea pig sperm capacitation time could be shortened by using lysolecithin in the medium [7], but that type of experiment was omitted by this study, because spermatozoa becomes immotile immediately in presence of lysolecithin when the medium does not contain albumin. When spermatozoa were preincubated for 1.5 h in Ca2+-freemedium (in presence of albumin) containing lysolecithin and then exposed to Ca2'-containing regular (with albumin) and test (without albumin) mT medium many spermatozoa underwent acrosome reaction (Table 2). However, albumin containing mT medium was the best in supporting the acrosome reaction of guinea pig spermatozoa, although most of the spermatozoa in the test medium show progressive-type motility. Hyperactivate-type motility occurs only in albumin-containing medium. The spermatozoa that had undergone the acrosome reaction following lysolecithin pretreatment were capable of fertilizing the zona-free eggs regardless of whether albumin was present in the medium (Table 3). However, the albumin-containing mT medium was most effective in supporting the fusion process.
DISCUSSION Our results show that albumin is an obligatory component for the guinea pig sperm capacitation, confirming the results of earlier studies [7, 101. The hyperactivated type of motility and the acrosome-reacted spermatozoa was observed only in medium containing albumin (Table l), suggesting that the action of albumin in enhancing the sperm capacitation involves more than a macromolecular effect. According to Barros et al. [2], guinea pig spermatozoa were able to undergo capacitation in a simple medium without serum albumin. This may be due to a slow increase of intracellular Ca" during the incubation process or the pH of the medium used [lo]. The mechanisms whereby albumin can promote the capacitation of spermatozoa are un-
A. Bhattacharyya
238
TABLE 2 Effect of Different Polymers on the Acrosome Reaction of Guinea Pig Spermatozoa
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Sperm 15-20 min After in Regular mT Media Contents (0.3%)
% Motile
Albumin PVP PVA Methocel Dextran Ficol None
82 58 77 71 52 53 58
f 5.2 f 11.3 f 4.2 f 5.8 f 12.9 k 13.7 f 11.3
Quality
3 2 2 2 2 1 1
% Acrosome Reacted
74.8 50.5 64.0 59.6 39.4 34.3 46.4
f 4.5 & 9.8 f 6.2
f 6.3
f 14.3 f 13.8 f 14.8
Note. Values are means f SD of six experiments using spermatozoa from different males. Spermatozoa were incubated in the lysolecithin containing CaZ+-free mT medium (containing 0.3 albumin) for capacitation. After 1- 1.5 h of incubation spermatozoa were washed with saline twice and incubated in Ca2+ containing various test mT medium for acrosome reaction.
clear, although it is hypothesized that albumin alters the sperm plasma membrane lipid composition through lipid exchange or hydrolysis [5, 91. Furthermore, lipid removed from the outer plasma membrane of spermatozoa may bind with extracellular albumin, since it has unfilled binding sites for fatty acids [11]. It would appear that sperm capacitation involves, in part, the removal of an inhibitory factor from the sperm surface [ 191. It is also possible that the addition of serum albumin to guinea pig spermatozoa incubated in medium could contribute to sperm capacitation by removing such an inhibitory factor from the cells. The importance of albumin for acrosome reaction in mouse [8] and hamster [ l , 121 has been documented. Unlike mouse and hamster spermatozoa, guinea pig spermatozoa are able to acrosome react and fertilize the zona-free egg in the absence of albumin or other synthetic macromolecules. Albumin-free media supporting the membrane-fusion events (both acrosome reaction and fusion with eggs) could be explained by the fact that once the spermatozoa capacitate properly, the controlling mechanism by which Ca2+ enters the spermatozoa is activated. The intimate relationship between membrane fusion and the rise of intracellular Ca2+ is TABLE 3 Effect of Different Polymers on the Sperm-Egg Fusion Media Contents (0.3%) Albumin PVP PVA Methocel Dextran Ficol None
Number of Eggs Inseminated
Number (%) of Eggs Fertilized
26 27 28 19 24 28 20
25 (96) 14 (51) 15 (53) 9 (47) 13 (54) 17 (61) 11 (55)
Mean f SD of Decondensed Sperm HeadIFertilized Egg
7.9 4.1 3.6 2.9 4.6 5.0 4.0
k 4.0 f 1.8 f 1.0
f 1.9 f 1.6 f 2.5 & 1.7
Nore. Results are means f SD of three separate experiments using different males. Acrosome-reacted guinea pig spermatozoa were washed with saline twice and incubated with zona-free guinea pig eggs 1-1.5 h in various test media for insemination.
Capacitation of Guinea Pig Sperm
239
well documented [ 191. However, for sperm-egg fusion, synthetic macromolecules can mimic some, but not all, of the actions of albumin. Acknowledgments. This study was supported by the Rockefeller Foundation, USA. I a m grateful to R. Yanagimachi, University of Hawaii School of Medicine, USA, for his valuable guidance during the study. I also thank A. Pakrashi, Indian Institute of Chemical Biology, India, for her encouragement in preparation of the manuscript.
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REFERENCES 1. Andrews JC, Bavister BD (1989):Hamster zone Pellucidae cannot induce physiological acrosome reactions in chemically capacitated hamster spermatozoa in the absence of albumin. Biol Reprod 40:117-122 2. Barros C, Berrios M, Herrera E (1973):Capacitation in vitro of guinea pig spermatozoa in saline solution. J Reprod Fertil 34:547-549 3. Bhattacharyya A, Yanagimachi R (1988):Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer. Gamete Res 19:123-129 4. Davis BK (1976):Influence of serum albumin on the fertilizing ability in vitro of rat spermatozoa. Proc SOCExp Biol Med 15 1:240-243 5. Davis BK, Byrne R, Bedigian K (1979):Studies on the mechanism of capacitation: albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm. Proc Natl Acad Sci USA 668:257-266 6. Edwards RG (1974):Follicular fluid. J Reprod Fertil 37:189-219 7. Fleming AD, Yanagimachi R (1981):Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acromsome reaction. Gamete Res 4:253-273 8. Fraser LR (1985):Albumin is required to support the acrosome reaction but not capacitation in mouse spermatozoa in vitro. J Reprod Fertil 74:185-196 9. Go KJ, Wolf DP (1985):Albumin-mediated changes in sperm sterol content during capacitation. Biol Reprod 32:145-153 10. Hyne RV, Garbers DL (1981):Requirement of serum factors for capacitation and the acrosome reaction of guinea pig spermatozoa in buffered medium below pH 7.8.Biol Reprod 24:257-266 11. Langlais J, Roberts KD (1985):A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 12:185-224 12. Lui CW, Meizel S (1977):Biochemical studies of the in vitro acrosome reaction inducing activity of bovine serum albumin. Differentiation 9:59-66 13. Mahi CA, Yanagimachi R (1978):Capacitation, acrosome reaction and egg penetration by canine spermatozoa in a simple defined medium. Gamete Res 1:IOl-109 14. Murphy SJ, Yanagimachi R (1984):The pH dependence of motility and the acrosome reaction of guinea pig spermatozoa. Gamete Res 10:1-8 15. Oliphant G (1976):Removal of sperm bound seminal plasma component as a prerequisite to induction of rabbit sperm acrosome reaction. Fertil Steril 27:28-38 16. Rogers BJ (1978):Mammalian sperm capacitation and fertilization in vitro: a critique of methodology. Gamete Res 1 : 165-223 17. Yanagimachi R (1970):In vitro capacitation of golden hamster spermatozoa by homologous and heterologous blood sera. Biol Reprod 3:147-153 18. Yanagimachi R (1972):Fertilization of guinea pig eggs in vitro. J Reprod Fertil 28:477-480 19. Yanagimachi R (1981):Mechanism of fertilization in mammals. In: Fertilization and Embryonic Development in Vitro, Mastoroianni L, Biggers JD (Eds.). New York: Plenum, pp 81-82 20. Yanagimachi R (1988): Mammalian fertilization. In: Physiology of reproduction, Vol. 1, Knobil E, Neil JD, Ewing LL, Markert CL, Greenwald GS, Pfaff DW (Eds). New York: Raven, pp 135-185 21. Yanagimachi R, Usui N (1974):Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp Cell Res 89:161-174 22. Yanagimachi R, Yanagimachi H, Rogers BJ (1976): The use of zona-free animal ova as a test system for assessment of the fertilizing capacity of human spermatozoa. Biol Reprod 15:471-476
Syst Biol Reprod Med Downloaded from informahealthcare.com by Chulalongkorn University on 12/28/14 For personal use only.
A. BHATTACHARYYA
The importance of serum albumin in supporting guinea pig sperm capacitation, acrosome reaction, and fusion with eggs in vitro was studied by incubating the spermatozoa in albumin-free medium containing different synthetic polymers. Serum albumin was found to be an obligatory component in the incubating medium for the capacitation of guinea pig spermatozoa. Albumin in the medium is not essential for the acrosome reaction and fusion with the eggs, but these phenomena take place most efficiently in presence of albumin. Key Words: Sperm; Capacitation; Guinea pig; Macromolecules; Factors.
INTRODUCTION In acquiring the ability to fuse with egg plasma membrane, mammalian spermatozoa must undergo the process of capacitation and acrosome reaction, in vivo, in the presence of follicular fluid [ 19, 201. Biochemical and immunochemical studies have revealed that the follicular fluid of most mammals contains the blood plasma proteins and that serum albumin is the major protein in this fluid [ 6 ] . Under in vitro conditions, mammalian sperm fertilization appears to be a complex process [16, 201 and the components of the medium definitely influence the process. The importance of protein, specifically serum albumin, in the fertilization process of hamsters [17], rabbits [15], rats [4], dogs [13], guinea pigs [lo], mice [8], and humans [22] is well documented. However, its exact site of action in the different components of fertilization is unclear and appears to vary, depending on the species studied. The guinea pig is an ideal model for evaluating the effects of albumin on the capacitation, acrosome reaction, and fusion with the eggs because the processes can be differentiated easily [21].
MATERIALS AND METHODS Media. The standard (regular) mT medium used in the present study had the following composition: 108.76 mM NaCl, 2.7 mM KCI, 2.0 mM CaCI,, 0.49 mM MgCl,, 6H,O, 25.07 mM NaHCO,, 5.56
From the Department of Anatomy and Reproductive Biology, University of Hawaii, School of Medicine, Honolulu, HI 96822. Address correspondence to Dr. Amitabha Bhattacharyya, Reproductive Biology Research Section, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India. ARCHIVES OF ANDROLOGY 28:235-239 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
235
236
A. Bhattacharyya
Syst Biol Reprod Med Downloaded from informahealthcare.com by Chulalongkorn University on 12/28/14 For personal use only.
mM D-glucose, 10 mM Na-lactate; 1 mM Na-pyruvate, and 3 mg/mL bovine serum albumin (Fr.V fatty acid poor). The osmolality and pH of the medium were 308 mosm and 7.8 (gas phase, pure air), respectively. The Ca2’ -free mT was prepared without CaCl, from the regular mT medium. The osrnolality of the Ca2’-free mT was kept constant by adjusting the concentration of NaCl. Albumin-free test mT medium were prepared by replacing bovine serum albumin in the regular or Ca2+-free medium with 3 mg/mL of one of the macromolecules purchased from Sigma Chemical Co. (St. Louis, MO): polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), dextran, Ficoll, and methocel. Sperm Cupacifation. Spermatozoa were collected from one cauda epididymis and vas deferens of mature male guinea pig (body weight 600 g) and were suspended in the 12 mL saline (0.9% NaCl solution). The sperm suspension was centrifuged at 280g for 3-4 min and the pellet was suspended in 2 mL saline. The sperm concentration at this stage was 4-5 x lo7 cells/mL. An aliquot (0.5 mL) of this suspension was mixed with 3 mL Ca2’-free test mT media which had been previously placed under mineral oil (Squibb and Sons, Princeton, NJ) in a plastic petri dish (10 X 35 mm; Falcon Plastics, Oxnard, CA). The concentration of spermatozoa at this stage was 4-6 x 106 cells/mL. After incubation for 16-18 h at 37°C (gas phase, pure air), the sperm suspensions were diluted to 12 mL in a centrifuge tube and centrifuged at 280g for 3-4 min. After discardation of the supernatent, the sperm pellet was diluted at 250 pL saline. An aliquot of this sperm suspension was mixed with 0.3 mL regular mT medium for acrosome reaction. The acrosome reaction of spermatozoon occurs only if they capacitate properly. The percentage of acrosome-related spermatozoa in the entire population was calculated as described by Murphy and Yanagimachi [ 141. Sperm Acrosome Reaction. For the acrosome reaction, capacitated guinea pig spermatozoa were prepared by incubating them at Ca2’ -free mT medium containing a low concentration (85 pg/mL) of lysolecithin (Avanti Biochemical, Birmingham, AL) at 37°C for 1-1.5 h [7]. Following incubation, the sperm suspension containing capacitated spermatozoa was diluted to 12 mL in a centrifuged tube and was centrifuged at 280g for 3-4 min. The pellet was resuspended in 0.25 mL saline. An aliquot (10 pL) of this sperm suspension was mixed with 0.3 mL of regular and albumin-free test mT medium that had been previously placed under mineral oil at 37°C in gas phase of pure air. The sperm concentration at this stage was 1.5-2 x lo6 cells/mL. After incubation for 15-20 min the overall percentage of motile spermatozoa and quality of motility were visually estimated and the acrosome-reacted spermatozoa were examined by phase contrast microscopy ( x 450) [ 141. Sperm-Egg Fusion. To determine the ability of fusion of spermatozoa with the zona-free eggs, fully acrosome-reacted guinea pig spermatozoa were prepared by incubating the same in regular mT medium following capacitation in Ca” -free lysolecithin containing capacitation medium (see above). Insemination was performed by introducing washed acrosome-reacted sperm suspension in 0.3 mL of regular or test mT medium containing zona-free guinea pig eggs. The zona-free guinea pig eggs were prepared following the method of Bhattacharyya and Yanagimachi [3]. After incubation of the eggs with spermatozoa (sperm concentration was 1 X lo5 cells/mL) for 1-1.5 h at 37°C under gas phase, pure air, the eggs were removed from the medium, cleaned, and mounted in between a slide and cover slip. The fusion was evaluated by phase contrast microscopy [ 181. An egg was considered fertilized when at least one decondensed sperm head was visible within its cytoplasm [3].
RESULTS When incubated continuously in Ca2‘-free mT medium (which contained 0.3% albumin) for 16-18 h, about 65-75 % spermatozoa underwent “synthronous” acrosome reaction after transfer into Ca2’ -containing medium (Table 1). Among the acrosome-reacted spermatozoa, many were vigorously motile (hyperactivated type). As shown in Table 1, guinea pig sper-
Capacitation of Guinea Pig Sperm
237
TABLE 1 Effect of Different Polymers on the Capacitation of Guinea Pig Spermatozoa ~
~
~
~
~
Sperm 15-20 After in Regular mT
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Media Contents (0.3%) Albumin PVP PVA Methocel Dextran Ficol None
% Motile Sperm After Incubation
85 51 65 36 60 78 21
5.6 12.8 10.4 18.5 12.7 16.8 f 19.7
f f f f 2 f
Quality
% Acrosome Reacted
f 5.2
3
f 15.9 f 15.9 17.7 f 1.5 k 9.9 f 30.1
1 1 1 1 1
69.7 f 3.8 0 0 0 0 0
1
0
% Motile
82 43 66 54 52 52 37
*
Nore. Values are means f SD of six experiments using spermatozoa from different males. Washed spermatozoa were incubated in 3 mL Ca2+-free different test mT medium for 16-18 h. After washing with saline, 10 pl of sperm suspension (in saline) was added to 0.3 mL regular mT medium.
matozoa are unable to undergo acrosome reaction when the capacitating medium contains synthetic macromolecules instead of albumin. This is due to inhibition of capacitation, since acrosome reaction occurs only in presence of Ca2+,after a successful completion of capacitation. Guinea pig sperm capacitation time could be shortened by using lysolecithin in the medium [7], but that type of experiment was omitted by this study, because spermatozoa becomes immotile immediately in presence of lysolecithin when the medium does not contain albumin. When spermatozoa were preincubated for 1.5 h in Ca2+-freemedium (in presence of albumin) containing lysolecithin and then exposed to Ca2'-containing regular (with albumin) and test (without albumin) mT medium many spermatozoa underwent acrosome reaction (Table 2). However, albumin containing mT medium was the best in supporting the acrosome reaction of guinea pig spermatozoa, although most of the spermatozoa in the test medium show progressive-type motility. Hyperactivate-type motility occurs only in albumin-containing medium. The spermatozoa that had undergone the acrosome reaction following lysolecithin pretreatment were capable of fertilizing the zona-free eggs regardless of whether albumin was present in the medium (Table 3). However, the albumin-containing mT medium was most effective in supporting the fusion process.
DISCUSSION Our results show that albumin is an obligatory component for the guinea pig sperm capacitation, confirming the results of earlier studies [7, 101. The hyperactivated type of motility and the acrosome-reacted spermatozoa was observed only in medium containing albumin (Table l), suggesting that the action of albumin in enhancing the sperm capacitation involves more than a macromolecular effect. According to Barros et al. [2], guinea pig spermatozoa were able to undergo capacitation in a simple medium without serum albumin. This may be due to a slow increase of intracellular Ca" during the incubation process or the pH of the medium used [lo]. The mechanisms whereby albumin can promote the capacitation of spermatozoa are un-
A. Bhattacharyya
238
TABLE 2 Effect of Different Polymers on the Acrosome Reaction of Guinea Pig Spermatozoa
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Sperm 15-20 min After in Regular mT Media Contents (0.3%)
% Motile
Albumin PVP PVA Methocel Dextran Ficol None
82 58 77 71 52 53 58
f 5.2 f 11.3 f 4.2 f 5.8 f 12.9 k 13.7 f 11.3
Quality
3 2 2 2 2 1 1
% Acrosome Reacted
74.8 50.5 64.0 59.6 39.4 34.3 46.4
f 4.5 & 9.8 f 6.2
f 6.3
f 14.3 f 13.8 f 14.8
Note. Values are means f SD of six experiments using spermatozoa from different males. Spermatozoa were incubated in the lysolecithin containing CaZ+-free mT medium (containing 0.3 albumin) for capacitation. After 1- 1.5 h of incubation spermatozoa were washed with saline twice and incubated in Ca2+ containing various test mT medium for acrosome reaction.
clear, although it is hypothesized that albumin alters the sperm plasma membrane lipid composition through lipid exchange or hydrolysis [5, 91. Furthermore, lipid removed from the outer plasma membrane of spermatozoa may bind with extracellular albumin, since it has unfilled binding sites for fatty acids [11]. It would appear that sperm capacitation involves, in part, the removal of an inhibitory factor from the sperm surface [ 191. It is also possible that the addition of serum albumin to guinea pig spermatozoa incubated in medium could contribute to sperm capacitation by removing such an inhibitory factor from the cells. The importance of albumin for acrosome reaction in mouse [8] and hamster [ l , 121 has been documented. Unlike mouse and hamster spermatozoa, guinea pig spermatozoa are able to acrosome react and fertilize the zona-free egg in the absence of albumin or other synthetic macromolecules. Albumin-free media supporting the membrane-fusion events (both acrosome reaction and fusion with eggs) could be explained by the fact that once the spermatozoa capacitate properly, the controlling mechanism by which Ca2+ enters the spermatozoa is activated. The intimate relationship between membrane fusion and the rise of intracellular Ca2+ is TABLE 3 Effect of Different Polymers on the Sperm-Egg Fusion Media Contents (0.3%) Albumin PVP PVA Methocel Dextran Ficol None
Number of Eggs Inseminated
Number (%) of Eggs Fertilized
26 27 28 19 24 28 20
25 (96) 14 (51) 15 (53) 9 (47) 13 (54) 17 (61) 11 (55)
Mean f SD of Decondensed Sperm HeadIFertilized Egg
7.9 4.1 3.6 2.9 4.6 5.0 4.0
k 4.0 f 1.8 f 1.0
f 1.9 f 1.6 f 2.5 & 1.7
Nore. Results are means f SD of three separate experiments using different males. Acrosome-reacted guinea pig spermatozoa were washed with saline twice and incubated with zona-free guinea pig eggs 1-1.5 h in various test media for insemination.
Capacitation of Guinea Pig Sperm
239
well documented [ 191. However, for sperm-egg fusion, synthetic macromolecules can mimic some, but not all, of the actions of albumin. Acknowledgments. This study was supported by the Rockefeller Foundation, USA. I a m grateful to R. Yanagimachi, University of Hawaii School of Medicine, USA, for his valuable guidance during the study. I also thank A. Pakrashi, Indian Institute of Chemical Biology, India, for her encouragement in preparation of the manuscript.
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