A follicular fluid chondroitin sulfate proteoglycan improves the retention of motility and velocity of human spermatozoa*

Transcription

1 FERTILITY AND STERILITY Copyright 1994 The American Fertility Society Vol. 62, No.3, September 1994 Printed on acid-free paper in U. S. A. A follicular fluid chondroitin sulfate proteoglycan improves the retention of motility and velocity of human spermatozoa* Gitte V. Eriksen, M.D.t:j: Anders Malmstrom, Ph.D. Niels Uldbjerg, M.D., Ph.D.t Gabor Huszar, M.D. II University of Aarhus, Kommunehospitalet, Aarhus, Denmark; University of Lund, Lund, Sweden; and Yale University School of Medicine, New Haven, Connecticut Objective: To examine the effects of two proteoglycans of different structure, isolated from human follicular fluid (FF), on the motility of human spermatozoa. Design: Normozoospermic semen samples and their swim-up sperm fractions were incubated in the presence of 0.4 mg/ml of a larger chondroitin sulfate proteoglycan (CS-PG) for 0, 3, 7, and 16 hours. The effects of a smaller heparan-cs-pg and the chondroitin sulfate side chains of the larger proteoglycan were also investigated in the same conditions. Sperm motility parameters were analyzed using a computer-aided sperm analysis system (CASA; Cryo Research Inc., New York, NY) Results: The larger CS-PG caused an immediate increase in sperm linearity. After 3 and 7 hours, the retention of sperm motility, velocity, linearity, and amplitude oflateral head displacement have increased by an average of 13% compared with the control samples. After a 16-hour incubation, the retention of the motility properties was improved by approximately 40% (range, 27% to 50%) in the samples containing proteoglycan. The effects of the isolated glycosaminoglycan side chains were much lower than those of the intact proteoglycan. The heparan-cs-pg did not affect sperm motility. Conclusion: A CS-PG from FF increases retention of motility and velocity of human sperm. These physiological effects may enhance the fertilizing efficiency of spermatozoa in the female reproductive tract. Fertil Steril 1994;62: Key Words: Proteoglycan, follicular fluid, human spermatozoa, sperm motility, fertilization Received October 11, 1993; revised and accepted April 13, *Supported by grant HD to (G.H.) from the National Institutes of Health, Bethesda, Maryland; by grant to (G.E.) from the Sven and Ina Hansen Foundation, Odense, Denmark, and by grant from the Danish Medical Research Council, Copenhagen, Denmark; by grant 7239 to (A.M.) from the Swedish Medical Research Council, Stockholm, Sweden; and by the Medical Schools, University of Aarhus, Aarhus, Denmark and University of Lund, Lund, Sweden. t Department of Obstetrics and Gynecology, University of Aarhus, Kommunehospitalet. t Reprint requests: Gitte V. Eriksen, M.D., Department of Obstetrics and Gynecology, University of Aarhus, Kommunehospitalet, DK-8000 Aarhus C, Denmark (FAX: ). Department of Physiological Chemistry, University of Lund. It is well documented that follicular fluid (FF) from women affects sperm function including the stimulation of capacitation (1), induction of the acrosome reaction (AR) (2), chemotatic effects on the spermatozoa (3), and promotion ofthe hyperactivated motility pattern (4). Furthermore, pretreatment of sperm with FF preceding tubal transfer during G 1FT improved the pregnancy rates ( 5). Toward identifying the underlying factors, various physiologically occurring components of FF, such as relaxin and prostaglandin E 2 (6) and creatine- II The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, Yale University School of Medicine. 618 Eriksen et al. Proteoglycans improve sperm motility Fertility and Sterility

2 phosphate (7) were shown to stimulate sperm motility. Glycoproteins also play an essential role in the regulation of sperm transport through the genital tract and in conditioning of spermatozoa before fertilization (2, 8). For instance, heparin (9) and heparan sulfate glycosaminoglycans from bovine oviductal fluid (10) increased the rate of oocyte fertilization during IVF when added to the medium. In addition, the unsulfated glycosaminoglycan hyaluronan improved retention of human sperm motility and velocity in vitro (11). The effects ofproteoglycans, however, have not yet been evaluated in such studies. Proteoglycans are macromolecules with 80 to 5,000 kd MW. They are composed of core proteins to which one or more highly negatively charged sulfated glycosaminoglycan side chains are attached (12). The properties of the individual proteoglycans are determined by the composition of the core proteins as well as the degree of side chain modifications. Two different proteoglycans, both with a high molecular weight, have been identified in the FF of women undergoing IVF (13). The larger of these is a chondroitin sulfate proteoglycan (CS PG) with Mr of 3,000 kd that consists of a core protein complex of 600 kd with 40 to 45 chondroitin sulfate side chains attached. The smaller is a heparan-cs-pg with an Mr of 1,100 kd that includes a core protein of 3 to 400 kd and approximately 20 hepar an -sulfate and chondroitin sulfate side chains. Both core proteins are substituted with numerous mainly 0-linked oligosaccharides ( 14). Similar proteoglycans are synthesized by cultured rat granulosa cells (15) and have been isolated from the extracellular matrix of the mouse cumulus-cell-oocyte complex (16). The aim of the present study was to investigate whether the two different proteoglycans isolated from human FF affect the maintenance of sperm motility during short-term and long-term incubations. MATERIALS AND METHODS Ham's F -10 medium was purchased from GIBCO (Grand Island, NY). Ethiodol was obtained from Sauvage Laboratories (Melville, NY), Chondroitin ABC lyase (proteus vulgaris EC ) from Seikagaky Kogyo, Tokyo, and diethylaminoethyl cellulose (DEAE-cellulose) ion-exchange chromatography material (DE-52} from Whatman (Maidstone, Kent, United Kingdom). Chemicals were from Sigma Chemical Co. (St. Louis, MO), and disposable test tubes, pipettes, and other laboratory Vol. 62, No.3, September 1994 equipment were purchased from American Scientific Products (Edison, NJ). Human FF Proteoglycans Follicular fluid was obtained from patients participating in the IVF Program at the Department of Gynecology and Obstetrics, University of Aarhus, Denmark. The women (n = 55} were stimulated with a clomiphene citrate and hmg protocol. Follicular fluid was aspirated transvaginally 34 hours after hcg injection. The FF was pooled and mixed with a solution containing 7 M guanidin hydrochloride, M sodium acetate, ph 5.8, and proteinase inhibitors (17) to a final concentration of 4 M guanidin hydrochloride, 0.05 M sodium acetate. The two proteoglycans were isolated by a combination of CsCl-density gradient centrifugation and gel-permeation chromatography (13). The proteoglycans were stored in 4 M guanidin hydrochloride, ph 7.0 at -18 C. Before the experiments, proteoglycans were dialyzed against several changes of Ham's F-10 medium (supplemented with 257 mm of calcium lactate, 1 mg/ml erythromycin, 0.5 mg/ ml streptomycin, 15 mm sodium bicarbonate ph 7.2), and bovine serum albumine (BSA) was added to a concentration of 0.3%. All incubation experiments were carried out at 36 C. The glycosaminoglycan side chains were prepared from the CS-PG after alkali treatment with sodium borohydride (18} with ion-exchange chromatography on a DEAE-cellulose ion-exchange column (0.5 X 2.5 em). The column was equilibrated and washed with 6 M urea, 0.05 M acetic acid, ph 5.8, and eluted with 4 M GuHCl, 0.05 M acetic acid, ph 5.8. The recovered glycosaminoglycan side chains were dialyzed against sodium chloride and water and were stored lyophylized. The glycosaminoglycan side chains were dissolved in Ham's F-10 medium before the sperm experiments, and BSA was added to a concentration of 0.3%. It was not possible to solubilize the protein core fraction in physiological buffers. Sperm Preparation The normozoospermic semen samples (concentration >20 X 10 6 sperm/ml, motility >40%, and <40% abnormal sperm forms) were provided by men who presented for semen analysis at the Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut without any preselection. Semen samples were collected by mastur- Eriksen et al. Proteoglycans improve sperm motility

3 bation after 2 days of abstinence. The liquefied semen and the swim-up sperm fractions were analyzed by the CellSoft (Version 3.5) computer-aided sperm analysis system (CASA; Cryo Research Inc., New York, NY). For the analysis, the semen samples with sperm concentrations >60 X 10 6 sperm/ ml were diluted to the 30 to 60 X 10 6 sperm/ml range using Ham's F-10 medium. For the preparation of the swim-up sperm fractions, the semen was diluted by five volumes of Ham's F-10 medium and layered slowly onto 1 ml Ethiodol (oil contrast medium, specific gravity 1.28 g/ml) in 15-mL conical tubes and centrifuged (400 X g for 10 minutes). The supernatant with exception of the lower 1.5 ml (which contains the sperm and other particulate matter) was removed gently and discharged. The tube was then allowed to incubate for 30 minutes at 36 C (19), and the motile sperm that had migrated into the supernatant was recovered and used for the swim-up sperm fraction experiments. All sperm motility and the morphology parameters in these migrated fractions were significantly improved when compared with the initial semen. Analysis of Sperm Motility Using a 10-~m deep Makler chamber in all analyses (Zygotek Systems, Inc., Springfield, MA) and an Olympus microscope (Olympus, Lake Success, NY) equipped with a phase-contrast objective, at each time point two drops of semen (3 fields in each) per sample were studied by CASA measurements. The CASA system parameters were as follows: 15 frames at 30 frames/s, threshold velocity 8 ~m/s, and maximum velocity 150 ~m/s. The analyses of the migrated sperm fractions were carried out at a maximum velocity of250 ~m/s. We have monitored sperm concentrations, motility(%), curvilinear velocity (VCL, ~m/s), linearity, and mean amplitude of lateral head displacement (ALH, ~m). Statistical Analysis Statistical analyses were carried out according to the t-tests or to the nonparametric Wilcoxon statistical test using the IBM Statistical Package for Social Sciences (SPSS) PC-system (SPSS-Scandinavia, Sollentuna, Sweden, site license to the University of Aarhus). All data are expressed as means ±SEM. Table 1 Effects of the CS-PG* Incubation time Oh 3h 7h 16 ht Motility(%) Control 51.5 ± 2.5:j: 42.6 ± ± ± 3.2 CS-PG 49.6 ± ± ± ± 2.9 Probability Velocity (VCL,!'m/s) Control 47.1 ± ± ± ± 1.2 CS-PG 46.9 ± ± ± ± 1.5 Probability Linearity Control 5.0 ± ± ± ± 0.1 CS-PG 5.5 ± ± ± ± 0.3 Probability <0.001 < <0.001 ALH(!'m) Control 2.8 ± ± ± ± 0.1 CS-PG 2.6 ± ± ± ± 0.1 Probability < * In 18 samples, initial semen parameters: concentration, 76.3 ± 10.5 X 10 6 sperm/ml; motility, 52.9% ± 3.6%; VCL, 41.5 ± 2.5 l'm/s; linearity, 5.3 ± 0.1; ALH, 2.6 ± 0.2 I'm. tall 16-hour data are based on 12 samples. :j: Values are means± SEM. Initial Studies RESULTS The effects of human FF proteoglycans on sperm motility and the question of concentration dependence were examined in five samples. In line with the proteoglycan concentrations in FF of 0.2 to 1.5 mg/ml for the CS-PG and 0.2 to 0.8 mg/ml for the heparan-cs-pg (Eriksen G, Malmstrom A, Uldbjerg N, unpublished data), the proteoglycans were added to the aliquots of sperm in concentrations of 0 (control), 0.1, 0.3, and 0.6 mg/ml, and the samples were incubated for 16 hours. A general improvement of several motility parameters was noted with the CS-PG (data not shown). Motility and lateral head displacement increased proportional to the concentration of proteoglycan, and at 0.3 mg/ml a twofold increase in motility was recorded. Linearity increased significantly (P < 0.05) already at 0.1 mg/ml. The heparan-cs-pg had no significant effect on the sperm motility parameters in the initial studies (data not shown). On the basis of these results, a concentration of 0.4 mg/ml was selected of the chondroitin sulfate or the heparan-cs-pg for the further studies. Effects of the CS-PG on Sperm We tested the effects of the CS-PG on 18 specimens at 0 time (actually within <7 minutes after addition, the duration of measurements of 6 fields originating in the 2 drops of semen) and after 3, 7, 620 Eriksen et al. Proteoglycans improve sperm motility Fertility and Sterility

4 and 16 hours' incubation. The CS-PG caused significant short-term and long-term improvements in the motility parameters and in the retention of sperm motility, which were detectable in three aspects (Table 1). First, in the 0 time samples, there was an immediate increase in sperm linearity (P < 0.001). This effect is similar to that of hyaluronan reported previously (11). Second, after 3 and 7 hours of incubation, a decrease of motility, velocity, linearity, and lateral head displacement was noted in both the control and study samples. However, in the presence of the proteoglycan, a significant improvement in the retention of motility parameters (range, 8% to 19%) compared with the control samples was obtained (except motility at 7 hours). Third, the long-term, 16-hour incubation with the CS-PG caused a major improvement in the retention of motility. In 6 of the 18 control samples, sperm motility was lost, whereas the sperm motility was sustained in all 18 samples that contained the CS-PG. The data representing the 12 samples with motile sperm in both groups showed a substantial and significant improvement in the retention of sperm motility, velocity, linearity and lateral head displacement parameters (range, 27% to 50%). Effects of the Heparan-CS-PG on Sperm The effects of incubation with the heparan-cs PG in a concentration of 0.4 mg/ml proteoglycan on 12 specimens were investigated as a function of time. The heparan-cs-pg caused no immediate effect on the sperm motility parameters (data not shown). After 7 hours of incubation, approximately an 8% increase in velocity (38.6 ± 2.0 versus 35.2 ± 2.1 ~-tm/s, P = 0.39) and linearity (4.5 ± 0.1 versus 4.2 ± 0.1, P = 0.01) was observed, whereas motility and lateral head displacement were not improved. After 16 hours of incubation, the retention of sperm motility was not improved because only 8 of 12 samples contained motile sperms in both the control and proteoglycan-treated specimens. Furthermore, in the semen samples that retained motility, no significant improvement in any of the sperm motility parameters were observed (data not shown). Effects of the CS-PG on the Swim-up Sperm Fractions To investigate whether the effects of proteoglycan are directed to the relative slow sperm population or if all sperm benefit, the effects of the CS-PG on the swim-up sperm fractions were studied in five samples at 0 time and 7 hours (Table 2). After the Table 2 Effects of the CS-PG on the Swim-up Sperm Fractions* Velocity Motility (VCL) Linearity ALH % p..m/s 0-h incubation Control 66.9 ± ± ± ± 0.2 CS-PG 72.9 ± ± ± ± 0.2 Probability h incubation Control 47.3 ± ± ± ± 0.1 CS-PG 63.4 ± ± ± ± 0.3 Probability * In five samples, initial semen parameters: concentration, 76.4 ± 9.6 X 10 6 sperm/ml; motility, 55.4% ± 5.7%; VCL, 41.6 ± 3.4 J.Lm/s; linearity, 5.4 ± 0.2; ALH, 2.4 ± 0.2 J.Lm. t Values are means ± SEM. addition of the proteoglycan, there was an immediate improvement in motility and linearity (P = 0.02). At 7 hours, there was a significant improvement in the motility (34%), velocity (53%), and lateral head displacement (78% ), whereas linearity was not affected. Effects of the CS Side Chains We also examined whether both the core protein and the glycosaminoglycan side chain component of the proteoglycan is necessary to enhance sperm motility, or if the isolated glycosaminoglycan side chains may also elicit the improvement. Aliquots of five semen samples were exposed to a concentration of 0.4 mg/ml CS side chains prepared by chemical degradation of the proteoglycan. Unlike the proteoglycan containing samples, incubation with the side chains did not cause the immediate effect at 0 time or the increase in the retention of motility parameters after 7 hours (data not shown). After the 16- hour incubation, the retention of sperm motility was increased by 34% (27.4% ± 4.7% versus 20.5% ± 4.0%, P = 0.05). The retention of other motility Table 3 Effects of CS Side Chains* Velocity Motility (VCL) Linearity ALH % 11m!s ~m Control 20.5 ± ± ± ± 0.2 CS side chains 27.4 ± ± ± ± 0.2 Probability * In five samples, initial semen parameters: concentration, 79.1 ± 15.1 X 10 6 sperm/ml; motility, 64.4% ± 8.2%; VCL, 54.0 ± 5.4 J.Lm/s; AHL, 5.0 ± 0.4; lateral head displacement, 3.2 ± 0.4 J.Lm. All data are 16 hours' incubation. Vol. 62, No. 3, September 1994 Eriksen et al. Proteoglycans improve sperm motility 621

5 parameters were also improved by approximately 10%, although without statistical significance (Table 3). The protein core fraction was insoluble; thus, its effects on sperm motility were not investigated. DISCUSSION We have studied the effects of two proteoglycans of different molecular weights and structure isolated from FF of women on the motility properties of human spermatozoa using a CASA system. The larger of these proteoglycans contains CS side chains, whereas the smaller proteoglycan is substituted with both heparan sulfate and CS side chains. The concentrations of the CS-PG that affected sperm motility were within the physiological range (0.2 to 1.5 mg/ml) measured in human FF using an ELISA technique (Eriksen G, Malmstrom A, Uldbjerg N, unpublished data). The CS-PG caused short-term and long-term improvements on theretention of sperm motility. First, we found an immediate increase in sperm linearity after the exposure to the larger proteoglycan. In this respect, the effects of the proteoglycan on sperm are very similar to that of the hyaluronan studied previously (11). After the midrange, 3- and 7-hour incubation, there was an increase by approximately 10% to 15% in the retention of the motility, velocity, linearity, and lateral head displacement parameters, whereas after the long-term 16-hour incubation, the various motility parameters showed approximately a 40% improvement (P ~ 0.001). In addition, in all proteoglycan-treated samples, there were more motile sperm present, whereas in 6 of 18 untreated samples, sperm motility ceased by 16 hours (note that using 8 f.lm/s threshold velocity, only the physiologically meaningful sperm motility was detected). Similar immediate and long-term effects were also observed in the proteoglycan-treated swim-up sperm fractions. Thus, the effects of the proteoglycan are directed to all sperm populations in the ejaculate. The results suggest that the CS-PG may be one of the components that is responsible for the physiological effects of FF toward enhancing the fertilization potential of spermatozoa. Indeed, in the samples containing proteoglycan after a 16-hour incubation, the combined effects of increased retained sperm motility along with the velocity, linearity, and lateral head displacement parameters were 70%, 100%, and 90% higher, respectively (P < in all 3 cases). This provided a substantial advantage; because of the increased rate of sperm- oocyte encounters, the overall fertilizing efficiency of the sperm in the specimen has been enhanced. The increased retained ALH brings up another point. It is known that sperm undergoes hyperactivated motility in connection with the capacitation and AR. The most characteristic CASA parameters that are related to hyperactivated motility are the increased VCL and ALH. Thus, in addition to the retention of motility and velocity, the spermatozoa is likely to be functionally sound because the elevated levels of ALH indicate that some of the sperm population may in the hyperactivated motility mode (20). Because the CS-PG is composed of a protein core to which chondroitin sulfate side chains are attached, we have also addressed the question whether both components are necessary for the sperm motility effects. After chemical degradation of the proteoglycan, the isolated CS side chains did not cause the immediate increase in linearity, and the long-term improvement in the retention of motility parameters was also diminished. This indicates that not only the glycosaminoglycan side chains but also their distribution along the protein core or the presence of specific steric configurations may be of importance. This hypothesis is supported by the fact that the heparan-cs-pg does not have similar effects on sperm motility. We previously have found that hyaluronan, an unsulfated glycosaminoglycan caused effects similar to the CS-PG on short-time and long-time sperm motility (11). Hyaluronan is present in the ovulatory mucus of the human cervix (21) and in the extracellular matrix of the mouse cumulus-oocyte complex (16) but not in the human FF (13). Considering the mechanism of the proteoglycan action, it is noted that the sperm surface contains receptors that are specific for heparin, another glycosaminoglycan (22, 23), and that heparan sulfate substances from bovine oviductal fluid promote sperm capacitation (10). Furthermore, a correlation was reported between heparin-binding capacity of sperm and their efficiency in hamster-egg penetration (24, 25). Thus, the heparan-cs-pg isolated from human FF, which because of its heparin sulfate content share many characteristics with heparin, may enhance functions other than motility of the sperm. Although FF appears to increase the fertilizing capacity of sperm by increasing sperm motility and the rate of AR (2), it is not clear at the present time whether any particular component or a combination of components are specifically responsible. 622 Eriksen et al. Proteoglycans improve sperm motility Fertility and Sterility

6 The CS-PG may certainly have a role because in physiological conditions, FF is transported to the fallopian tubes along with the oocyte, and the constituent proteoglycan may improve the chances for fertilization. Acknowledgment. The authors thank the staff at the In Vitro Fertilization Laboratory, Department of Obstetrics and Gynecology, Aarhus Kommunehospital, Denmark, and colleagues at the Department of Physiological Chemistry, University of Lund, Sweden, and at The Sperm Physiology Laboratory, Department Obstetrics and Gynecology, Yale University, New Haven, Connecticut fore technical assistance. REFERENCES 1. Yee B, Cummings LM. Modification of the sperm penetration assay using human follicular fluid to minimize false negative results. Fertil Steril 1988;50: Suarez SS, Wolf DP, Meizel S. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 1986;14: Ralt D, Goldenberg M, Fetterolf P, Thompson D, Dor J, Mashiach S, et a!. Sperm attraction to a follicular fluid factor(s) correlates with human egg fertilizability. Proc Nat! Acad Sci USA (Med Sci) 1991;88: Mbizvo MT, Burkman LJ, Alexander NJ. Human follicular fluid stimulates hyperactivated motility in human sperm. Fertil Steril1990;54: Fakih H, Vijayakumar R. Improved pregnancy rates and outcome with gamete intrafallopian transfer when follicular fluid is used as a sperm capacitation and gamete transfer medium. Fertil Steril 1990;53: Colon JM, Ginsburg F, Lessing JB, Schoenfeld C, Goldsmith LT, Amelar RD, eta!. The effect of relaxin and prostaglandin E 2 on the motility of human spermatozoa. Fertil Steril 1986;46: Fakih H, MacLusky N, DeCherney A, Wallimann T, Huszar G. Enhancement of human sperm motility and velocity in vitro: effects of calcium and creatine phosphate. Fertil Steril 1986;46: Siiteri JE, Dandekar P, Meizel S. Human sperm acrosome reaction-initiating activity associated with human cumulus oophorous and mural granulosa cells. J Exp Zoo! 1988;246: Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Bioi Reprod 1988;38: Parrish LL, Susko-Parrish JL, Handrow RR, Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Bioi Reprod 1989;40: Huszar G, Willetts M, Corrales M. Hyaluronic acid (Sperm Select) improves retention of sperm motility and velocity in normospermic and oligospermic specimens. Fertil Steril 1990;54: Kjellen L, Lindahl U. Proteoglycans: structures and interactions. Annu Rev Biochem 1991;60: Eriksen GV, Malmstrom A, Carlstedt I, Uldbjerg N. Human follicular fluid contains two large proteoglycans. In: Leppert PC, Woessner F, editors. The extracellular matrix of the uterus, cervix and fetal membranes: synthesis, degradation and hormonal regulation. Ithaca (NY): Perinatology Press, 1991: Yanagishita M, Rodbard D, Hascall VC. Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. J Bioi Chern 1979;254: Yanagishita M, Hascall VC. Biosynthesis of proteoglycan by rat granulosa cells cultured in vitro. J Bioi Chern 1979;254: Salustri A, Yanagishita M, Hascall VC. Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone induced mucification. J Bioi Chern 1989;264: Uldbjerg N, Malmstrom A, Ekman G, Sheehan J, Ulmsten U, Wingerup L. Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix. Biochem J 1983;209: Carlson DM. Structures and immunochemical properties of oligosaccharids isolated from pig submaxillary mucins. J Bioi Chern 1968;243: Makler A, Morillo 0, Huszar G, Tarlatzis B, DeCherney A, Naftolin F. Improved techniques for separation motile spermatozoa from human semen. II. An atraumatic centrifugation method. Int J Androl1984;7: Burkman LJ. Hyperactivated motility of human spermatozoa during in vitro capacitation and implications for fertility. In: Gagnon C, editor. Controls of sperm motility: biological and clinical aspects. Boston: CRC Press, 1990: Wikland M, Wik o; Steen Y, Qvist K, Soderlund B, Janson PO. A self-migration method for preparation of sperm for in vitro fertilization. Hum Reprod 1987;2: Hurst RE, Byum RL, Einfeldt SE. The identification of a heparin-binding protein on the surface of bovine sperm. Biochem Biophys Res Commun 1989;153: Handrow RR, Boehm SK, Lenz RW, Robinson JA, Ax RL. Specific binding of the glycosaminoglycan 3 H-heparin to bull, monkey and rabbit spermatozoa. J Androl 1985;5: Lalich RA, Vedantham S, McCormick N, Wagner C, Prins GS. Relationship between heparin binding characteristics and ability to penetrate hamster ova. J Reprod Fertil 1985;86: Vasquez JM, Winer MA, Ax RL, Boone WR. Correlation of human spermatozoa heparin binding with the zona free hamster egg in vitro penetration assay. Am J Obstet Gynecol 1989;160:20-6. Vol. 62, No. 3, September 1994 Eriksen et al. Proteoglycans improve sperm motility 623