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a Unitat d'Immunologia de la Reproducció, Institut de Biologia Fonamental,
b Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A time-dependent cholesterol removal from spermatozoa is observed in the presence of serum, which has a beneficial effect on capacitation and fertilization as has been observed for ovine in vitro fertilization [10]. Several cholesterol acceptors have been tested in vitro, albumin being one of the most prominent proteins supporting in vitro capacitation by accepting cholesterol. Sterol depletion by albumin is highly dependent on the phospholipid content in the type of albumin used. Commercial preparations of serum albumin contain variable amounts of fatty acids and other contaminants, which prevent albumin from manifesting its full steroid-binding affinity. Through elimination of BSA contaminants, AR is enhanced [11].
Components that act as cholesterol acceptors are present in the female tract, the major protein in the uterus and oviduct being albumin. Because of the long residency of spermatozoa in the isthmus prior to ovulation, the oviduct is considered the principal site for completion of sperm capacitation. High-density lipoproteins present in bovine oviductal secretions appear to support cholesterol efflux from bovine spermatozoa [12]. Follicular fluid, also present at the site of fertilization, may promote spontaneous human sperm AR, although by nonspecific induction, as described by Mortimer and Camenzind [13]. All these biological fluids have been used for supporting in vitro capacitation and AR.
Other newly discovered nonphysiological cholesterol acceptors have been used to alter the membrane cholesterol content in several cell types. Three different cyclodextrins (
-, ß-, and 
-cyclodextrin) have been used to alter the lipid composition of erythrocytes. ß-Cyclodextrin (ßCD), a cyclic oligosaccharide consisting of 7 ß(1-4)-glucopyranose units, was found to selectively extract cholesterol from the plasma membrane in preference to other membrane lipids [14]. Cyclodextrins enhance the solubility of nonpolar substances by incorporating them in their hydrophobic cavity and forming inclusion complexes. The very high efficiency of cyclodextrins in stimulating cholesterol efflux [15] makes them valuable in studying the influence of cholesterol on membrane protein function. Modification of the cholesterol content of the isolated plasma membranes has been used to study the effect of cholesterol on the binding function of the myometrial oxytocin receptor [16]. These results suggest a direct interaction between the oxytocin receptor and cholesterol, inducing a high-affinity state in the receptor. Although ßCD is not a biological molecule found in the female reproductive tract or in oocyte envelopes, it can be used as a highly efficient cholesterol acceptor to investigate the role of cholesterol release as an early event of in vitro sperm capacitation and AR. We present a study of the kinetics of both cholesterol efflux and AR in the presence of ßCD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following chemicals were used: ß-cyclodextrin and glutaraldehyde (Fluka, Madrid, Spain); BSA (fraction V, fatty acid free; Boehringer Mannheim, Mannheim, Germany); heparin (Rovi, Madrid, Spain), cholesterol (ICN, Aurora, OH); chloroform and methanol (Scharlau, Barcelona, Spain). M-199 H incubation medium was from Biological Industries, Beit Haemek, Israel. All other chemicals were obtained from Sigma (Alcobendas, Spain).
Sperm Preparation
Spermatozoa from the goat (Capra irca) were obtained from two fertile males by artificial vagina technique. The quality of the sample was checked before every experiment. Spermatozoa were washed three times in M-199 incubation medium and centrifuged at 300 x g for 10 min to remove seminal plasma. The number of spermatozoa in the ejaculate was counted by means of a Neubauer chamber and divided into 2-ml aliquots (8 x 107 cells/ml). One aliquot of washed goat spermatozoa was separated without any treatment and used as a control at time zero. The other aliquots were incubated under capacitation conditions, 37°C, 10% CO2, in M-199 medium for various times in the presence or absence of ßCD, as cholesterol acceptor, at several concentrations. Alternatively, the following components were dissolved in M-199 medium: 20 µg/ml heparin; 10 mg/ml BSA; 10 mg/ml BSA+20 µg/ml heparin; 8 mM ßCD; 8 mM ßCD+20 µg/ml heparin. The incubation process was stopped by addition of 8 ml Tris-buffered saline, pH 7.4 (TBS), centrifuged, and washed twice by resuspension in the same buffer.
Different determinations were performed in parallel for each sperm suspension after incubation. For lipid analysis, 150 x 106 cells were taken, and centrifugation was performed at 3300 x g for 10 min to ensure quantitative recovery of cells; the last pellet was resuspended in 0.5 ml TBS and stored at -20°C until analyzed. For acrosomal status and vitality measurements, 10 x 106 cells were taken; centrifugation was performed at 1000 x g for 10 min, to avoid cell damage, and the last pellet was resuspended in 50 µl TBS.
Analytical Procedures
Lipids were extracted both from intact and incubated spermatozoa using a modification of the method of Bligh and Dyer [17] as described by Wolf et al. [18]. The concentration of unesterified cholesterol was determined in the lipid extracts from 100 x 106 spermatozoa by a modification of a commercially available enzymatic serum cholesterol assay (HDL Cholesterol; BioSystems, Barcelona, Spain), which was free of cholesterol esterase (EC 3.1.1.13). Briefly, cholesterol oxidase (EC 1.1.3.6) oxidizes free cholesterol, but not cholesterol esters, to cholest-4-en-3-one with H2O2 release. The peroxide oxidatively couples with 4-aminoantipyrine and dichlorophenolsulfonate in the presence of peroxidase (EC1.11.1.7) to yield quinoneimine, a chromogen with maximum absorption at 500 nm [19]. Dried lipid extracts were used as the material to be analyzed. To ensure solubilization of the lipid extracts during the assay procedure, detergent Triton X-100 was added to the Reagent A solution (35 mM phosphate, 0.5 mM sodium cholate, 4 mM dichlorophenolsulfonate, pH 7.0) to a final concentration of 7.7 mM [20]. We estimated that the lowest level of sensitivity for this cholesterol assay was around 1 µg.
Phospholipids were measured colorimetrically in the lipid extract of 50 x 106 spermatozoa according to the procedure of Stewart [21]. Since sensitivity of this method differs depending on the phospholipid headgroups [22], this assay was used only to monitor possible changes in total phospholipid content. In the case of dipalmitoyl lecithin, the lowest sensitivity level was 5 µg [21]. Sperm cholesterol and phospholipid content were expressed per number of spermatozoa and were normalized within each experiment with respect to the cholesterol or phospholipid content of the 0-h control (100%). The results were expressed as percentages with respect to the control.
Acrosomal Status and Vitality
AR and vitality were measured by the triple-stain technique of Talbot and Chacon [23]. Briefly, the sample was incubated with 1% trypan blue for 15 min at 37°C to evaluate sperm vitality. Sperm were washed for removal of excess stain and fixed in 1.5% glutaraldehyde for 15 min. After two washing cycles, sperm were stained with Bismarck Brown and Bengale Rose. We observed the acrosomal status under a Zeiss AxioPlan (Carl Zeiss, Thornwood, NY) epifluorescence microscope after washing. Approximately 400 spermatozoa were scored for each sperm preparation, the samples being scored randomly. The advantage of this technique is that live acrosome-reacted spermatozoa are distinguishable from dead spermatozoa with degenerative acrosomal loss. To validate the triple-stain technique, we compared the results obtained by this technique and the lectin Pisum sativum-fluorescein isothiocyanate technique [24]. There were no significant differences in the percentage of AR (AR%) as measured by the two techniques.
Statistical Analysis
Statistical analyses were performed with the SPSS for Windows software package (version 7.5.2S; SPSS Inc., Chicago, IL; 1997). The mean comparison was performed by ANOVA (the a posteriori method of Gabriel was used to compare the mean values of different samples, and Dunnett's method was used to compare each sample with the control). The sample size, n, was 3 to 6. Significance was indicated by P < 0.05 or P < 0.01.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ßCD-induced AR was also studied after 150-min incubation of sperm with increasing concentrations of ßCD, from 2 mM to 16 mM (Fig. 2). In contrast to the hyperbolic saturation curve displayed by cholesterol desorption, the AR% displayed a sigmoidal dependence on cyclodextrin concentration. At concentrations below 2 mM, the AR percentage was extremely low, whereas it rose steeply at higher concentration.
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To test the possible toxic effect of ßCD on sperm, the percentage of vitality was assessed by trypan blue staining after the 150-min incubation period. Samples showed high vitality percentages ranging between 83% and 97% for all the ßCD concentrations between 0 and 8 mM (Fig. 3). Noticeably decreased vitality (68%) appeared at 16 mM ßCD. Therefore, 8 mM was taken as the optimal ßCD concentration to be used in the kinetic study of cholesterol removal and AR induction.
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ßCD was added at a concentration of 8 mM ßCD to the incubation medium (M-199) to study its efficiency on cholesterol removal in terms of time dependency. In Figure 4 we show the cholesterol remaining in sperm membranes after this treatment. Cholesterol efflux, analyzed as described above, displayed exponential kinetics with a half-time of around 10 min. The effect of 8 mM ßCD was to promote cholesterol efflux up to 47%. In contrast, sperm phospholipid content after different times of incubation in 8 mM ßCD was not significantly different (P > 0.05) from the control level (data not shown).
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When we analyzed the percentage of acrosome-reacted cells in the presence of 8 mM ßCD, we observed a roughly sigmoidal curve, with no significant change in the AR% during the first 60 min and a steep change in the slope of the curve around 90 min of incubation (Fig. 5). Interestingly, there was no significant change in the cholesterol levels between 60 min and 180 min of incubation (Fig. 4).
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In order to understand the mechanism by which ßCD promotes both a rapid cholesterol efflux and an increase in the AR%, we investigated the effect of the presence of heparin in the sperm incubation medium. Heparin is considered by several authors to be an inducer of the AR through removal of coating proteins from the sperm membrane surface [25,26]. In our experiments, incubation of goat spermatozoa in the presence of 20 µg/ml heparin for 2 h was unable to induce AR (Fig. 6). On the other hand, the coincubation of goat spermatozoa with 10 mg/ml albumin and 20 µg/ml heparin showed a synergistic effect on the AR%, although the cholesterol content did not change. Both AR and membrane cholesterol removal were increased in the sole presence of ßCD. The coincubation of spermatozoa with 8 mM ßCD and 20 µg/ml heparin, however, did not significantly modify the effect of ßCD alone. Thus, ßCD was able both to remove cholesterol from sperm membranes and to promote AR without the cooperation of heparin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Albumin has been used extensively in the incubation medium for an in vitro induction of the AR [27–31], although the results are very controversial. Using albumin, some authors obtained cholesterol efflux [27,32,33] but others did not [31,34]. It may be worth considering the origin of the albumin used in the experiments. Commercial albumin has several impurities, and among them there are lipids. When the albumin source is serum or other biological fluids, inducers of the AR are present. This may explain the broad range of cholesterol efflux percentages described in the literature.
It has been reported that extensive release of cholesterol from erythrocyte membranes can be achieved upon incubation of the cells with ßCD. There was a rapid cholesterol exchange (< 1 min) [35] from erythrocytes to ßCD, which is in contrast with the cholesterol transfer from these cells to serum or isolated lipoproteins (1–8 h) [36]. The very fast exchange rate of ßCD makes this molecule highly useful as a cholesterol acceptor in capacitating medium.
Our results indicate that at 2 mM ßCD, 30% of cholesterol was released from sperm membranes, but this concentration did not induce any AR. Only when more cholesterol was lost were membranes susceptible to initiating all the changes leading to capacitation and AR. When cholesterol efflux had increased to 50%, at a concentration of 4 mM ßCD, we observed more than half of the maximal AR percentage. Cholesterol removal between 50% and 65% was able to induce the AR. The use of concentrations of ßCD higher than 8 mM resulted in only a moderate increase in cholesterol efflux and AR%. Our results suggest that there is a direct effect of cholesterol efflux on the AR induction rather then any kind of binding of ßCD to membranes. In fact, some authors have demonstrated in erythrocytes that cholesterol removal by ßCD occurs with negligible binding to the cell membrane [14]. That is, ßCD extracts cholesterol from membranes into a new compartment located in the aqueous phase, while phospholipid levels remain unchanged. This agrees with the fact that there was no membrane loss from spermatozoa, so we could consider only the cholesterol efflux.
The sigmoidal shape of the curve (Fig. 2), indicating the AR% versus ßCD concentration, can be interpreted as a cooperative effect in which the early molecular events in a restricted area of sperm membrane may facilitate further changes in a broader zone, thus inducing a quick AR in a short period of time.
Cholesterol efflux kinetics due to ßCD (t1/2 around 10 min) is much more rapid than the reported kinetics (t1/2 > 1 h) of sperm membrane cholesterol efflux in the presence of estrous sheep serum [32] or human female serum or follicular fluid [10]. Small-particle cholesterol acceptors show more rapid kinetics of cholesterol uptake from cells than macromolecular acceptors [37]. This rapid loss of cholesterol from sperm membrane, which follows an exponential decay (Fig. 4), contrasts with the S-shaped kinetics of AR increase (Fig. 5). After 30 min of incubation in the presence of 8 mM of ßCD, all the exchangeable cholesterol has been released from the membranes, but a negligible AR is obtained. Incubation for 1–2 h is required to reach the maximal AR value. Cholesterol efflux has been proposed as an early event previous to the AR [7,27,38,39]. Our kinetic study of both cholesterol efflux and AR suggests that acrosome responsiveness is triggered by a major cholesterol loss.
Several authors have reported significant cholesterol removal through addition of various components to the incubation medium or through use of biological fluids. Huneau et al. [10] obtained a time-dependent cholesterol removal ranging from 54% to 67% after 5 h of incubation in DM-H medium containing 20% estrous sheep serum, whereas removal was limited to 14 ± 3% in the absence of serum. Female biological fluids have been extensively used, and similar cholesterol efflux was obtained. Langlais et al. [32] incubated spermatozoa for 4 h at 37°C in Ham's F-10 medium supplemented with human female serum or follicular fluid and obtained cholesterol efflux in a time-dependent manner (57% and 42%, respectively). With spermatozoa incubated in protein-free medium, low cholesterol efflux (14%) occurs.
The role of albumin in the uptake of cholesterol has been discussed. There is some evidence that albumin impurities can modify the ability of albumin to bind cholesterol. For Langlais et al. [32], after a period of 2-h incubation with 3.5% HSA (fraction V, Calbiochem-Boehring; La Jolla, CA) the uptake of cholesterol was 38% versus 12% for cells incubated in medium alone. In contrast, using similar concentrations of albumin, others found that cholesterol efflux from sperm membranes was not statistically significant [31]. As shown by Sugkraroek et al. [34], the cholesterol level from fertile donors' sperm declines < 5% when incubated in BWW medium supplemented with 3.5% HSA for 5 h. Even after long periods of incubation (18–20 h), Benoff et al. [28] obtained a cholesterol decrease of only 15% in swim-up spermatozoa incubated in Ham's F-10 with 30 mg/ml HSA.
Interestingly, the sample some studies used was ejaculated spermatozoa [31,32,34], whereas others used epididymal spermatozoa [27,33]; this would confer on the sample a differential accessibility for cholesterol acceptors, probably because of the presence of coating proteins in ejaculated spermatozoa. At low concentrations of albumin in the medium, a relatively high amount of cholesterol could be removed from the membranes of epididymal spermatozoa, these lacking all the coating molecules that are adsorbed from seminal plasma molecules. Go and Wolf [27] obtained a 20–59% decrease in mouse epididymal sperm cholesterol with several commercially purified BSA fractions (20 mg/ml). Davis et al. [33] also gave evidence of a 12% cholesterol transfer from cauda epididymal rat sperm to albumin after incubation in 4 mg/ml BSA for 5 h.
Ehrenwald et al. [40] did not detect any acrosome-reacted spermatozoa after 31% cholesterol efflux in 90 min. They obtained acrosome responsiveness only after the addition of lysophosphatidylcholine. Our results show that if insufficient cholesterol is removed from the membrane (around 30%), as detected in 2 mM ßCD, no AR is induced. However, if 40–60% of cholesterol was removed, as occurs in 4 mM ßCD, we observed that AR was induced.
It has been determined both in vivo and in vitro that heparin or glycosaminoglycans capacitate sperm, probably due to their ability to sequester coating proteins, some of them, for this reason, named heparin-binding proteins [25,40]. We do not find a synergistic effect on the cholesterol efflux or on the AR percentage when adding heparin to ßCD. This could indicate that the removal of coating proteins is not necessary for cholesterol uptake by ßCD.
In our study we provide evidence that with use of ßCD in the medium, a high percentage of cells undergo the AR, the magnitude of the response being in the range of the induced AR obtained by other authors with different inducers. Cross [8] reported 30% AR in human spermatozoa when induced with progesterone in the presence of 26 mg/ml of BSA in the medium. Alternatively, a fusogenic lipid, lysophosphatidylcholine (LC), may be used in the capacitation medium as a potent inducer of AR. With use of LC, as much as an 80% increase in the percentage of acrosome-reacted cells was obtained by Parrish et al. [25], but spermatozoa in which the AR is not induced by LC achieve only a 15% spontaneous AR among the sperm population. Our results might suggest that merely removing sufficient cholesterol from sperm membranes would trigger a series of molecular events leading to AR.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Support for this research was provided by grant SAF95-0268 from CICYT (P.M.). 
2 Correspondence: Antoni Morros, Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Edifici M, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. FAX: 34–93–5811907; amorros{at}cc.uab.es
Accepted: October 6, 1999.
Received: August 13, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, ß- and - cyclodextrins on human erythrocytes. Eur J Biochem 1989; 186:17–22.[Medline]This article has been cited by other articles:
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  K. Muller, P. Muller, G. Pincemy, A. Kurz, and C. Labbe Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa Biol Reprod, March 1, 2008; 78(3): 390 - 399. [Abstract] [Full Text] [PDF]
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T. Takeo, T. Hoshii, Y. Kondo, H. Toyodome, H. Arima, K.-i. Yamamura, T. Irie, and N. Nakagata Methyl-Beta-Cyclodextrin Improves Fertilizing Ability of C57BL/6 Mouse Sperm after Freezing and Thawing by Facilitating Cholesterol Efflux from the Cells Biol Reprod, March 1, 2008; 78(3): 546 - 551. [Abstract] [Full Text] [PDF]
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J. R. Herrick, P. Bartels, and R. L. Krisher Postthaw Evaluation of In Vitro Function of Epididymal Spermatozoa from Four Species of Free-Ranging African Bovids Biol Reprod, September 1, 2004; 71(3): 948 - 958. [Abstract] [Full Text] [PDF]
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 M. Cotman, D. Jezek, K. F. Tacer, R. Frangez, and D. Rozman A Functional Cytochrome P450 Lanosterol 14{alpha}-Demethylase CYP51 Enzyme in the Acrosome: Transport through the Golgi and Synthesis of Meiosis-Activating Sterols Endocrinology, March 1, 2004; 145(3): 1419 - 1426. [Abstract] [Full Text] [PDF]
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 K. Fukami, M. Yoshida, T. Inoue, M. Kurokawa, R. A. Fissore, N. Yoshida, K. Mikoshiba, and T. Takenawa Phospholipase C{delta}4 is required for Ca2+ mobilization essential for acrosome reaction in sperm J. Cell Biol., April 14, 2003; 161(1): 79 - 88. [Abstract] [Full Text] [PDF]
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 K. F. Tacer, T. B. Haugen, M. Baltsen, N. Debeljak, and D. Rozman Tissue-specific transcriptional regulation of the cholesterol biosynthetic pathway leads to accumulation of testis meiosis-activating sterol (T-MAS) J. Lipid Res., January 1, 2002; 43(1): 82 - 89. [Abstract] [Full Text] [PDF]
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4 µM. Data are expressed as mean ± SEM (n = 4)
