Enhancement of zona binding using 2-hydroxypropyl- ß-cyclodextrin

Jean Parinaud1,3, Gérard Vieitez1, Claude Vieu2, Xavier Collet2 and Bertrand Perret2

1 Laboratoire de Biologie de la Reproduction, CHU La Grave, 31052 Toulouse Cedex and 2 INSERM U326, CHU Purpan, Toulouse Cedex, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since membrane cholesterol depletion is known to play an important role in sperm capacitation, we have investigated the effect of 2-hydroxypropyl-ß-cyclodextrin, a cyclic oligosaccharide that mediates cholesterol efflux, on sperm functions. Sperm treatment with cyclodextrin did not affect the motility patterns but induced an increase in sperm binding to zona pellucida (24 ± 5 versus 13 ± 4 in control, P < 0.01). Cyclodextrin treatment was associated with an increase in spontaneous acrosome reaction (32 ± 8% versus 22 ± 4% in controls after a 4 h incubation, P < 0.05; 61 ± 10% versus 50 ± 11% in controls after a 24 h incubation, NS) but with a decrease in acrosome response to ionophore challenge (44 ± 5% versus 51 ± 3% in controls, P < 0.05). Concerning cell sterols, cyclodextrin induced a rapid and dramatic fall in the cholesterol and desmosterol content of spermatozoa. We conclude that cyclodextrin is a powerful capacitating agent, but since it induces an increase in spontaneous acrosome loss, it needs to be further evaluated before routine use in assisted reproductive technology media.

Key words: acrosome/cholesterol/cyclodextrin/spermatozoa/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dramatic changes in membrane lipid contents have been reported to occur during sperm capacitation (Harrison and Gadella, 1995Go; Muller and Ravnik, 1995Go). Indeed, this event, which plays a key role in fertilization processes, is associated with a loss of membrane cholesterol and a decrease in the cholesterol/phospholipid ratio (Davis, 1981Go; Hoshi et al., 1990Go; Benoff, 1993Go; Cross, 1998Go). These modifications of the lipid content increase the fluidity of the membranes and therefore induce the exposure of binding sites for zona pellucida (Gamzu et al., 1997Go) and for D-mannose (Benoff et al., 1993Go) and the ability to undergo acrosome reaction (Davis, 1981Go). Furthermore, incubations in cholesterol-enriched medium prevent acrosome reaction in response to progesterone (Cross, 1996aGo) and the inhibitory effect of seminal plasma on acrosome response to progesterone is highly correlated with the cholesterol content of ejaculates (Cross, 1996bGo). Human follicular fluid, which is known as a capacitating agent (Kulin et al., 1994Go), acts, at least in part, through a depletion of sperm cholesterol (Hamamah et al., 1995Go). The cholesterol acceptors in follicular fluids have been identified as high density lipoproteins (HDL) and albumin (Langlais et al., 1988Go), as well as the lipid transfer protein-I (Ravnik et al., 1993Go). From a clinical point of view, the cholesterol/phospholipid ratio has been shown to be higher in spermatozoa from infertile patients than from fertile donors (Sugkraroek et al., 1991Go) and this ratio was significantly decreased by Percoll preparation (Sugkraroek et al., 1991Go), a procedure used for sperm capacitation before in-vitro fertilization (IVF).

The aim of the present study was to evaluate, on sperm function in vitro, the effect of 2-hydroxypropyl-ß-cyclodextrin, a cyclic oligosaccharide that has been demonstrated to mediate cholesterol efflux in various cell models (Kilsdonk et al., 1995Go) and to induce capacitation of mouse spermatozoa (Choi and Toyoda, 1998Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Media
SMART1 (Parinaud et al., 1998Go) was obtained from Bio-Media (Boussens, France). This albumin-free medium, based on Earle's salt solution, contains glucose, amino acids, synthetic and plant molecules and is devoid of any serum-extracted compound.

2-hydroxypropyl-ß-cyclodextrin (Sigma Chemical Co., St Louis, MO, USA) was dissolved in SMART1 (1 mg/ml) (Choi and Toyoda, 1998Go).

Sperm preparation
Ten semen samples from 10 healthy fertile volunteers (mean age ± SD: 35 ± 4 years) were used. After 30 min liquefaction at 37°C, motile spermatozoa were isolated using a discontinuous (60, 80, 90%) PureSperm® (NidaCon International AB, Gothenburg, Sweden) gradient made with SWIM medium (Bio-Media). Following centrifugation for 30 min at 200 g, the 90% PureSperm® fraction was recovered and sperm cells were resuspended in 4 ml SWIM. This suspension was split into two equal parts and then centrifuged. One pellet was resuspended in control SMART1 medium (control), the other in cyclodextrin (1 mg/ml) containing SMART1 medium (cyclodextrin). The mean ± SD (range) sperm concentration of the 10 samples was 65 ± 23x106 (36–105x106).

Sperm kinematics
Motility parameters were assessed in 20 µl of semen for each sample in a 2-well microcell (Cryo Bio System, L'Aigle, France), after incubation for 30 min, 60 min, 4 h and 24 h, at 37°C using a Hamilton Thorn Motility Analyzer® version 10.8 (Hamilton Thorn Research, Beverly, MA, USA). Machine parameters were frame rate of 25/s, minimum contrast 8, minimum size 6, low/high size gates 0.5/1.7, low/high intensity gates 0.4/1.7, non-motile head size 9, non-motile intensity 200, a path velocity >5 µm/s to be counted as motile. The variables measured included the percentage motile, percentage progressive motility, straight line velocity (VSL), mean path velocity (VAP), curvilinear velocity (VCL), mean linearity, mean amplitude of lateral head displacement (ALH) and percentage of motile spermatozoa with a path velocity >25 µm/s defined as the rapid motility. Hyperactivated motility was defined as follows: VCL >100 µm/s, linearity <65% and ALH >7.5 µm (Burkman, 1991Go).

Hemizona assay
Hemizona assay was performed according to a previously described method (Burkman et al., 1988Go). Briefly, for each sample, three hemizonae, obtained from mature unfertilized and non-activated oocytes after intracytoplasmic sperm injection, were incubated with control and cyclodextrin for 4 h at 37°C, with 5% CO2 to achieve maximal binding. Spermatozoa were not pre-incubated and their concentration was adjusted to 0.1x106 (motile cells). The zonae were then washed twice in Tyrode's solution (GIBCO BRL, Cergy Pontoise, France) and the number of bound spermatozoa was recorded. The results are expressed as the mean of the number of bound spermatozoa from three hemizonae.

Spontaneous acrosome reaction
The spontaneous acrosome reaction was determined as the percentage of reacted spermatozoa after incubation for 30 min, 4 h and 24 h at 37°C with 5% CO2.

Calcium ionophore challenge
After 4 h incubation at 37°C with 5% CO2 in either control or cyclodextrin, spermatozoa were incubated for 30 min in control and cyclodextrin containing 10 µmol/l calcium ionophore A 23187 (Sigma Chemical Co.). Response ({Delta} A23187) was assessed by the difference between percentages of acrosome-reacted spermatozoa before and after A 23187 addition.

Assessment of acrosomal status
Acrosomal status and viability of spermatozoa were monitored as previously described (Mortimer et al., 1990Go), combining the use of fluorescent peanut (Arachis hypogea) agglutinin lectin (Sigma Chemical Co.) and ethidium homodimer (Interchim, Montlucion, France). Spermatozoa were considered to be reacted when they were classified as equatorial segment or dark (Mortimer et al., 1990Go). In each sample, 200 spermatozoa were observed.

Extraction and analysis of neutral lipids by gas liquid chromatography (Vieu et al., 1996Go)
Lipids from each sample were extracted according to a previously published method (Bligh and Dyer, 1959Go) after acidification of the aqueous phase with formic acid (12 µl/ml). Since in previous studies, about 500 pmol of free cholesterol was extracted from 106 spermatozoa, four internal standards were added before extraction on this basis: 0.5 µg of stigmasterol, 0.125 µg of 1.3 dimyristoyl-sn-glycerol (DG14:0), 0.05 µg of heptadecanoyl cholesterol (CE 17:0), and 0.05 µg of triheptadecanoyl glycerol (TG 17:0), obtained from Sigma Chemical Co. A small quantity of the following lipid compounds, analysed under their natural formulae, was added: sn-1–3 dimyristoyl glycerol, cholesteryl heptadecanoate and triheptadecanoyl glycerol. This allowed us to see that quantification of other neutral lipids was not possible due to the very low amounts involved. After evaporation to dryness of the chloroform phase, extracts were dissolved in 100 µl ethyl acetate. Lipids were analysed by gas liquid chromatography (Intersmat, Paris, France, model 120 DFL), using an Ultra 1 Hewlett Packard (Evry, France) fused silica capillary column (5 mx0.31 mm internal diameter) coated with cross-linked methyl silicone. Oven temperature was programmed from 205°C to 345°C at a rate of 6°C/min and the carrier gas was hydrogen (0.5 bar). The response factors for the different lipid classes were determined using a mixture of internal standards. The intra- and interassay coefficients of variation were less than 6%.

Phospholipids were measured according to their phosphorus content (Böttcher et al., 1961Go) following lipid extraction.

Statistical analysis
Data are means ± SEM. Statistical comparisons were performed using the non-parametric Wilcoxon paired test.


    Results
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 Materials and methods
 Results
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No difference was found in the percentage of motile forms or in the kinematic parameters between control- and cyclodextrin-incubated sperm cells at any incubation time. However, treatment with cyclodextrin was associated with a significant increase in the number of zona-bound spermatozoa (24 ± 5 versus 13 ± 4 in control, P < 0.01) (Figure 1Go). Concerning the acrosome function, it appeared that the response to ionophore challenge was significantly reduced with cyclodextrin (44 ± 5% versus 51 ± 3% in control, P < 0.05), while the spontaneous acrosome reaction (Figure 2Go) was significantly increased (32 ± 8% in cyclodextrin versus 22 ± 4% in control) after a 4 h incubation (P < 0.05); 61 ± 10% in cyclodextrin versus 50 ± 11% in control after a 24 h incubation (NS). Measurements of the cholesterol content of the spermatozoa (Figure 3Go) indicated that, in all cases, incubation with cyclodextrin was associated with a lower cholesterol content than controls (392 ± 258, 175 ± 92, 250 ± 101 and 144 ± 63 pmol/106 spermatozoa after a 30, 60, 120 and 240 min incubation for control versus 120 ± 24, 73 ± 18, 109 ± 34 and 75 ± 19 pmol/106 spermatozoa after a 30, 60, 120 and 240 min incubation with cyclodextrin). The overall cholesterol content was significantly lower in cyclodextrin-incubated spermatozoa than in control (261 ± 97 versus 90 ± 13 pmol/106 spermatozoa, P < 0.01). Over the incubation period, it appeared that, even in controls, there was a dramatic decrease in the cholesterol content, but this phenomenon was more rapid and important in the cyclodextrin-treated samples. Similar findings were obtained for desmosterol (160 ± 117, 56 ± 33, 91 ± 40 and 53 ± 29 pmol/106 spermatozoa after a 30, 60, 120 and 240 min incubation for control versus 34 ± 4, 27 ± 5, 39 ± 12 and 21 ± 9 pmol/106 spermatozoa after a 30, 60, 120 and 240 min incubation with cyclodextrin). The overall demosterol content was significantly lower in cyclodextrin-incubated spermatozoa than in control (34 ± 6 pmol/106 spermatozoa versus 95 ± 35 pmol/106 spermatozoa, P < 0.05). Moreover, the cholesterol to phospholipid ratio was reduced in the presence of cyclodextrin (0.77 ± 0.23, 0.62 ± 0.15, 0.95 ± 0.22 and 0.63 ± 0.11 after a 30, 60, 120 and 240 min incubation for control versus 0.56 ± 0.16, 0.41 ± 0.08, 0.59 ± 0.07 and 0.55 ± 0.08 after a 30, 60, 120 and 240 min incubation with cyclodextrin). The overall cholesterol to phospholipid ratio was significantly lower in cyclodextrin-incubated spermatozoa than in control (0.52 ± 0.05 versus 0.71 ± 0.33, P < 0.05).



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Figure 1. Results of hemizona assay in control and cyclodextrin-treated samples. Data are means ± SEM of 10 samples from 10 individual donors. *P < 0.01.

 


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Figure 2. Percentage of spontaneously acrosome-reacted spermatozoa as a function of incubation length in control and cyclodextrin treated samples. Data are means ± SEM from 10 samples. *P < 0.05 versus control at the same time.

 


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Figure 3. Cholesterol content of spermatozoa as a function of incubation time in control and cyclodextrin-treated samples. Data are means ± SEM from four samples.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of the present study show that cyclodextrin induces both a fall in the sterol content of spermatozoa and an increase in the binding to the zona pellucida.

These findings are in good agreement with others (Choi and Toyoda, 1998Go), who have shown that, in the mouse, pre-incubation of spermatozoa with cyclodextrin enhanced capacitation, as evaluated by the chlortetracycline fluorescence assay, and in IVF. These effects were related to the release of cholesterol and desmosterol from sperm membranes (Choi and Toyoda, 1998Go; Visconti et al., 1999Go). Cholesterol content of spermatozoa is known to play a key role in capacitation since it has been related to mannose-ligand receptor expression (Benoff et al., 1993Go). Moreover, Cross (1996) has shown that incubation in cholesterol-enriched medium prevents spermatozoa from responding to progesterone stimulation.

The kinetics of cholesterol and desmosterol content loss appeared to be very rapid since, after a 30 min incubation, the lowest cholesterol content of spermatozoa was reached and prolonged incubations did not modify this content. These results agree with those of others (Yancey et al., 1996Go) who have demonstrated, in mouse L-cells and on GM3468A human skin fibroblasts, the existence of two kinetic pools of cholesterol, the slowest having a half-life of 15–30 min. However, it must be pointed out that in control medium there was also a decrease in cholesterol content, although less deep and rapid. Since acrosome response to ionophore necessitates a pre-incubation period of 3–4 h (Cummins et al., 1991Go) and since hemizona assay is assessed after a 4 h incubation of the spermatozoa with the zonae (Burkman et al., 1988Go), the immediate biological consequences of the rapidity of cholesterol loss in the presence of cyclodextrin cannot really be monitored.

In the present study, we have used SMART1, a medium devoid of protein and therefore of cholesterol acceptors (Parinaud et al., 1998Go). We can thus postulate that cyclodextrin enhances and accelerates a spontaneous cholesterol loss which normally occurs in a cholesterol-free medium. The choice of SMART1 medium instead of an albumin-supplemented medium was made in order to avoid the problem of cholesterol efflux mediated by albumin and the batch to batch variations due to the lipid content of albumin (Dröge et al., 1982Go). Since SMART1 is completely synthetic, this medium is perfectly chemically defined and constitutes a better tool to study effects of molecules on capacitation than albumin-supplemented ones.

The use of cyclodextrin enhances zona binding but also spontaneous acrosome reaction. Similar results were previously obtained (Choi and Toyoda, 1998Go) using mouse spermatozoa. This finding indicates that cholesterol depletion induces better availability of ZP3 receptors but destabilizes membranes allowing acrosome reaction to occur. This is not surprising since capacitation is an intermediary step before acrosome reaction and a high capacitation state may lead to acrosome exocytosis. It could seem paradoxical that cyclodextrin enhances both zona binding and spontaneous acrosome loss, since spontaneous acrosome reaction has been shown to be associated with low fertilization rates (Fénichel et al., 1991Go). However, it must be pointed out that at the beginning of incubation with zonae, the percentages of reacted spermatozoa were almost identical with or without cyclodextrin (23.4 ± 15.4 versus 21.4 ± 11.2, Figure 2Go). It was only in longer incubations that an increase in acrosome-reacted spermatozoa was observed. Moreover, since in the hemizona assay zonae are not surrounded by cumulus oophorus, the sperm binding probably occurs rapidly. This point is of great importance for the clinical use of capacitating agents. Indeed, if capacitation is necessary for fertilization, spontaneous acrosome reaction is deleterious (Parinaud et al., 1995Go). Since the present study was performed using normal semen samples with a low rate of spontaneous acrosome reaction, we may suspect that the use of cyclodextrin could exacerbate the fertilizing defect of samples displaying a high rate of spontaneous acrosome reaction in conventional conditions. Therefore, the use of cholesterol acceptors with such an efficiency must be done with care in routine IVF protocols.


    Notes
 
3 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on October 28, 1999; accepted on January 20, 2000.