1 Laboratoire de Biologie de la Reproduction, CHU La Grave, 31052 Toulouse Cedex and 2 INSERM U326, CHU Purpan, Toulouse Cedex, France
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Abstract |
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Key words: acrosome/cholesterol/cyclodextrin/spermatozoa/zona pellucida
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Introduction |
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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., 1995) and to induce capacitation of mouse spermatozoa (Choi and Toyoda, 1998
).
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Materials and methods |
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2-hydroxypropyl-ß-cyclodextrin (Sigma Chemical Co., St Louis, MO, USA) was dissolved in SMART1 (1 mg/ml) (Choi and Toyoda, 1998).
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 (36105x106).
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, 1991).
Hemizona assay
Hemizona assay was performed according to a previously described method (Burkman et al., 1988). 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 ( 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., 1990), 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., 1990
). In each sample, 200 spermatozoa were observed.
Extraction and analysis of neutral lipids by gas liquid chromatography (Vieu et al., 1996)
Lipids from each sample were extracted according to a previously published method (Bligh and Dyer, 1959) 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-13 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., 1961) following lipid extraction.
Statistical analysis
Data are means ± SEM. Statistical comparisons were performed using the non-parametric Wilcoxon paired test.
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Results |
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Discussion |
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These findings are in good agreement with others (Choi and Toyoda, 1998), 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, 1998
; Visconti et al., 1999
). 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., 1993
). 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., 1996) 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 1530 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 34 h (Cummins et al., 1991
) and since hemizona assay is assessed after a 4 h incubation of the spermatozoa with the zonae (Burkman et al., 1988
), 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., 1998). 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., 1982
). 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, 1998) 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., 1991
). 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 2
). 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., 1995
). 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.
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Notes |
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References |
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Submitted on October 28, 1999; accepted on January 20, 2000.