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d -Mannose-binding sites are putative sperm determinants of human oocyte recognition and fertilization
RBMOnline - Vol 15. No 2. 2007 182-190 Reproductive BioMedicine Online; www.rbmonline.com/Article/2786 on web 5 June 2007
Article D-Mannose-binding
sites are putative sperm determinants of human oocyte recognition and fertilization Dr Maria Jose Munuce is a Staff Professor in the Clinical Biochemistry Department of the School of Biochemical and Pharmaceutical Sciences at the University of Rosario, Argentina. She was a Fellow of the World Health Organization’s Human Reproduction Programme and the Puigvert Foundation at Barcelona, Spain. She has published over 15 scientific papers and given over 50 presentations at both national and international scientific meetings. Her research is focused on the study of the effect of female genital tract secretions on sperm function.
Dr Maria Jose Munuce Germán Rosano, Adriana M Caille, Marlene Gallardo-Ríos and María José Munuce1 Laboratorio de Estudios Reproductivos, Cátedra de Bioquímica Clínica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, San Lorenzo 939-9A (2000), Rosario, Argentina 1 Correspondence: Fax: +54 414251738; e-mail: [email protected]
Abstract The aim of the present study was to further evaluate the participation of D-mannose in the process of human sperm–egg interaction. Zona pellucida binding competitive assays in the presence of D-mannose were carried out using discarded oocytes from IVF. Spermatozoa were capacitated and D-mannose-binding site (MBS) expression, sperm viability and follicular fluidinduced acrosome reaction (AR) were evaluated. MBS were visualized using a fluorescein-neoglycoprotein probe. The capacity of free D-mannose and mannosylated albumin to induce the AR was also tested. MBS and the IVF outcome were also analysed. The involvement of D-mannose in sperm binding to the zona pellucida was verified by the inhibitory effect produced when the sugar was present during binding assays. MBS expression increased during capacitation, in parallel with the ability to undergo the induced AR. Mannosylated albumin, but not the free sugar, induced the AR. In acrosome-reacted spermatozoa, the MBS was located at the plasma membrane, as shown by confocal analysis. No significant difference in the increase in MBS expression was observed among the different IVF groups of patients. The data show that D-mannose is involved in the sperm–zona pellucida interaction, and that the expression of MBS on the sperm surface occurs during the acquisition of in-vitro sperm fertilizing ability. Keywords: acrosome reaction, D-mannose-binding site, hemizona assay, zona pellucida binding
Introduction
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For fertilization to occur, spermatozoa must first recognize and bind to the zona pellucida, triggering the exocytotic release of the acrosomal contents, and allowing spermatozoa to penetrate the zona pellucida and fertilize the egg (Yanagimachi, 1988). There is compelling evidence indicating that carbohydrates mediate the sperm–egg interaction. The proposed mechanism for this event is based on the specific binding of sperm-surface carbohydrate-binding proteins to glycoconjugates present in the zona pellucida, and this sets off the signal transduction pathways that result in the acrosome reaction (AR) (for reviews see Benoff, 1997; Töpfer-Petersen, 1999; Oehningher, 2003). The exact sugar
composition of zona glycoproteins and the molecular structure of the recognition/binding site(s) for human spermatozoa have not yet been determined. The detection of D-mannose-binding sites (MBS) on the sperm surface was first described by Tesarik et al. (1991) using a fluoresceinated neoglycoprotein containing D-mannose residues. The increase in the population of spermatozoa expressing MBS during capacitation has been proposed as the basis of a clinical test to discriminate between ‘fertile’ and ‘infertile’ patients undergoing IVF procedures (Benoff et al., 1993a, Hershlag et al., 1998).
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
In humans, it has been demonstrated that the fertile days are the 5 days previous to ovulation (Wilcox et al., 1995); thus spermatozoa have to wait in the female genital tract before encountering the oocyte. This interval provides an opportunity for female genital tract secretions to modulate sperm function (Barratt and Cooke, 1991). We have reported that both human follicular fluid and peritoneal fluids, which are present in the ampullary microenvironment, regulate sperm binding to the zona pellucida by decreasing the number of available MBS on the sperm head (Munuce et al., 2003, 2004). Furthermore, it has been reported that a family of glycoproteins called glycodelins present in human follicular fluid bind to the acrosomal region of intact spermatozoa and inhibit zona pellucida binding (Chiu et al., 2003), maybe through the terminal D-mannose residues in the protein (Chiu et al., 2004). In addition, the authors of this study have recently reported that levonorgestrel, a progestagen used in a contraceptive device placed in the female uterine cavity, increases the proportion of spermatozoa showing a MBS distribution associated with a lower ability to interact with the zona pellucida, maybe as part of its antifertility activity (Munuce et al., 2006). Considering that MBS could be an expression of in-vitro sperm fertilizing capacity and a target for its regulation, the participation of D-mannose in the sperm–egg interaction was investigated.
Materials and methods The present study was approved by the Institutional Review Board of School of Biochemical and Pharmaceutical Sciences of the National University of Rosario, Argentina. All human samples used in this study were obtained with the donors’ written consents.
Human follicular fluid Human follicular fluids were collected during oocyte retrieval from hormone-stimulated women (n = 6) participating in a local IVF programme. Only human follicular fluids with no blood contamination and obtained from follicles containing a mature oocyte (metaphase II) were collected. Follicular fluids from each sample were centrifuged (10 min, 600 g) to remove cellular debris, filtered through a 0.22 μm membrane (Millipore Corporation, Bedford, MA, USA), mixed into a pool and stored at –20°C (<12 months). The activity of the pool of human follicular fluid was checked monthly by analysing its ability to induce the acrosome reaction on capacitated spermatozoa.
Semen samples, sperm processing and capacitation Fresh semen specimens were obtained from normozoospermic donors after 3 to 5 days of sexual abstinence (n = 28). Semen analysis was performed according to World Health Organization guidelines (WHO, 1999) and morphological strict criteria (Kruger et al., 1988). Motile spermatozoa were selected by layering 1 ml of semen on top of a discontinuous 90–50% Percoll gradient (ICN Biomedicals, Aurora, OH, USA). Following centrifugation for 20 min at 275 g, the supernatant was discarded by gentle aspiration and the sperm RBMOnline®
pellet was then washed with 1 ml of Ham’s F-10 medium (ICN Biomedicals). Spermatozoa were resuspended in Ham’s F-10 medium supplemented with 35 mg/ml bovine serum albumin (BSA; ICN Biomedicals) at a concentration of 4 × 106 to 5 ×106 spermatozoa per ml and incubated for up to 22 h at 37°C in 5% CO2 in air. Capacitation status was evaluated by incubating capacitated spermatozoa for 30 min with 20% v/v human follicular fluid according to the method of Calvo et al. (1989) at 37°C in 5% CO2 in air, and determining the acrosomal status by the Pisum sativum technique (as described below).
Participation of D-mannose in the sperm– egg interaction Unfertilized human oocytes, donated by women undergoing IVF, were stored in a salt solution containing 1.5 mol/l MgCl2, 0.1% polyvinylpyrrolidone (MW 360 000) and 40 mmol/l HEPES in phosphate-buffered saline (PBS; pH 7.2). It has been previously shown that under these conditions, the zona pellucida retains its normal sperm-binding capacity, while the ooplasm and nucleus degenerate (Yanagimachi et al., 1979). For whole-oocyte–zona-binding assays, desalted oocytes were washed and randomly placed in a 100 μl droplet of mineraloil-overlaid Ham’s F-10 containing 35 mg/ml BSA and 0, 30, 100 or 300 mmol/l D-mannose, and inseminated with 1 × 105 motile progressive spermatozoa per ml for 4 h at 37°C under 5% CO2 in air. Oocytes were then washed by repeated pipetting to remove loosely attached spermatozoa and then placed on a slide under a coverslip supported by paraffin/Vaseline bases (80:20% v/v). For the hemizona assays, desalted oocytes were immobilized with a holding pipette and separated into equal halves using a microscalpel. Each hemizona was then placed in a 100-μl drop of Ham’s F-10 supplemented with 35 mg/ml BSA, with and without 30 mmol/l D-mannose. Hemizonas were then incubated for 4 h with 1 × 105 progressively motile spermatozoa per ml at 37°C under 5% CO2 in air, and treated as described for the whole-oocyte–zona-binding assay. Results were expressed as a hemizona index (HZI) = [(number of spermatozoa bound in treated hemizona) / (number of spermatozoa bound in control hemizona)] × 100.
Localization of D-mannose binding sites Spermatozoa were labelled with fluorescein-isothiocyanateconjugated mannosylated BSA (Man–FITC–BSA; Sigma Chemical Co., St Louis, USA) according to the protocol of Benoff et al. (1993a). Briefly, spermatozoa were washed twice with core buffer (30 mmol/l HEPES, 0.5 mmol/l MgCl2, 150 mmol/l NaCl, 10 mg/ml BSA, pH 7.0) supplemented with 20 mmol/l Ca2+ and incubated for 30 min with 100 μg/ml Man– FITC–BSA at 37°C and 5% CO2 in air. Following labelling, spermatozoa were pelleted by centrifugation and washed twice with core buffer without Ca2+. Sperm viability was assessed by eosin Y exclusion (WHO, 1999) before and after every labelling reaction. Spermatozoa were immobilized on glass slides, air-dried and mounted with glycerol/PBS (9:1, pH 9.0). Specimens were evaluated at ×1000 magnification using a Leica microscope (Leica Microsystems, Wetzlar, Germany) equipped with epifluorescence optics specific for fluorescein excitation (BP490 excitation, 500 dichroic mirror and LP515 barrier).
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Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al. Two hundred spermatozoa were counted for each individual experiment and the fluorescence signal was classified as: neck/ midpiece and tail (pattern I); entire acrosomal cap plus neck/ midpiece (pattern II); or equatorial/post-equatorial regions plus neck/midpiece (pattern III). Each slide was conserved at 4°C and analysed within a 2-week period.
Ligand specificity To investigate sugar specificity of MBS, 4 h capacitated spermatozoa were incubated for 60 min with 30 mmol/l D-glucose, D-galactose, N-acetyl-D-glucosamine, D-fucose or D-mannose (Sigma Chemical Co.), at 37°C and 5% CO2 in air, and then the proportion of cells expressing MBS was evaluated as described above.
Effect of D-mannose and mannosylatedBSA on sperm acrosomal status To investigate the ability of D-mannose to induce AR, overnightincubated spermatozoa were treated with one of the following: 30 mmol/l D-mannose, 100 μg/ml mannosylated BSA (D-Man– BSA; Fertility Technologies Inc., Natick, MA, USA), 20% v/v human follicular fluid, or control medium, for 2 h at 37°C under 5% CO2 in air. The acrosomal status was then determined using the Pisum sativum technique.
Acrosomal status evaluation by the Pisum sativum technique Spermatozoa were washed twice in 1 ml PBS and immobilized on slides, air-dried, permeabilized by exposure for 30 s to cold absolute methanol, and stained with fluoresceinated Pisum sativum agglutinin (50 μg/ml; Sigma Chemical Co.) for 30 min at room temperature. At least 200 spermatozoa were evaluated and classified as follows: (i) acrosome-intact, if the acrosomal cap was uniformly labelled; (ii) acrosome-reacting, if the acrosomal region of the head was labelled in a patchy pattern; or (iii) acrosome-reacted, if only the equatorial segment was labelled. The percentage of acrosome-reacted cells was calculated as b + c, unless otherwise noted.
Confocal laser scanning microscopy In order to determine the localization and topography of MBS after the occurrence of the AR, spermatozoa showing pattern III (acrosome-reacted) were evaluated by the use of a confocal laser scanning microscope (Carl Zeiss, Jena, Germany) at ×880. Serial confocal sections (stacking) were recorded at 0.35 μm focus steps. Image analysis and three-dimensional reconstruction were carried out using LSM Image Browser software (Carl Zeiss), while Scion Image (Scion Corporation, Frederick, USA) was used for histogram analysis of signal intensity and localization.
Detection of D-mannose binding sites and IVF outcome 184
A prospective, double-blind analysis of patients (n = 26) participating in the IVF programme at the Programa de
Asistencia Reproductiva de Rosario was undertaken. Conventional IVF inseminations were used in all cases. On the day of the oocyte retrieval, motile spermatozoa were selected by the swim-up technique (WHO, 1999). The remains of the semen sample were divided and use to determine MBS in fresh and overnight capacitated spermatozoa, as described above. Results were normalized as proposed by Hershlag et al. (1998) to represent the increase in MBS pattern II after incubation: percentage of incubated spermatozoa showing MBS pattern II minus the percentage of fresh cells with MBS pattern II divided by the percentage of incubated spermatozoa showing binding Man–FITC–BSA. Fertilization rate was defined as the ratio between oocytes that developed into normal two-pronuclear embryos 16–18 h after insemination and the total number of mature (metaphase II) oocytes inseminated. Patients were assigned to three groups according the rate of fertilization achieved: group 1 (n = 5), ≤50%; group 2 (n = 7), 50–75%; and group 3 (n = 14), >75%; and the average increase in MBS pattern II calculated for each group.
Statistical analysis Data processing was performed using the GraphPad InStat program (GraphPad Software, San Diego, CA, USA). Differences between treatments or groups were determined by one-way analysis of variance for matched samples and the Tukey–Kramer multiple comparison post-test. The difference in the number of spermatozoa bound to treated versus control oocytes was analysed using the non-parametric Kruskal– Wallis test and Dunn’s multiple comparisons post-tests. The non-parametric Wilcoxon sign ranked test for paired samples was applied for the analysis of the hemizona assays. Data are expressed as means ± SEM and a P-value <0.05 was considered significant.
Results The participation of D-mannose in the process of sperm–zona pellucida interaction was first evaluated by incubating both gametes in the presence of this monosaccharide. Using the whole-oocyte test, 300 mmol/l D-mannose was required to observe a significant reduction in the number of spermatozoa bound to the zona pellucida (P < 005, n = 6, Table 1). It is remarkable that at this concentration, sperm vitality was reduced to 70% viable cells. Given the high variability in the number of spermatozoa bound per oocyte, the hemizona assay was also used. In this case, the presence of 30 mmol/l D-mannose was enough to significantly reduce the number of bound spermatozoa (P < 003, n = 6, hemizona assay) with a HZI of 55 ± 13% (Table 2). When MBS on the sperm head were evaluated by fluorescent labelling, three characteristic fluorescence patterns, consistent with those reported by Benoff et al., (1993a), could be detected. All cells presented a nonspecific labelling in the neck/midpiece region (pattern I). In addition, some cells also showed a uniform distribution of the fluorescent signal either over the acrosomal cap (pattern II) or in the equatorial segment and/or post-acrosomal region (pattern III), considered as a specific head labelling (Figure 1a). The percentage of spermatozoa showing specific patterns of Man–FITC–BSA binding increased in a time-dependent manner. After 4 h RBMOnline®
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
incubation under capacitating conditions, this percentage was significantly different (P < 0.05; n = 5) from t = 0 h, reaching even higher values at 22 h (Figure 1b). In order to determine whether the increase in MBS was associated with capacitation, at the end of the incubation period spermatozoa were exposed to 20% v/v human-follicular-fluid. Data showed that there was a significant increase in the proportion of acrosome-reacted spermatozoa in the human-follicularfluid-treated group with respect to controls: human follicular fluid 41 ± 4% versus control 17 ± 1% (P < 0.003, n = 5). To exclude any deleterious effect of the procedure, viability was evaluated before and after the labelling protocol. Exposure of spermatozoa to Man–FITC–BSA did not affect the percentage of viable spermatozoa since the difference in live cell numbers before and after the labelling reaction did not exceed 10% (data not shown). To determine whether other monosaccharides that are structurally similar to D-mannose could also bind to the MBS, 4 h capacitated spermatozoa were exposed to five different hexoses before the labelling reaction. Data showed that only the preincubation of spermatozoa with D-mannose significantly decreased the percentage of cells displaying specific patterns (Figure 2, P < 0.001, n = 4). The percentage of viable cells at the end of the procedure was unaffected (>85% viable cells in all treatments). To investigate whether D-mannose was related to the AR, capacitated spermatozoa were exposed to D-mannose or mannosylated BSA. Results indicated that only when spermatozoa were exposed to D-mannose coupled to a protein matrix (D-Man–BSA) was there a significant increase in the percentage of acrosome-reacted cells (Figure 3, P < 0.001, n = 7). However, the magnitude of the AR induced by D-Man-BSA was significantly lower (P < 0.05) than the
Table 1. Effects of D-mannose on the whole-oocyte–zonabinding assay (n = 6). Treatment
Number of oocytes
Number of bound spermatozoaa
Control D-Mannose (30 mmol/l) D-Mannose (100 mmol/l) D-Mannose (300 mmol/l)
24 5 12 8
17 ± 5 26 ± 3 11 ± 4 4 ± 1b
one produced by human follicular fluid (considered to be the positive control). In order to investigate the location of MBS after the occurrence of the AR, in some experiments spermatozoa showing pattern III were further analysed by confocal microscopy. The analysis of three-dimensional reconstruction by confocal laser scan microscopy generated after the incubation of spermatozoa with Man–FITC–BSA showed that the fluorescence signal was located at the plasma membrane on the equatorial segment (Figure 4a). The histogram of the fluorescent signal visualized in five representative serial sections is showed in Figure 4b. The diagrams on the left represent transverse slices of the sperm head. Each box represents the section being analysed and the whole fluorescent signal is indicated in grey. On the right, the x-axis represents the length of the bar and the y-axis represents an arbitrary normalized value of the signal intensity. Results from a typical spermatozoon are shown. It has previously been observed (Munuce et al., 2006) in double-stained spermatozoa that those showing pattern III of MBS have lost their acrosomes, suggesting that both events are associated. Finally, the capacity of the MBS assay to discriminate between failed and successful IVF was investigated. Table 3 shows clinical variables in the three groups of patients included in the study. No differences in sperm count, motility or the percentage of normal forms were found. In addition, no differences in the women’s ages or in the number of mature (metaphase II) oocytes retrieved were observed between groups. Although there was a progressive increase in the mean value of MBS-pattern II increment along the groups, no statistical significance was achieved (Figure 5, n = 26, not significant). Sperm viability among groups did not differ at the end of the labelling procedure (>80% viable cells).
Table 2. Effects of D-mannose on the hemizona assay (n = 6). Treatment
Control
D-Mannose
Number of bound spermatozoa/ hemizona (30 mmol/l)
10.0 ± 1.0 5.0 ± 0.4a
Values are means ± SEM. a P < 0.03 vs. control.
Values are means ± SEM. P < 0.05 versus control.
a
b
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Figure 1a. Photomicrograph of human spermatozoa labelled with fluorescein-isothiocyanate-conjugated mannosylated bovine serum albumin (Man–FITC–BSA). There is a spermatozoon labelled in pattern III with Man–FITC–BSA; the remaining spermatozoa exhibit pattern II (×880).
Figure 1b. Expression of mannose-binding sites (MBS) during sperm incubation under capacitating conditions. The percentage of spermatozoa displaying head-directed MBS (II and III) was obtained for non-capacitated, and 4 h and 22 h capacitated spermatozoa. Results are means ± SEM, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0 h.
Figure 2. Effect of exposure to monosaccharides on the detection of sperm mannose-binding sites (MBS). Capacitated spermatozoa were preincubated for 60 min with 30 mmol/l D-mannose (D-Man), D-glucose (D-Glc), N-acetyl glucosamine (NAG), D-galactose, (D-Gal), D-fucose (D-Fuc) or control medium (C) and then the expression of MBS was detected with fluorescein-isothiocyanate-conjugated mannosylated bovine serum albumin. Results are means ± SEM, n = 4. ***P < 0.001 versus other treatments.
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Figure 3. Induction of the acrosome reaction by a D-mannose bovine serum albumin. Capacitated spermatozoa were incubated 2 h with 100 μg/ml mannosylated bovine serum albumin (D-Man–BSA), 30 mmol/l D-mannose (D-Man), 20% v/v human follicular fluid (hFF), or control medium (C). The percentage of acrosome-reacted sperm was determined by Pisum sativum agglutinin labelling. Results are means ± SEM, n = 7. *P < 0.05 versus D-Man-BSA, ***P < 0.001 versus C and D-Man. RBMOnline®
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
Figure 4a. Three-dimensional reconstruction by confocal laser scan microscopy of fluorescence of a spermatozoon showing a pattern III of mannose-binding sites. Serial optical sections were recorded in focus steps of 0.35 μm. The fluorescent area is localized on the equatorial segment (×880).
Figure 4b. Distribution of mannose-binding sites in a pattern III spermatozoon. Histogram analysis of the fluorescence signal visualized in four representative serial sections obtained using a laser confocal scanning microscope. Images of a representative cell: left, transverse cut of the sperm head (fluorescence signal in grey); bars represent each serial section. Histograms: the x-axis represents the length of the bar; the y-axis represents an arbitrary normalized value of signal intensity. RBMOnline®
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Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al. Table 3. Clinical variables in IVF patients. Parameter
Group 1 (n = 5)
Group 2 (n = 7)
Group 3 (n = 14)
Sperm count (×106/ml) Sperm motility (%) Normal forms (%) Maternal age (years) Mature oocyte (%)
132 ± 36 56 ± 3 7±1 37 ± 2 89 ± 8
123 ± 20 58 ± 4 6.7 ± 0.5 31 ± 1 88 ± 5
152 ± 17 60 ± 1 7.5 ± 0.4 36 ± 1 92 ± 5
Values are means ± SEM. Group 1, ≤50% fertilization; group 2, 50–75% fertilization; group 3, >75% fertilization. Results were not significant between groups.
Figure 5. Detection of mannose-binding sites (MBS) and IVF outcome. Patients were assigned to three groups according the rate of fertilization achieved: group 1 (n = 5), ≤50%; group 2 (n = 7), 50–75%; and group 3 (n = 14), >75%; and compared with the increase in MBS pattern II in spermatozoa capacitated for 22 h. Results are the means ± SEM, n = 26, not significant.
Discussion
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The participation of D-mannose in sperm–egg interaction was first investigated using a competitive assay. When the wholeoocyte test was used, high sugar concentrations (300 mmol/l) were required to significantly decrease the number of bound spermatozoa. These results are in agreement with those of Mori et al. (1989) who did not observe inhibition of sperm binding in the presence of up to 50 mmol/l D-mannose. However, the high osmotic pressure at 300 mmol/l decreases sperm viability and could be responsible for the lower number of bound spermatozoa observed in our study. To bypass this limitation and the oocyte variability in their capacity to bind spermatozoa, the hemizona assay was used. Since the hemizona assay has an internal control, 30 mmol/l D-mannose was enough to detect a significant inhibition in sperm binding, confirming the participation of the residue in the process of sperm–egg interaction (Miranda et al., 1997). Since eggs obtained from IVF may have serious problems for the evaluation of sperm–egg interaction (including carbohydrate moieties on zona pellucida), the identification of putative sperm–zona pellucida ligands using proprietary whole-
cell combinatorial library analysis seems to be encouraging in identifying new binding sites (Malter et al., 2005; Pieczenik et al., 2006). In parallel, three characteristic fluorescent patterns of MBS on the sperm head were observed according to previous descriptions (Tesarik et al., 1991; Benoff et al., 1993a). Despite the fact that there is no ideal method to determine ‘capacitation status’ (de Lamirande et al., 1997), the human follicular fluid challenge has been adopted as a marker (Calvo et al., 1989). In the present study, at the end of the incubation under capacitating conditions there was a significant increase (P < 0.003) in the percentage of human follicular fluid-induced acrosome-reacted spermatozoa, suggesting that the process of capacitation had taken place concomitantly with the increase in expression of MBS (Benoff et al., 1993b). The inhibition of sperm binding by D-mannose observed in this study could be the result of: (i) a steric obstruction of the sperm receptor on the sperm head; or (ii) an increase in the proportion of acrosome-reacted cells with no ability to bind to RBMOnline®
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
the zona pellucida (Liu and Baker, 1990). Both alternatives were investigated in the present study. Specificity for the site was clearly demonstrated since only in the presence of D-mannose was there a 65% decrease in the detection of MBS, suggesting that the receptor was blocked for further detection with the Man–FITC–BSA. It is important to note that before exposure to the different hexoses, spermatozoa were capacitated in fresh medium for 4 h, and this latency in the exposition of the MBS is consistent with the time required to observe maximum sperm binding to the zona pellucida (Burkman et al., 1988). The results presented strongly support the hypothesis that in the presence of D-mannose the sugar competes with the zona pellucida for the MBS on the sperm head, decreasing the affinity for the oocyte. An increase in the proportion of reacted spermatozoa with lower affinity for the zona pellucida was excluded since we showed evidence that the free monosaccharide was not able to induce the AR unless linked to a macromolecule such as BSA. These data are in accordance with those reported in the mouse by Loeser and Tulsiani (1999), and also in humans by Brandelli et al. (1994) and Benoff et al. (1997), who demonstrated that spermatozoa undergo the AR in the presence of synthetic glycoproteins containing a monosaccharide covalently linked to BSA, mimicking the authentic zona pellucida induction. Using dual-colour labelling we have shown previously that while those spermatozoa displaying the MBS in pattern II were acrosome-intact, spermatozoa showing pattern III were those in which the AR was in progress or had already occurred (Munuce et al., 2006). In those acrosome-reacting spermatozoa, MBS has just migrated to the equatorial region. With the aid of the zona-free hamster oocyte test, Gabriele et al. (1998) observed that in the presence of D-Man–BSA the rate of penetration was decreased. Since this test evaluates sperm fusion capacity, it is possible to speculate that after the occurrence of the AR, MBS may still play a role in the interaction and fusion with the oolema. Because MBS redistribution to the equatorial segment seems to be an early step in the induction of the AR, and considering that fusion with the oolema begins by equatorial domain of human sperm head (Yanagimachi, 1988), the participation of MBS in sperm–egg fusion was considered. By threedimensional reconstruction after image acquisition, we showed here that the fluorescence signal on living non-permeabilized spermatozoa showing pattern III is located in a restricted area of the equatorial or post-equatorial segment, suggesting that the receptor is associated with the remaining plasma membrane. The data presented in this work support the model proposed by Benoff (1997), in which MBS could be localized in alleged subplasmalemmal stores in spermatozoa. During capacitation, the cholesterol efflux from the plasma membrane would promote their appearance on the sperm surface. Initially uniformly distributed on the acrosomal cap, upon receiving the AR-inducing signal, receptors would aggregate and migrate to the equatorial segment previous to the complete loss of the acrosomal contents. This rapid movement of MBS at the onset of the AR has been proposed to be associated with the rapid depolymerization of actin filaments, an early event of the AR (Spungin et al., 1995). Finally, the evaluation of MBS would be an easy and low-cost bioassay for clinical purposes, which could help to identify those patients whose spermatozoa fail to express the receptors and thus have low probability of contacting the zona pellucida, where intracytoplasmic sperm injection might be recommended.
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Although we observed an increase in the mean values of MBS accompanying fertilization rate, no statistical significance was achieved. The individual variability observed among patients could be responsible for this, since in the three groups 30–40% of the samples showed no increase in pattern II of MBS after incubation, although fertilization had taken place. Patients with no change in MBS expression in association with a successful fertilization were also found in the study of Hershlag et al. (1998); however, maybe a larger sample size is required before it is possible to discriminate between spermatozoa from those patients with a good or a poor chance of interacting with the zona pellucida. The present results confirm that the D-mannose/MBS system participates in sperm–egg interaction and that there is an association between the detection of MBS and acquisition of in-vitro sperm fertilizing ability. However, other moieties and other sperm-binding sites are probably involved in order to guarantee a successful interaction between gametes.
Acknowledgements The authors are very grateful for the skilful help of Dr José Pellegrino and Carl Zeiss, Argentina S.A., for their support in confocal laser scanning microscopy. We appreciate the collaboration of Dr Carlos Morente from the Programa de Asistencia Reproductiva de Rosario for performing IVF and providing IVF-semen samples, non-living human oocytes and human follicular fluid. The authors wish to thank to Dr Patricia Miranda (Instuto de Biología y Medicina Experimental) for the constructive criticism in revising the manuscript.
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RBMOnline®
Article D-Mannose-binding
sites are putative sperm determinants of human oocyte recognition and fertilization Dr Maria Jose Munuce is a Staff Professor in the Clinical Biochemistry Department of the School of Biochemical and Pharmaceutical Sciences at the University of Rosario, Argentina. She was a Fellow of the World Health Organization’s Human Reproduction Programme and the Puigvert Foundation at Barcelona, Spain. She has published over 15 scientific papers and given over 50 presentations at both national and international scientific meetings. Her research is focused on the study of the effect of female genital tract secretions on sperm function.
Dr Maria Jose Munuce Germán Rosano, Adriana M Caille, Marlene Gallardo-Ríos and María José Munuce1 Laboratorio de Estudios Reproductivos, Cátedra de Bioquímica Clínica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, San Lorenzo 939-9A (2000), Rosario, Argentina 1 Correspondence: Fax: +54 414251738; e-mail: [email protected]
Abstract The aim of the present study was to further evaluate the participation of D-mannose in the process of human sperm–egg interaction. Zona pellucida binding competitive assays in the presence of D-mannose were carried out using discarded oocytes from IVF. Spermatozoa were capacitated and D-mannose-binding site (MBS) expression, sperm viability and follicular fluidinduced acrosome reaction (AR) were evaluated. MBS were visualized using a fluorescein-neoglycoprotein probe. The capacity of free D-mannose and mannosylated albumin to induce the AR was also tested. MBS and the IVF outcome were also analysed. The involvement of D-mannose in sperm binding to the zona pellucida was verified by the inhibitory effect produced when the sugar was present during binding assays. MBS expression increased during capacitation, in parallel with the ability to undergo the induced AR. Mannosylated albumin, but not the free sugar, induced the AR. In acrosome-reacted spermatozoa, the MBS was located at the plasma membrane, as shown by confocal analysis. No significant difference in the increase in MBS expression was observed among the different IVF groups of patients. The data show that D-mannose is involved in the sperm–zona pellucida interaction, and that the expression of MBS on the sperm surface occurs during the acquisition of in-vitro sperm fertilizing ability. Keywords: acrosome reaction, D-mannose-binding site, hemizona assay, zona pellucida binding
Introduction
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For fertilization to occur, spermatozoa must first recognize and bind to the zona pellucida, triggering the exocytotic release of the acrosomal contents, and allowing spermatozoa to penetrate the zona pellucida and fertilize the egg (Yanagimachi, 1988). There is compelling evidence indicating that carbohydrates mediate the sperm–egg interaction. The proposed mechanism for this event is based on the specific binding of sperm-surface carbohydrate-binding proteins to glycoconjugates present in the zona pellucida, and this sets off the signal transduction pathways that result in the acrosome reaction (AR) (for reviews see Benoff, 1997; Töpfer-Petersen, 1999; Oehningher, 2003). The exact sugar
composition of zona glycoproteins and the molecular structure of the recognition/binding site(s) for human spermatozoa have not yet been determined. The detection of D-mannose-binding sites (MBS) on the sperm surface was first described by Tesarik et al. (1991) using a fluoresceinated neoglycoprotein containing D-mannose residues. The increase in the population of spermatozoa expressing MBS during capacitation has been proposed as the basis of a clinical test to discriminate between ‘fertile’ and ‘infertile’ patients undergoing IVF procedures (Benoff et al., 1993a, Hershlag et al., 1998).
© 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
In humans, it has been demonstrated that the fertile days are the 5 days previous to ovulation (Wilcox et al., 1995); thus spermatozoa have to wait in the female genital tract before encountering the oocyte. This interval provides an opportunity for female genital tract secretions to modulate sperm function (Barratt and Cooke, 1991). We have reported that both human follicular fluid and peritoneal fluids, which are present in the ampullary microenvironment, regulate sperm binding to the zona pellucida by decreasing the number of available MBS on the sperm head (Munuce et al., 2003, 2004). Furthermore, it has been reported that a family of glycoproteins called glycodelins present in human follicular fluid bind to the acrosomal region of intact spermatozoa and inhibit zona pellucida binding (Chiu et al., 2003), maybe through the terminal D-mannose residues in the protein (Chiu et al., 2004). In addition, the authors of this study have recently reported that levonorgestrel, a progestagen used in a contraceptive device placed in the female uterine cavity, increases the proportion of spermatozoa showing a MBS distribution associated with a lower ability to interact with the zona pellucida, maybe as part of its antifertility activity (Munuce et al., 2006). Considering that MBS could be an expression of in-vitro sperm fertilizing capacity and a target for its regulation, the participation of D-mannose in the sperm–egg interaction was investigated.
Materials and methods The present study was approved by the Institutional Review Board of School of Biochemical and Pharmaceutical Sciences of the National University of Rosario, Argentina. All human samples used in this study were obtained with the donors’ written consents.
Human follicular fluid Human follicular fluids were collected during oocyte retrieval from hormone-stimulated women (n = 6) participating in a local IVF programme. Only human follicular fluids with no blood contamination and obtained from follicles containing a mature oocyte (metaphase II) were collected. Follicular fluids from each sample were centrifuged (10 min, 600 g) to remove cellular debris, filtered through a 0.22 μm membrane (Millipore Corporation, Bedford, MA, USA), mixed into a pool and stored at –20°C (<12 months). The activity of the pool of human follicular fluid was checked monthly by analysing its ability to induce the acrosome reaction on capacitated spermatozoa.
Semen samples, sperm processing and capacitation Fresh semen specimens were obtained from normozoospermic donors after 3 to 5 days of sexual abstinence (n = 28). Semen analysis was performed according to World Health Organization guidelines (WHO, 1999) and morphological strict criteria (Kruger et al., 1988). Motile spermatozoa were selected by layering 1 ml of semen on top of a discontinuous 90–50% Percoll gradient (ICN Biomedicals, Aurora, OH, USA). Following centrifugation for 20 min at 275 g, the supernatant was discarded by gentle aspiration and the sperm RBMOnline®
pellet was then washed with 1 ml of Ham’s F-10 medium (ICN Biomedicals). Spermatozoa were resuspended in Ham’s F-10 medium supplemented with 35 mg/ml bovine serum albumin (BSA; ICN Biomedicals) at a concentration of 4 × 106 to 5 ×106 spermatozoa per ml and incubated for up to 22 h at 37°C in 5% CO2 in air. Capacitation status was evaluated by incubating capacitated spermatozoa for 30 min with 20% v/v human follicular fluid according to the method of Calvo et al. (1989) at 37°C in 5% CO2 in air, and determining the acrosomal status by the Pisum sativum technique (as described below).
Participation of D-mannose in the sperm– egg interaction Unfertilized human oocytes, donated by women undergoing IVF, were stored in a salt solution containing 1.5 mol/l MgCl2, 0.1% polyvinylpyrrolidone (MW 360 000) and 40 mmol/l HEPES in phosphate-buffered saline (PBS; pH 7.2). It has been previously shown that under these conditions, the zona pellucida retains its normal sperm-binding capacity, while the ooplasm and nucleus degenerate (Yanagimachi et al., 1979). For whole-oocyte–zona-binding assays, desalted oocytes were washed and randomly placed in a 100 μl droplet of mineraloil-overlaid Ham’s F-10 containing 35 mg/ml BSA and 0, 30, 100 or 300 mmol/l D-mannose, and inseminated with 1 × 105 motile progressive spermatozoa per ml for 4 h at 37°C under 5% CO2 in air. Oocytes were then washed by repeated pipetting to remove loosely attached spermatozoa and then placed on a slide under a coverslip supported by paraffin/Vaseline bases (80:20% v/v). For the hemizona assays, desalted oocytes were immobilized with a holding pipette and separated into equal halves using a microscalpel. Each hemizona was then placed in a 100-μl drop of Ham’s F-10 supplemented with 35 mg/ml BSA, with and without 30 mmol/l D-mannose. Hemizonas were then incubated for 4 h with 1 × 105 progressively motile spermatozoa per ml at 37°C under 5% CO2 in air, and treated as described for the whole-oocyte–zona-binding assay. Results were expressed as a hemizona index (HZI) = [(number of spermatozoa bound in treated hemizona) / (number of spermatozoa bound in control hemizona)] × 100.
Localization of D-mannose binding sites Spermatozoa were labelled with fluorescein-isothiocyanateconjugated mannosylated BSA (Man–FITC–BSA; Sigma Chemical Co., St Louis, USA) according to the protocol of Benoff et al. (1993a). Briefly, spermatozoa were washed twice with core buffer (30 mmol/l HEPES, 0.5 mmol/l MgCl2, 150 mmol/l NaCl, 10 mg/ml BSA, pH 7.0) supplemented with 20 mmol/l Ca2+ and incubated for 30 min with 100 μg/ml Man– FITC–BSA at 37°C and 5% CO2 in air. Following labelling, spermatozoa were pelleted by centrifugation and washed twice with core buffer without Ca2+. Sperm viability was assessed by eosin Y exclusion (WHO, 1999) before and after every labelling reaction. Spermatozoa were immobilized on glass slides, air-dried and mounted with glycerol/PBS (9:1, pH 9.0). Specimens were evaluated at ×1000 magnification using a Leica microscope (Leica Microsystems, Wetzlar, Germany) equipped with epifluorescence optics specific for fluorescein excitation (BP490 excitation, 500 dichroic mirror and LP515 barrier).
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Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al. Two hundred spermatozoa were counted for each individual experiment and the fluorescence signal was classified as: neck/ midpiece and tail (pattern I); entire acrosomal cap plus neck/ midpiece (pattern II); or equatorial/post-equatorial regions plus neck/midpiece (pattern III). Each slide was conserved at 4°C and analysed within a 2-week period.
Ligand specificity To investigate sugar specificity of MBS, 4 h capacitated spermatozoa were incubated for 60 min with 30 mmol/l D-glucose, D-galactose, N-acetyl-D-glucosamine, D-fucose or D-mannose (Sigma Chemical Co.), at 37°C and 5% CO2 in air, and then the proportion of cells expressing MBS was evaluated as described above.
Effect of D-mannose and mannosylatedBSA on sperm acrosomal status To investigate the ability of D-mannose to induce AR, overnightincubated spermatozoa were treated with one of the following: 30 mmol/l D-mannose, 100 μg/ml mannosylated BSA (D-Man– BSA; Fertility Technologies Inc., Natick, MA, USA), 20% v/v human follicular fluid, or control medium, for 2 h at 37°C under 5% CO2 in air. The acrosomal status was then determined using the Pisum sativum technique.
Acrosomal status evaluation by the Pisum sativum technique Spermatozoa were washed twice in 1 ml PBS and immobilized on slides, air-dried, permeabilized by exposure for 30 s to cold absolute methanol, and stained with fluoresceinated Pisum sativum agglutinin (50 μg/ml; Sigma Chemical Co.) for 30 min at room temperature. At least 200 spermatozoa were evaluated and classified as follows: (i) acrosome-intact, if the acrosomal cap was uniformly labelled; (ii) acrosome-reacting, if the acrosomal region of the head was labelled in a patchy pattern; or (iii) acrosome-reacted, if only the equatorial segment was labelled. The percentage of acrosome-reacted cells was calculated as b + c, unless otherwise noted.
Confocal laser scanning microscopy In order to determine the localization and topography of MBS after the occurrence of the AR, spermatozoa showing pattern III (acrosome-reacted) were evaluated by the use of a confocal laser scanning microscope (Carl Zeiss, Jena, Germany) at ×880. Serial confocal sections (stacking) were recorded at 0.35 μm focus steps. Image analysis and three-dimensional reconstruction were carried out using LSM Image Browser software (Carl Zeiss), while Scion Image (Scion Corporation, Frederick, USA) was used for histogram analysis of signal intensity and localization.
Detection of D-mannose binding sites and IVF outcome 184
A prospective, double-blind analysis of patients (n = 26) participating in the IVF programme at the Programa de
Asistencia Reproductiva de Rosario was undertaken. Conventional IVF inseminations were used in all cases. On the day of the oocyte retrieval, motile spermatozoa were selected by the swim-up technique (WHO, 1999). The remains of the semen sample were divided and use to determine MBS in fresh and overnight capacitated spermatozoa, as described above. Results were normalized as proposed by Hershlag et al. (1998) to represent the increase in MBS pattern II after incubation: percentage of incubated spermatozoa showing MBS pattern II minus the percentage of fresh cells with MBS pattern II divided by the percentage of incubated spermatozoa showing binding Man–FITC–BSA. Fertilization rate was defined as the ratio between oocytes that developed into normal two-pronuclear embryos 16–18 h after insemination and the total number of mature (metaphase II) oocytes inseminated. Patients were assigned to three groups according the rate of fertilization achieved: group 1 (n = 5), ≤50%; group 2 (n = 7), 50–75%; and group 3 (n = 14), >75%; and the average increase in MBS pattern II calculated for each group.
Statistical analysis Data processing was performed using the GraphPad InStat program (GraphPad Software, San Diego, CA, USA). Differences between treatments or groups were determined by one-way analysis of variance for matched samples and the Tukey–Kramer multiple comparison post-test. The difference in the number of spermatozoa bound to treated versus control oocytes was analysed using the non-parametric Kruskal– Wallis test and Dunn’s multiple comparisons post-tests. The non-parametric Wilcoxon sign ranked test for paired samples was applied for the analysis of the hemizona assays. Data are expressed as means ± SEM and a P-value <0.05 was considered significant.
Results The participation of D-mannose in the process of sperm–zona pellucida interaction was first evaluated by incubating both gametes in the presence of this monosaccharide. Using the whole-oocyte test, 300 mmol/l D-mannose was required to observe a significant reduction in the number of spermatozoa bound to the zona pellucida (P < 005, n = 6, Table 1). It is remarkable that at this concentration, sperm vitality was reduced to 70% viable cells. Given the high variability in the number of spermatozoa bound per oocyte, the hemizona assay was also used. In this case, the presence of 30 mmol/l D-mannose was enough to significantly reduce the number of bound spermatozoa (P < 003, n = 6, hemizona assay) with a HZI of 55 ± 13% (Table 2). When MBS on the sperm head were evaluated by fluorescent labelling, three characteristic fluorescence patterns, consistent with those reported by Benoff et al., (1993a), could be detected. All cells presented a nonspecific labelling in the neck/midpiece region (pattern I). In addition, some cells also showed a uniform distribution of the fluorescent signal either over the acrosomal cap (pattern II) or in the equatorial segment and/or post-acrosomal region (pattern III), considered as a specific head labelling (Figure 1a). The percentage of spermatozoa showing specific patterns of Man–FITC–BSA binding increased in a time-dependent manner. After 4 h RBMOnline®
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
incubation under capacitating conditions, this percentage was significantly different (P < 0.05; n = 5) from t = 0 h, reaching even higher values at 22 h (Figure 1b). In order to determine whether the increase in MBS was associated with capacitation, at the end of the incubation period spermatozoa were exposed to 20% v/v human-follicular-fluid. Data showed that there was a significant increase in the proportion of acrosome-reacted spermatozoa in the human-follicularfluid-treated group with respect to controls: human follicular fluid 41 ± 4% versus control 17 ± 1% (P < 0.003, n = 5). To exclude any deleterious effect of the procedure, viability was evaluated before and after the labelling protocol. Exposure of spermatozoa to Man–FITC–BSA did not affect the percentage of viable spermatozoa since the difference in live cell numbers before and after the labelling reaction did not exceed 10% (data not shown). To determine whether other monosaccharides that are structurally similar to D-mannose could also bind to the MBS, 4 h capacitated spermatozoa were exposed to five different hexoses before the labelling reaction. Data showed that only the preincubation of spermatozoa with D-mannose significantly decreased the percentage of cells displaying specific patterns (Figure 2, P < 0.001, n = 4). The percentage of viable cells at the end of the procedure was unaffected (>85% viable cells in all treatments). To investigate whether D-mannose was related to the AR, capacitated spermatozoa were exposed to D-mannose or mannosylated BSA. Results indicated that only when spermatozoa were exposed to D-mannose coupled to a protein matrix (D-Man–BSA) was there a significant increase in the percentage of acrosome-reacted cells (Figure 3, P < 0.001, n = 7). However, the magnitude of the AR induced by D-Man-BSA was significantly lower (P < 0.05) than the
Table 1. Effects of D-mannose on the whole-oocyte–zonabinding assay (n = 6). Treatment
Number of oocytes
Number of bound spermatozoaa
Control D-Mannose (30 mmol/l) D-Mannose (100 mmol/l) D-Mannose (300 mmol/l)
24 5 12 8
17 ± 5 26 ± 3 11 ± 4 4 ± 1b
one produced by human follicular fluid (considered to be the positive control). In order to investigate the location of MBS after the occurrence of the AR, in some experiments spermatozoa showing pattern III were further analysed by confocal microscopy. The analysis of three-dimensional reconstruction by confocal laser scan microscopy generated after the incubation of spermatozoa with Man–FITC–BSA showed that the fluorescence signal was located at the plasma membrane on the equatorial segment (Figure 4a). The histogram of the fluorescent signal visualized in five representative serial sections is showed in Figure 4b. The diagrams on the left represent transverse slices of the sperm head. Each box represents the section being analysed and the whole fluorescent signal is indicated in grey. On the right, the x-axis represents the length of the bar and the y-axis represents an arbitrary normalized value of the signal intensity. Results from a typical spermatozoon are shown. It has previously been observed (Munuce et al., 2006) in double-stained spermatozoa that those showing pattern III of MBS have lost their acrosomes, suggesting that both events are associated. Finally, the capacity of the MBS assay to discriminate between failed and successful IVF was investigated. Table 3 shows clinical variables in the three groups of patients included in the study. No differences in sperm count, motility or the percentage of normal forms were found. In addition, no differences in the women’s ages or in the number of mature (metaphase II) oocytes retrieved were observed between groups. Although there was a progressive increase in the mean value of MBS-pattern II increment along the groups, no statistical significance was achieved (Figure 5, n = 26, not significant). Sperm viability among groups did not differ at the end of the labelling procedure (>80% viable cells).
Table 2. Effects of D-mannose on the hemizona assay (n = 6). Treatment
Control
D-Mannose
Number of bound spermatozoa/ hemizona (30 mmol/l)
10.0 ± 1.0 5.0 ± 0.4a
Values are means ± SEM. a P < 0.03 vs. control.
Values are means ± SEM. P < 0.05 versus control.
a
b
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Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
Figure 1a. Photomicrograph of human spermatozoa labelled with fluorescein-isothiocyanate-conjugated mannosylated bovine serum albumin (Man–FITC–BSA). There is a spermatozoon labelled in pattern III with Man–FITC–BSA; the remaining spermatozoa exhibit pattern II (×880).
Figure 1b. Expression of mannose-binding sites (MBS) during sperm incubation under capacitating conditions. The percentage of spermatozoa displaying head-directed MBS (II and III) was obtained for non-capacitated, and 4 h and 22 h capacitated spermatozoa. Results are means ± SEM, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0 h.
Figure 2. Effect of exposure to monosaccharides on the detection of sperm mannose-binding sites (MBS). Capacitated spermatozoa were preincubated for 60 min with 30 mmol/l D-mannose (D-Man), D-glucose (D-Glc), N-acetyl glucosamine (NAG), D-galactose, (D-Gal), D-fucose (D-Fuc) or control medium (C) and then the expression of MBS was detected with fluorescein-isothiocyanate-conjugated mannosylated bovine serum albumin. Results are means ± SEM, n = 4. ***P < 0.001 versus other treatments.
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Figure 3. Induction of the acrosome reaction by a D-mannose bovine serum albumin. Capacitated spermatozoa were incubated 2 h with 100 μg/ml mannosylated bovine serum albumin (D-Man–BSA), 30 mmol/l D-mannose (D-Man), 20% v/v human follicular fluid (hFF), or control medium (C). The percentage of acrosome-reacted sperm was determined by Pisum sativum agglutinin labelling. Results are means ± SEM, n = 7. *P < 0.05 versus D-Man-BSA, ***P < 0.001 versus C and D-Man. RBMOnline®
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
Figure 4a. Three-dimensional reconstruction by confocal laser scan microscopy of fluorescence of a spermatozoon showing a pattern III of mannose-binding sites. Serial optical sections were recorded in focus steps of 0.35 μm. The fluorescent area is localized on the equatorial segment (×880).
Figure 4b. Distribution of mannose-binding sites in a pattern III spermatozoon. Histogram analysis of the fluorescence signal visualized in four representative serial sections obtained using a laser confocal scanning microscope. Images of a representative cell: left, transverse cut of the sperm head (fluorescence signal in grey); bars represent each serial section. Histograms: the x-axis represents the length of the bar; the y-axis represents an arbitrary normalized value of signal intensity. RBMOnline®
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Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al. Table 3. Clinical variables in IVF patients. Parameter
Group 1 (n = 5)
Group 2 (n = 7)
Group 3 (n = 14)
Sperm count (×106/ml) Sperm motility (%) Normal forms (%) Maternal age (years) Mature oocyte (%)
132 ± 36 56 ± 3 7±1 37 ± 2 89 ± 8
123 ± 20 58 ± 4 6.7 ± 0.5 31 ± 1 88 ± 5
152 ± 17 60 ± 1 7.5 ± 0.4 36 ± 1 92 ± 5
Values are means ± SEM. Group 1, ≤50% fertilization; group 2, 50–75% fertilization; group 3, >75% fertilization. Results were not significant between groups.
Figure 5. Detection of mannose-binding sites (MBS) and IVF outcome. Patients were assigned to three groups according the rate of fertilization achieved: group 1 (n = 5), ≤50%; group 2 (n = 7), 50–75%; and group 3 (n = 14), >75%; and compared with the increase in MBS pattern II in spermatozoa capacitated for 22 h. Results are the means ± SEM, n = 26, not significant.
Discussion
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The participation of D-mannose in sperm–egg interaction was first investigated using a competitive assay. When the wholeoocyte test was used, high sugar concentrations (300 mmol/l) were required to significantly decrease the number of bound spermatozoa. These results are in agreement with those of Mori et al. (1989) who did not observe inhibition of sperm binding in the presence of up to 50 mmol/l D-mannose. However, the high osmotic pressure at 300 mmol/l decreases sperm viability and could be responsible for the lower number of bound spermatozoa observed in our study. To bypass this limitation and the oocyte variability in their capacity to bind spermatozoa, the hemizona assay was used. Since the hemizona assay has an internal control, 30 mmol/l D-mannose was enough to detect a significant inhibition in sperm binding, confirming the participation of the residue in the process of sperm–egg interaction (Miranda et al., 1997). Since eggs obtained from IVF may have serious problems for the evaluation of sperm–egg interaction (including carbohydrate moieties on zona pellucida), the identification of putative sperm–zona pellucida ligands using proprietary whole-
cell combinatorial library analysis seems to be encouraging in identifying new binding sites (Malter et al., 2005; Pieczenik et al., 2006). In parallel, three characteristic fluorescent patterns of MBS on the sperm head were observed according to previous descriptions (Tesarik et al., 1991; Benoff et al., 1993a). Despite the fact that there is no ideal method to determine ‘capacitation status’ (de Lamirande et al., 1997), the human follicular fluid challenge has been adopted as a marker (Calvo et al., 1989). In the present study, at the end of the incubation under capacitating conditions there was a significant increase (P < 0.003) in the percentage of human follicular fluid-induced acrosome-reacted spermatozoa, suggesting that the process of capacitation had taken place concomitantly with the increase in expression of MBS (Benoff et al., 1993b). The inhibition of sperm binding by D-mannose observed in this study could be the result of: (i) a steric obstruction of the sperm receptor on the sperm head; or (ii) an increase in the proportion of acrosome-reacted cells with no ability to bind to RBMOnline®
Article - D-Mannose-binding sites in human oocyte recognition and fertilization - G Rosano et al.
the zona pellucida (Liu and Baker, 1990). Both alternatives were investigated in the present study. Specificity for the site was clearly demonstrated since only in the presence of D-mannose was there a 65% decrease in the detection of MBS, suggesting that the receptor was blocked for further detection with the Man–FITC–BSA. It is important to note that before exposure to the different hexoses, spermatozoa were capacitated in fresh medium for 4 h, and this latency in the exposition of the MBS is consistent with the time required to observe maximum sperm binding to the zona pellucida (Burkman et al., 1988). The results presented strongly support the hypothesis that in the presence of D-mannose the sugar competes with the zona pellucida for the MBS on the sperm head, decreasing the affinity for the oocyte. An increase in the proportion of reacted spermatozoa with lower affinity for the zona pellucida was excluded since we showed evidence that the free monosaccharide was not able to induce the AR unless linked to a macromolecule such as BSA. These data are in accordance with those reported in the mouse by Loeser and Tulsiani (1999), and also in humans by Brandelli et al. (1994) and Benoff et al. (1997), who demonstrated that spermatozoa undergo the AR in the presence of synthetic glycoproteins containing a monosaccharide covalently linked to BSA, mimicking the authentic zona pellucida induction. Using dual-colour labelling we have shown previously that while those spermatozoa displaying the MBS in pattern II were acrosome-intact, spermatozoa showing pattern III were those in which the AR was in progress or had already occurred (Munuce et al., 2006). In those acrosome-reacting spermatozoa, MBS has just migrated to the equatorial region. With the aid of the zona-free hamster oocyte test, Gabriele et al. (1998) observed that in the presence of D-Man–BSA the rate of penetration was decreased. Since this test evaluates sperm fusion capacity, it is possible to speculate that after the occurrence of the AR, MBS may still play a role in the interaction and fusion with the oolema. Because MBS redistribution to the equatorial segment seems to be an early step in the induction of the AR, and considering that fusion with the oolema begins by equatorial domain of human sperm head (Yanagimachi, 1988), the participation of MBS in sperm–egg fusion was considered. By threedimensional reconstruction after image acquisition, we showed here that the fluorescence signal on living non-permeabilized spermatozoa showing pattern III is located in a restricted area of the equatorial or post-equatorial segment, suggesting that the receptor is associated with the remaining plasma membrane. The data presented in this work support the model proposed by Benoff (1997), in which MBS could be localized in alleged subplasmalemmal stores in spermatozoa. During capacitation, the cholesterol efflux from the plasma membrane would promote their appearance on the sperm surface. Initially uniformly distributed on the acrosomal cap, upon receiving the AR-inducing signal, receptors would aggregate and migrate to the equatorial segment previous to the complete loss of the acrosomal contents. This rapid movement of MBS at the onset of the AR has been proposed to be associated with the rapid depolymerization of actin filaments, an early event of the AR (Spungin et al., 1995). Finally, the evaluation of MBS would be an easy and low-cost bioassay for clinical purposes, which could help to identify those patients whose spermatozoa fail to express the receptors and thus have low probability of contacting the zona pellucida, where intracytoplasmic sperm injection might be recommended.
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Although we observed an increase in the mean values of MBS accompanying fertilization rate, no statistical significance was achieved. The individual variability observed among patients could be responsible for this, since in the three groups 30–40% of the samples showed no increase in pattern II of MBS after incubation, although fertilization had taken place. Patients with no change in MBS expression in association with a successful fertilization were also found in the study of Hershlag et al. (1998); however, maybe a larger sample size is required before it is possible to discriminate between spermatozoa from those patients with a good or a poor chance of interacting with the zona pellucida. The present results confirm that the D-mannose/MBS system participates in sperm–egg interaction and that there is an association between the detection of MBS and acquisition of in-vitro sperm fertilizing ability. However, other moieties and other sperm-binding sites are probably involved in order to guarantee a successful interaction between gametes.
Acknowledgements The authors are very grateful for the skilful help of Dr José Pellegrino and Carl Zeiss, Argentina S.A., for their support in confocal laser scanning microscopy. We appreciate the collaboration of Dr Carlos Morente from the Programa de Asistencia Reproductiva de Rosario for performing IVF and providing IVF-semen samples, non-living human oocytes and human follicular fluid. The authors wish to thank to Dr Patricia Miranda (Instuto de Biología y Medicina Experimental) for the constructive criticism in revising the manuscript.
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