Molecular Human Reproduction, Vol. 7, No. 7, 617-624,
July 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Changes of the major sperm maturation-associated epididymal protein HE5 (CD52) on human ejaculated spermatozoa during incubation in capacitation conditions
1 Institute of Reproductive Medicine of the University, Münster, Germany and 2 Department of Cell Biology, University of València, Spain and 3 Institute for Hormone and Fertility Research, Hamburg, Germany
Abstract
HE5 (CD52) is a glycoprotein which is secreted by the epididymis and which becomes inserted onto maturing spermatozoa. We have previously shown that, in cynomolgus monkey spermatozoa, changes occur upon maturation rendering cryptic the epitope to the monoclonal antibody CAMPATH-1G; the recognition site is then re-exposed during incubation under capacitation conditions. The present study investigated human ejaculated spermatozoa during incubation under similar conditions, using monoclonal antibodies that recognize different epitopes of the HE5 molecule comprising parts of the N-glycan (2E5) or peptide segments, including (CAMPATH-1G) or excluding (097) the glycosylphosphatidylinositol (GPI) anchor, to reveal modifications of sperm surface HE5. Flow cytometric analysis showed equally high percentages (~90%) of viable spermatozoa cross-reacting with the antibodies before and after 6 h incubation. However, during incubation, the staining intensity increased 57% with CAMPATH-1G, 31% with 097, but remained unchanged with 2E5. The lymphocyte CD52 antibody CF1D12 stained only ~10% of spermatozoa either before or after incubation. Western blotting of sperm protein extracts using lectins indicated an increase in the exposure of sialic acid residues of HE5 after incubation. These results suggest that during incubation in capacitating conditions, there is an opening up of the HE5 glycoprotein molecule, increasing accessibility of some sialic acid residues and of the core peptide, particularly the GPI anchor.
cryptic epitope/epididymal secretion/flow cytometry/sperm protein/surface antigen
Introduction
While advances in assisted reproductive technologies have shown that fertilization can be achieved with spermatozoa obtained from the testis, the concept of post-testicular sperm maturation as a natural process should not become redundant since there is accumulating evidence for changes in the properties and functional aspects of spermatozoa occurring in the epididymis (Cooper and Yeung, 1997
; Cooper, 2000). Study of these processes could yield explanations for subfertility and infertility on the one hand and offer new leads to the development of male contraceptive methods on the other (Cooper and Yeung, 1999; Habenicht, 2000).
Among the few epididymal secretory products that have been cloned and shown to interact with maturing spermatozoa in the epididymal lumen, the major protein is HE5 which is identical to lymphocyte protein, CD52, in its cDNA sequence (Kirchhoff et al., 1993; Kirchhoff, 1996). HE5 transcripts are absent from the testis and are expressed in the corpus and cauda epididymis as well as in the vas deferens (Krull et al., 1993). The epididymis is the sole source of HE5 secretion in seminal plasma, as it is not detectable in ejaculates from vasectomized men (Rooney et al., 1996; Yeung et al., 1998). HE5 secreted into the epididymal lumen is associated either with particles above 0.1 µm in size, presumably aposomes, or with high molecular mass components. Such an association is required for the transfer of HE5 onto the surface of maturing spermatozoa. The uptake of HE5 is also dependent on the maturational status of the epididymal spermatozoa (Yeung et al., 1997a; Kirchhoff and Schröter, 2001). A correlation between ejaculated sperm motility and the amount of HE5 on the spermatozoa has been shown using monoclonal antibodies for detection and flow cytometry for quantification (Yeung et al., 1997b). However, the observation that a similar abundance of HE5 is present in the seminal plasma of normo-, astheno-, oligo- and terato-zoospermic ejaculates suggests that the lower amount of HE5 on spermatozoa in asthenozoospermia more likely reflects defects in its uptake onto sperm membranes than limiting amounts of epididymal secretion (Yeung et al., 1998).
For more than a decade, several laboratories have worked independently on sperm antigens which are derived from different sources and which have now been identified as part of the same glycoprotein that is a cell-type specific modification of CD52 (see Table I). Although the sperm isoform of CD52 has recently been shown to be identical to the antigen of a sperm-immobilizing antibody originating from an infertile woman (Isojima et al., 1987; Diekman et al., 1999), the physiological role of this sperm glycoprotein in reproduction is yet to be established.
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Both sperm HE5 and lymphocyte CD52 have only 12 amino acids in the mature protein peptide which is linked at its C-terminus to a glycosylphosphatidylinositol (GPI) anchor that is inserted into the plasma membrane (Figure 1
). The single N-glycosylation site is occupied by heterogeneous and complex glycans which are rich in terminal sialic acid residues (Schröter et al., 1999). The sperm and lymphocyte isoforms differ in the structure and heterogeneity of the N-linked glycans and have structurally different GPI anchors (Schröter et al., 1999). CAMPATH-1 monoclonal antibodies which were developed against human lymphocyte CD52 for therapeutic use in lymphocyte depletion (Xia et al., 1993) and which recognize the C-terminus of the molecule, including the GPI anchor as an epitope (Hale, 1995), have been used in our previous studies of HE5 on human spermatozoa. In a recent quantitative study of viable and non-fixed cynomolgus monkey spermatozoa using these antibodies, there were marked increases in the manifestation of the surface antigen both in the percentage of stained cells and in their staining intensity as the maturing cells moved from the caput to the corpus epididymidis. However, the detectable levels of HE5 were drastically reduced in mature spermatozoa from the cauda epididymidis and ejaculates, despite its undiminished presence in sperm extracts as detected by protein extraction and Western blotting (Yeung et al., 2000). Such maturational changes rendering the CAMPATH-1 epitopes cryptic were apparently reversed upon incubation of the mature spermatozoa under capacitating conditions (Yeung et al., 2000). The process of epitope re-exposure was temperature dependent, protease sensitive, and did not involve endogenous neuraminidase activity or deglycosylation. The present study was performed to investigate if any similar changes occur during the incubation of human spermatozoa in capacitating conditions. Various antibodies recognizing different epitopes of CD52 were used in order to determine the molecular nature of the changes.
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Materials and methods
Sperm preparation
Normozoospermic ejaculates were obtained by masturbation from 16 patients attending the fertility clinic at the Institute of Reproductive Medicine, Münster. After 30 min liquefaction at 37°C, the ejaculates were analysed according to published criteria (World Health Organization, 1999) in a routine procedure subjected to internal and external quality control (Cooper et al., 1992, 1999). Semen parameters (mean ± SD) were 3.7 ± 2.3 ml ejaculate volume, 81 ± 38x106 spermatozoa/ml, 62 ± 8% sperm motility with 22 ± 16, 30 ± 15 and 10 ± 6% a, b and c grades respectively, and 22 ± 7% normal morphology. One to 2 ml of ejaculate was layered on top of 3 ml 30% (v/v) and 3 ml 60% Percoll (Sigma, Deisenhofen, Germany) made up in BWW medium (Biggers et al., 1971). Sperm pellets obtained by centrifugation at 500 g for 20 min were washed again with 4 ml BWW containing 4 mg bovine serum albumin (BSA)/ml at 450g for 5min. Washed spermatozoa were resuspended to 20x106/ml and incubated at 37°C in 5% CO2 in humid air for 3 or 6 h, as conditions for capacitation.
Immunostaining of spermatozoa and analysis by flow cytometry
At 0, 3 and 6 h of incubation, aliquots of 2x106 spermatozoa were stained with each of the CD52 monoclonal antibodies, used as neat hybridoma solution or diluted in phosphate-buffered saline (PBS), by adding 5 µl to 95 µl sperm suspension at a final concentration of 1:200 for CAMPATH-1G (equivalent to 19.2 µg/ml; a gift from Prof. Geoff Hale, Therapeutic Antibody Centre, Sir William Dunn School of Pathology, Oxford University, UK), 1:200 for antibody 097 (a gift from Prof. Dr A.Bernard, University of Marseille, France), 1:20 for 2E5 (a gift from Prof. Shinzo Isojima, Hyogo Medical College, Nishinomiya, Japan) and 1:10 for CF1D12 (a gift from Dr Martin Hadam, Medical University of Hannover, Germany). In control samples, the primary monoclonal antibodies were replaced by the corresponding non-specific IgG (rat IgG for CAMPATH and mouse IgG for the others) at the same concentration (20 µg/ml) and processed in parallel. After incubation for 20 min at room temperature, the spermatozoa were washed twice with PBS containing 4 mg BSA/ml and incubated for 30 min at room temperature with the appropriate secondary antibody at 1:100 [rabbit anti-rat IgG-fluorescein isothiocyanate (FITC; Sigma F-1763) or goat anti-mouse IgG-FITC (Sigma F-8771)]. The spermatozoa were washed and suspended in 0.3 ml PBS containing the vital dye propidium iodide at 5 µg/ml for 5 min before analysis with the flow cytometer (Coulter Epics XL, system II, version 3.0; Beckman Coulter, Krefeld, Germany) as described (Yeung et al., 1997a). For light microscopy, spermatozoa were processed as for flow cytometry, except for the omission of propidium iodide, were pelleted by centrifugation and spread onto glass slides, mounted with anti-fade solution (Sigma) and examined as previously described (Yeung et al., 1997a).
For flow cytometric analysis, about 5000 individual sperm cells were included by their forward and side-scatter signal characteristics and analysed individually for fluorescence emissions from propidium iodide and FITC. Percentages of viable spermatozoa (propidium iodide negative cells) and their FITC signal intensities (in channel numbers) were analysed. Sperm samples stained by non-specific IgG as negative controls were used to set the threshold for specific CD52 staining (Figure 2), and in each analysis the small percentage of cells above the set background was subtracted from the result of the aliquot sample stained with the CD52 antibodies to obtain the net percentage of specifically stained spermatozoa.
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Western blot analysis and lectin binding studies
Human ejaculates were divided into two aliquots, and each aliquot was purified on a two-step Percoll gradient (2 ml 90% Percoll, 2 ml 47.5% Percoll, Amersham-Pharmacia, in PBS). The pellet was washed again in an IVF medium (MedicultTM; Gück, Berlin, Germany; containing 10 mg human serum albumin/ml) and spermatozoa were counted, dispensed in equal aliquots, and stored at –80°C as non-capacitated controls. The second portion of washed spermatozoa was incubated for 6 h at 37°C in 5% CO2 in Medicult before freezing without further washing. By taking advantage of the glycolipid-like properties of CD52, the antigen was enriched from a known number of spermatozoa by modified Folch extraction (Hale et al., 1993
). Methanol/chloroform (Folch) extracts of protein samples were prepared as previously described (Schröter et al., 1999). Samples of 2–10 µl Folch extract, depending on sperm number or protein content as determined with the Protein Assay Kit No. 1 (Biorad, München, Germany), were separated on 15% polyacrylamide Laemmli gels, and the proteins were transferred by semi-dry blotting to polyvinylidene difluoride (PVDF) membranes (Amersham, Braunschweig, Germany) as described (Schröter et al., 1999). The membrane was blocked overnight with Tris-buffered saline containing 1% blocking reagent (Roche Molecular Biochemicals) and incubated with the monoclonal antibodies 097 from mouse (ascites fluid 1:2000 in Tris-buffered saline containing 0.1% blocking reagent), CF1D12 from mouse (hybridoma supernatant 1:500), 2E5 from mouse (hybridoma supernatant 1:500) and CAMPATH-1G from rat (1:2500). Biotinylated Maackia amurensis lectin (MAA) and Sambucus nigra lectin (SNA; Oxford GlycoSystems, Abingdon, UK) were employed at a dilution of 1 µg/ml for detection of sialic acid residues in the N-linked glycans. Primary binding was detected by employing peroxidase-coupled second antibodies (0.4 µg/ml, Sigma). Alternatively, anti-DIG-Fab-POD (150 U/ml; Roche-Boehringer Mannheim, Germany) was employed at a dilution of 1:1000 in 100 mmol/l Tris-Cl, pH 7.5 with 150 mmol/l sodium chloride and 1% blocking reagent. Chemiluminescence film detection was carried out with the SuperSignalTM CL-HRP substrate system (Pierce Chemical Company, Rockford, USA) following the instructions of the supplier. Signals were detected by exposure to Fuji X-ray film.
Statistics
Changes in the percentages of positive spermatozoa and their staining intensities during capacitation-incubation at 3 and 6 h compared to preincubation, were statistically tested by one-way repeated measure analysis of variance followed by the Tukey test at a significance level of P < 0.05.
Results
Reactivities of spermatozoa with different CD52 antibodies and their staining patterns
The present study used monoclonal antibodies that recognize different epitopes of the molecule comprising parts of the N-glycan (2E5) or peptide segments including (CAMPATH-1G) or excluding (097) the glycosylphosphatidylinositol (GPI) anchor (see Figure 1
). Specific labelling of spermatozoa by these various CD52 antibodies was revealed by comparing the dual parameter analysis of each staining with that of the corresponding control run parallel in each experiment (Figure 2). With the monoclonal antibodies CAMPATH-1G, 097 and 2E5, nearly 90% of the freshly washed viable spermatozoa were already stained without incubation, and the percentages of stained spermatozoa remained unchanged during the 6 h incubation (Figure 3). CAMPATH-1G and 097 produced similar patterns of staining with the majority of spermatozoa being stained over the whole surface of the head and tail, all showing intense staining particularly at the post-acrosomal region, and some showing intense staining of the entire acrosomal cap (Figure 4a and b). Staining by CAMPATH-1G was more intense than 097 but less homogeneous. Staining by 2E5 was also consistent over the post-acrosomal region and very patchy or dotted over the tail (mainly mid-piece), with occasional patchy staining of the whole head (Figure 4c). Among the ejaculates studied, there was no general trend of changes in these staining patterns during incubation under capacitating conditions, except that the post-acrosomal patches of fluorescence became more prominent. Staining by CF1D12 was barely detectable throughout incubation with occasional weak green fluorescence over the head and along the tail (Figure 4d); staining intensity was just slightly above if not the same as the control (Figure 4e, see also Figure 2) and net staining was quantified to be only 10% of spermatozoa (Figure 3).
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Flow cytometric detection of changes in antibody staining intensity during sperm incubation
Although the staining intensity of viable spermatozoa with CAMPATH-1G varied among men, it consistently showed a progressive increase during incubation under capacitating conditions, reaching an average level of 57% above preincubation levels by 6 h (Figure 5
). Staining intensities with 097 also increased in parallel to that of CAMPATH-1G, but to a lesser extent of 31% on average above preincubation levels. In contrast, the staining intensities with 2E5 remained relatively constant over 6 h (Figure 5). The weak staining by CF1D12 in a low percentage of spermatozoa (Figure 3) remained barely above control levels throughout the 6 h incubation (data not shown).
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Another interesting feature of CD52 staining with CAMPATH-1G was the lower viability of the sperm sample compared to the aliquot stained with non-specific IgG as control, and this decreased viability became more marked during incubation (Figure 6
). The results represent findings in free individual spermatozoa since there was no appreciable agglutination at the antibody dilution used, and in the flow cytometric analysis aggregated spermatozoa were excluded by the forward and side-scatter signal gating. Furthermore, this effect on viability was not observed when aliquots were stained with the other CD52 antibodies (Figure 6).
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Western blot detection of changes in HE5 during sperm incubation
When extracts of equal numbers of spermatozoa were analysed by Western blotting, the multiple protein bands at 15–22 kDa, characteristic of HE5, showed the same cross-reactivities with CAMPATH-1G before and after 6 h incubation (Figure 7
). On the other hand, some increases were found in cross-reactivities of HE5 with the lectin MAA in the incubated samples, but no staining was detectable using the lectin SNA either before or after incubation of spermatozoa (Figure 7).
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The extra band showing MAA reactivity at ~46 kDa was probably some molecule which is unrelated to HE5 but is also extracted by methanol/chloroform and exhibits sialic acid residues upon incubation.
Discussion
We have previously shown that, in the monkey, caput spermatozoa readily take up epididymal CD52 during their passage to the corpus epididymidis where 85% of spermatozoa become coated. However, the CAMPATH-1 epitopes subsequently become cryptic when spermatozoa are stored in the cauda tubule (Yeung et al., 2000). Unlike in the monkey, there is progressive uptake of HE5 onto human spermatozoa along the length of the epididymis with 80–90% of cauda spermatozoa expressing the surface antigen (Yeung et al., 1997b). After ejaculation, monkey spermatozoa show increases upon capacitation-incubation both in the percentages of cells stained by the CAMPATH-1 antibodies and in the intensities of staining of individual spermatozoa (Yeung et al., 2000). The present study with human spermatozoa showed that, although the already high percentages of freshly washed ejaculated spermatozoa that stained with CAMPATH-1 showed no further increase in numbers upon incubation, staining intensities did increase progressively by an average of ~60% after 6 h, suggesting further exposure of the epitope. The monoclonal antibody 097, which recognizes only four amino acids and excludes the GPI anchor in its binding to the antigen, achieved an increase in staining intensity of only 30% over the basal level by 6 h of incubation. This suggests that at least part of the exposure of CAMPATH-1G epitope was caused by changes in the anchorage environment, possibly in properties of the lipid membrane including the clustering of sphingolipid and cholesterol in lipid rafts into which the GPI anchors are inserted. The association of GPI anchored proteins with raft microdomains can be affected by cholesterol removal (Simons and Ikonen, 1997). Cholesterol loss during sperm capacitation has been shown (Benoff, 1993; Cross, 1998) and changes in membrane architecture upon capacitation-incubation have been indicated by the increase in merocyanine stainability (Harrison et al., 1996; Harrison and Miller, 2000). Similarly, the present finding of the loss of sperm viability on interaction with CAMPATH-1G (but not with the other antibodies), with the loss of viability becoming more marked during incubation in step with the epitope exposure, may be explained by increases in the extent of membrane perturbation by CAMPATH-1G binding to the GPI anchor. Increased entry of DNA dye indicating loss of viability is probably via the post acrosomal membrane, as this region was most intensely stained by CAMPATH-1G and therefore most vulnerable. Despite higher percentages of dead cells, acrosome loss among dead spermatozoa was the same in control and CAMPATH-1G (C.H.Yeung, unpublished data) bound spermatozoa, as examined using dual parameter flow cytometry analysis (Cooper and Yeung, 1998). Nevertheless, CAMPATH-1G binding may render the acrosome less stable, since viable spermatozoa after incubation with CAMPATH-1G showed a tendency of more acrosomal loss (about 10%) compared to control (C.H.Yeung, unpublished data). Membrane perturbation in the tail is reflected by a previous finding that when spermatozoa are incubated with CAMPATH-1G in the absence of the secondary antibody, motility decreases by 10% at 5 min and by a further 20% after 3.5 h without agglutination (Yeung et al., 1997b).
The dependence of the interaction between the antigen and CAMPATH-1G on the accessibility of the GPI anchor is in accordance with the present finding of equal reactivity in the incubated and non-incubated sperm samples when analysed by Western blot using their methanol/chloroform extracts, as the GPI anchors would no longer be inserted in the plasma membrane as they are in the whole viable spermatozoa detected by flow cytometry.
The antibody 2E5 was developed as a sperm immobilizing monoclonal antibody whose epitope has been shown to be on the N-glycan of the SAGA-1 (sperm agglutinin antibody-1) glycoprotein, which is identical to sperm CD52 (HE5); additionally 2E5 has been shown to recognize lymphocyte CD52 (Isojima et al., 1990; Kameda et al., 1992; Diekmann et al., 1999). In the present study, although 2E5 bound to identical numbers of spermatozoa as CAMPATH-1G and 097, its staining intensity did not change on incubation in capacitation conditions, as did those of the two lymphocyte CD52 antibodies. This result indicates that the accessibility of the 2E5 carbohydrate epitope remained constant despite progressive exposure of the GPI anchor and the core peptide of the glycoprotein. This is in agreement with the absence of deglycosylation as a change leading to CAMPATH-1 epitope exposure during incubation of monkey spermatozoa (Yeung et al., 2000). During incubation of human spermatozoa, there was an increase in reactive sialic acid residues as detected by staining of protein blots with the lectin MAA. The tertiary oligosaccharide branches of the highly complex N-glycan of sperm CD52 (HE5) are rich in sialic acid terminal residues (Schröter et al., 1999) which should not be affected by the methanol/chloroform extraction procedure. The absence of staining by the lectin SNA, which is specific for 
-2,6-linked sialic acid, confirms the previous finding that these sugar residues of sperm CD52 (HE5) are mostly 2,3-linked instead of 2,6-linked as in lymphocyte CD52 (Schröter et al., 1999). The increased MAA signal of CD52 (HE5) probably reflects unfolding of some sialic acid-loaded oligosaccharide branches in the N-glycan complex without disturbing the unidentified 2E5 epitope. Such unfolding could also facilitate accessibility of antibodies to the GPI anchor and the peptide. It is unlikely to be due to unmasking of electrostatically bound coating protein, since we have shown in monkey spermatozoa that high salt treatment has no effect on CAMPATH-1 epitope exposure (Yeung et al., 2000). However, exogenous neuraminidase facilitates exposure (Yeung et al., 2000), suggesting some steric hindrance by the carbohydrates to antibody binding. Additionally, the possibility of a chemical modification of the HE5 molecule itself, or the unmasking of residues by neighbouring transmembrane molecules during incubation, cannot be entirely ruled out at this stage.
Unlike the other CD52 antibodies, CF1D12 did not bind to the majority of spermatozoa throughout incubation. CF1D12 is a monoclonal antibody which predominantly recognizes part of the N-glycan of the lymphocyte CD52 antigen. Although some binding on spermatozoa is still detectable by Western blotting after N-glycosidase F digestion (Schröter et al., 1999), the little staining detectable was probably non-specific since the flow cytometry data were largely similar to those of control IgG staining. This provides further evidence for differences between sperm and lymphocyte CD52 glycoforms in the structures of the oligosaccharide branches of the N-glycan, as has been shown by MALDI/TOF mass spectrometry analysis (Schröter et al., 1999).
Although antibody-induced sperm immobilization or agglutination is the phenomenon which initiated the studies of sperm CD52/HE5 in some laboratories, the physiological role of the sperm glycoprotein itself is still unclear. Sperm functions inhibited by various antibodies in the absence of agglutination include zona binding with the use of S19 (Mahony et al., 1991), zona-free hamster oocyte penetration using antisera of gp20 (Focarelli et al., 1998) and individual sperm swimming velocities and kinematic patterns using CAMPATH-1G (Yeung et al., 1997b). The last observation probably reflects perturbation of sperm membrane due to antibody-antigen interaction involving the GPI anchor as described above, rather than a direct involvement of the glycoprotein in the regulation of sperm flagellation, although the profile of HE5 expression along the length of the epididymis parallels the maturation profile of sperm motility (Krull et al., 1993; Yeung et al., 1993). The antibody effects on zona binding and oocyte penetration would agree with the antigen localization on the sperm head. However, the glycoprotein is also present on the sperm tail, as shown in this and studies using different antibodies, including the sperm-specific antibody S19 that recognizes carbohydrate moieties (Diekman et al., 1999). This could mean that there are different CD52 glycoforms on the head and the tail such that the moieties serving as zona or oolemma receptors on the head are absent from the N-glycan of the tail glycoforms, even though both interact with the same antibodies. Alternatively it could mean that zona and oolemma binding do not actually involve CD52 glycoforms but are affected by steric hindrance caused by antibody binding in the proximity of the true binding sites.
Structural molecular analysis has indicated that HE5 is a heterogeneous glycoprotein (Schröter et al., 1999). Our previous and the present electrophoresis studies using CAMPATH-1G have indicated five bands of different electrophoretic mobility. About 20 variants are found by 2D gel analysis using SAGA-1 (Diekman et al., 1999), and the gp20 antigen also shows variations in pI and molecular mass (Focarelli et al., 1999). The functional significance of these different isoforms needs further investigation. Various sugar residues in different animal species play important roles in the events leading to fertilization (see Yanagimachi, 1994; Suarez, 1999; Töpfer-Petersen, 1999). The present finding of changes in the glycoprotein occurring on the surface of viable spermatozoa during incubation is supportive of the speculation that sperm CD52 serves as a malleable scaffolding for carbohydrate residues. The molecule may undergo changes before fertilization occurs to facilitate survival and interaction with the female tract or with the oocyte.
Acknowledgements
We thank Barbara Hellenkemper for technical assistance and Prof. Eberhard Nieschlag for his encouragement. We also thank Raphaele Kürten, Heidi Kersebom, Sebine Rehr, Daniele Schmidt and Katherin Wardecki for the routine semen analysis. The work was supported by the Deutsche Forschungsgemeinschaft Confocal Research Group `The Male Gamete, Maturation and Function', Grant no. Ni 130/15 and Iv 4/7, and a Travel Grant from the University of València for F.P.
Notes
4 To whom correspondence should be addressed. E-mail: yeung{at}uni-muenster.de 
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Submitted on January 22, 2001; accepted on April 9, 2001.
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