Molecular Human Reproduction, Vol. 5, No. 1, 46-51,
January 1999
© 1999 European Society of Human Reproduction and Embryology
A sialoglycoprotein, gp20, of the human capacitated sperm surface is a homologue of the leukocyte CD52 antigen: analysis of the effect of anti-CD52 monoclonal antibody (CAMPATH-1) on capacitated spermatozoa
1 Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, 2 Department of Internal Medicine, University of L'Aquila and 3 Department of Molecular Biology, University of Siena, Pian dei Mantellini 44, Siena, Italy
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Abstract |
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In this study we performed N-terminal sequence analysis of gp20, a 20 kDa sialoglycoprotein on the human sperm surface previously identified by radiolabelling of the sialic acid residues of sperm surface. We found 100% identity with the N-terminus of CD52, an antigen expressed on almost all human leukocytes. We also show that, like CD52, gp20 behaves as a glycosylphosphatidylinositol (GPI)-anchored protein and that anti-gp20 antiserum reacts with an antigen on leukocytes of the same molecular weight as CD52. Using CAMPATH-1, the monoclonal antibody against CD52, in fluorescent staining of capacitated spermatozoa, Western blot analysis and the zona-free hamster egg penetration test, we found that the effect of this antibody was different from that of our anti-gp20. Western blot analysis revealed a well-defined 20 kDa band with anti-gp20, whereas a 14–20 kDa band was detected with CAMPATH-1. Anti-gp20 stained the equatorial region of the sperm head, whereas CAMPATH-1 stained the tail in immunofluorescence analysis of capacitated spermatozoa. A dose-dependent inhibitory effect was seen with CAMPATH-1, similar to that previously detected with anti-gp20, in a zona-free hamster egg penetration test. However, with CAMPATH-1 agglutination of motile spermatozoa was detected, and this was not present with anti-gp20. This suggests that the epitopes recognized by the two antibodies are different.
capacitation/CD52/glycoproteins/leukocytes/spermatozoa
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Introduction |
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Freshly ejaculated spermatozoa fail to fertilize eggs even though they are motile and structurally mature. The ability to fertilize eggs is acquired in the female reproductive tract, after a multistep maturation process known as capacitation. An important step in this process is the removal of coating substances derived from seminal fluid. In previous studies we have investigated the kinetics of release and the size of material removed from the sperm surface during capacitation. This process is associated with the removal of two classes of sialoglycoconjugates (Focarelli et al., 1990
). We also showed that a single sialoglycoprotein of ~20 kDa (gp20) was present on the sperm surface after capacitation (Focarelli et al., 1995). In a very recent paper we reported the partial purification of gp20, the production of a specific antibody against it, its use to localize the antigen in ejaculated spermatozoa before and after capacitation and its capacity to inhibit sperm penetration of hamster eggs (Focarelli et al., 1998). We also discovered that the antigen was expressed in the epididymis and not in the testis.
Here we report the results of microsequencing the protein N-terminus. One hundred per cent homology of gp20 with CD52, an antigen present on most human leukocytes (Hale et al., 1983, 1990; Xia et al., 1991) was found. We also report the results of experiments with capacitated spermatozoa using fluorescent staining, Western blot analysis and the zona-free hamster egg penetration test with CAMPATH-1, the monoclonal antibody against CD52. The behaviour as a glycosylphosphatidylinositol (GPI)-anchored protein of gp20 is also reported.
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Materials and methods |
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Cell preparation
Human ejaculates were obtained from healthy men and allowed to liquefy at room temperature for ~30 min. Sperm cells were separated from seminal plasma after dilution in phosphate-buffered saline (PBS; 50 mM KH2PO4, 150 mM NaCl, pH 7.4) by centrifuging at 500 g for 10 min at 10°C. Capacitation was achieved by incubating spermatozoa for 6 h in Biggers–Whitten–Whittingham medium (BWW; Biggers et al., 1971
) containing 35 mg/ml human serum albumin (HSA) as previously described (Focarelli et al., 1990). Leukocytes were obtained from blood samples after haemolysis with 0.87% NH4Cl and centrifuging at 500 g for 10 min at 4°C. Proteins were extracted from spermatozoa and leukocytes using a solubilization buffer containing 1% Triton X-114 in 10 mM Tris–HCl (pH 7.4), 150 mM NaCl and 1 mM EDTA.
Electrophoresis and Western blotting
Samples were separated by tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Schägger and von Jagow, 1987). The separation gel consisted of 10% acrylamide with a 3% stacking gel. The gels were either used for staining with Coomassie Brilliant Blue or transferring the proteins to nitrocellulose (Towbin et al., 1979). Nitrocellulose sheets were then blocked with 0.2% non-fat powdered milk in (TBS; 20 mM Tris–HCl, 500 mM NaCl, pH 7.5) and incubated for 4 h at room temperature with anti-gp20 at a dilution of 1:250 or with CAMPATH-1 (rat IgG2b, clone YTH34.5G2bb, Delta Biological SRL, Rome, Italy) at a dilution of 1:50 (final concentration: 20 µg/ml) in the same solution containing 0.1% Tween-20. After several washings with TBS containing 0.1% Tween-20, blots were incubated for 1 h with a goat anti-rabbit IgG conjugated with alkaline phosphatase (Biorad Microscience, Cambridge, MA, USA) or with a goat biotinylated anti-rat IgG followed by avidin conjugated with alkaline phosphatase. After extensive rinsing in TBS–Tween, the labelled proteins were developed using the Immun-star chemiluminescent protein detection systems (Biorad Microscience) according to the manifacturer's instructions. Preimmune serum was used as control.
Phase separation in solution of Triton X-114
To determine whether gp20 was amphiphilic or hydrophilic, experiments were conducted by the method of Bordier (1981). Briefly, sperm cells were solubilized with 1% Triton X-114, 10 mM Tris–HCl (pH 7.4) 150 mM NaCl, 1 mM EDTA, and the extracts layered over a cushion of the above buffer containing 0.06% Triton X-114 and 6% sucrose, warmed to 30°C for 3 min and then microfuged for 2 min. The aqueous phase was extracted twice more with 0.5% Triton X-114. The detergent phase was recovered as an oily droplet at the bottom of the tube. Aliquots of the detergent and aqueous phase and of the input sample were analysed by SDS–PAGE and immunoblotting as described above. The same protocol was also applied to human white blood cells obtained as described above.
Fluorescence microscopy
Aliquots of capacitated human spermatozoa were smeared onto ethanol-cleaned glass slides and allowed to attach at room temperature, taking care to keep them in a liquid phase. After repeated washings in PBS, the smears were blocked for 30 min with 2% bovine serum albumin (BSA) in PBS and then incubated for 1 h in the same buffer containing the anti-gp20 antibody diluted 1:20. After repeated washings, the smears were incubated with fluorescein-conjugated goat anti-rabbit IgG (Boehringer Mannheim, Germany) diluted to 20 µg/ml, washed, and mounted in PBS–glycerol. Other smears were instead incubated with the CAMPATH-1 antibody (final dilution 1:10) followed by fluorescein isothiocyanate (FITC)-labelled goat anti-rat IgG. Membrane integrity was checked in sperm smears using the impermeable nuclear dye propidium iodide (1 µg/ml) with and without permeabilization. Some untreated and ethanol-permeabilized smears were also double-labelled with rhodamine-conjugated PSA (Pisum sativum agglutinin) and acrosomal status was assessed by the method of Cross et al. (1986).
Blood smears obtained from volunteers were blocked for 30 min with 2% BSA in PBS and then incubated for 1 h in the same buffer containing the anti-gp20 antiserum at a dilution of 1:20. After repeated washings, the slides were incubated with a goat anti-rabbit IgG conjugated with fluorescein (Boehringer Mannheim, Germany) diluted to 20 µg/ml, washed and then mounted in PBS–glycerol.
All the samples were observed with a Laser Scanning Confocal Apparatus (BioRad Microscience).
N-terminal amino acid sequence
N-terminal amino acid sequence analysis of gp20 was performed using a pulsed liquid phase sequenator (Applied Biosystems 477A; Foster City, CA, USA) with on-line high-performance liquid chromatography (HPLC) analysis (Applied Biosystems 120A) of the resulting phenylthiohydantoin (PTH) amino acids as described: (i) HPLC fractions were applied to polybrene-coated fibreglass filters (manually precycled beforehand) and sequenced using the standard cartridge and standard cycles (NORMAL-1). (ii) Polyvinylidene difluoride (PVDF) (Biorad; Microscience, Cambridge, MA, USA) pieces of 2x5 mm were inserted into a Blot-cartridge without the fibreglass filter. The sequenator was then run using the BLOTT-2 cycle.
For the on-line PTH amino acid analysis we used a Shandon Hypersyl octadecylsilane (ODS) column (250 mmx1.6 mm, 5 mm) at 50°C with buffer A/3.5% tetrahydrofuran (Fluka, spectroscopy grade) in water adjusted to pH 4.0 with 14 ml of 3 M sodium acetate buffer (pH 3.8) and 2 ml of 3 M sodium acetate buffer (pH 4.6) and 200 ml triethylamine and buffer B/100% acetonitrile with 500 nM dimethylphenylthiourea at a flow rate of 125 ml/min. The gradient was: 10 min hold at 10% B, in 0.6 min to 14% B, in 18 min to 39% B, 9 min hold at 39% B, in 0.1 min to 90% B, and 7 min hold at 90% B.
Zona-free hamster egg penetration test
The effect of CAMPATH-1 on hamster egg penetration was evaluated by matching the same donor sperm suspensions exposed to scalar dilutions of CAMPATH-1. The hamster egg penetration assay (HEPT) enhanced with TES–Tris (TEST) yolk buffer (Sigma Chemical Co., St Louis, MO, USA) was performed as previously reported (Francavilla et al., 1997). Briefly, a motile sperm suspension from a normozoospermic donor was obtained by the swim-up procedure, mixed with an equal volume of TEST–yolk buffer and incubated at 4°C for 20 h. At the end of incubation, the mixture was washed twice in BWW containing 0.3% BSA and the pellets were resuspended in BWW containing 3% BSA. The sperm suspension was divided into aliquots, which were exposed to scalar dilutions of anti-gp20 immune serum and preimmune serum. Standard procedures were utilized for the recruitment and processing of hamster oocytes (World Health Organization, 1992). Sixteen to eighteen zona-free oocytes were added to 5.105 spermatozoa in 100 µl under paraffin oil. After 3 h of coincubation at 37°C in an atmosphere of 5% CO2/95% air, the oocytes were fixed in picric acid/formaldehyde overnight and stained with Giemsa. Ova were examined at x400 magnification for the evidence of swollen heads. The number of penetrations per oocyte (penetration index: PI) and the number of spermatozoa attached to the oolemma (bound spermatozoa) were recorded. The parameters of evaluation were expressed as ratios of test samples to control samples.
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Results |
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Triton X-100-extracted sperm components were fractioned by anion exchange Fast Performance Liquid Chromatography (FPLC) according to Focarelli et al. (1995) and the fractions corresponding to the peak including gp20 were pooled, dialysed extensively against distilled water, loaded on a tricine electrophoretic system and electroblotted (Focarelli et al., 1998
). A strip corresponding to the band reactive to anti-gp20 was then used for N-terminal sequence analysis. The following unique sequence of gp20 was obtained: GLY-GLN-ASN-ASP-THR-SER-GLN
A data bank search showed 100% identity with the aminoterminus of the mature form of CD52, a human leukocyte antigen (Hale et al., 1990). To determine whether gp20 was a GPI-anchored protein similar to CD52, we analysed its behaviour by temperature-induced phase separation in Triton X-114 of spermatozoa and leukocyte extracts. After treating washed spermatozoa with Triton X-114 and phase partitioning, the protein content of both the phases was analysed by SDS–PAGE on a tricine gel. Most proteins were detected in the aqueous phase; by this method no bands were found in the detergent phase (data not shown). However, immunoblot analysis revealed that gp20 was only present in the detergent phase (Figure 1). Immunoblot analysis of the same samples using the monoclonal antibody CAMPATH-1 was performed as control. As seen in Figure 1 the anti-CD52 reactive band had the same mobility as that identified by anti-gp20 but the signal was quite different. Indeed, anti-gp20 gave rise to a well-defined wide band (in other experiments, it often appears as a doublet, as already described in Focarelli et al. (1998) with an apparent mol. wt of 20 kDa, whereas anti-CD52 had a poorly defined profile and was associated with a component running as a broad smear from 20 to 14 kDa. Experiments were then carried out to investigate whether anti-gp20 reacted with CD52. Immunoblot analysis of the Triton X-114 leukocyte extract and of corresponding aqueous and detergent phases revealed only a very sharp reactive band with an apparent mol. wt of 20 kDa in whole extract and in the detergent phase (Figure 1).
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The CD52 antigen was also localized on leukocytes. Human blood smears were tested for reactivity with the anti-gp20 serum followed by FITC-labelled anti-rabbit IgG. All leukocytes showed bright fluorescence (Figure 2A
). Spotted superficial fluorescence was clearly observed by laser scanning confocal microscope (Figure 2B). To complete the immunological characterization of gp20, the CD52 monoclonal antibody (CAMPATH-1) was used in immunolocalization on capacitated spermatozoa and inhibition of the sperm penetration of zona-free hamster eggs. The two antibodies had very different effects in the immunofluorescence analysis of capacitated spermatozoa. Treatment with anti-CD52 produced high fluorescence on the tail and low fluorescence signal on the acrosomal region of the head (Figure 3A). Anti-gp20 used as control (Figure 3B) reacted mainly with components in the equatorial region of the head with minor positivity on the mid piece of the tail. This was in agreement with our previous results (Focarelli et al., 1998).
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Since we had shown that anti-gp20 caused dose-dependent inhibition of sperm penetration of zona-free hamster egg (Focarelli et al., 1998
), we also evaluated whether CAMPATH-1 had the same effect. This was done by matching the same donor sperm suspensions exposed to scalar dilutions of the antibody. Spermatozoa were preincubated in TEST–yolk buffer at 4°C which makes the cells highly fusogenic, enhancing sperm penetration (Johnson et al., 1984; Jacobs et al., 1995; Mortimer and Fraser, 1996). After coincubation of spermatozoa with eggs, sperm agglutinating activity and motility inhibition of the antibody were evaluated. As shown in Table I, CAMPATH-1 caused a significant inhibition of hamster egg penetration (P < 0.000005), with 20% inhibition persisting at an antibody dilution of 1:800 (1.2 µg/ml). The inhibitory effect was dose-dependent and involved both egg penetration and sperm binding to the oolemma. However, as indicated in Table I, the antibody also caused a concomitant dose-dependent agglutination effect on motile spermatozoa. With anti-gp20, no agglutination was observed at the antibody dilution used. It therefore cannot be excluded that the agglutination effect was responsible for the inhibition of sperm penetration in the presence of CAMPATH-1.
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Discussion |
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We recently reported that a sialoglycoprotein with an apparent mol. wt of 20 kDa (gp20) is synthesized by epididymal cells, acquired by human spermatozoa during their transit in this organ and becomes prevalently localized in the equatorial region of the head after capacitation (Focarelli et al., 1998
). Its involvement in sperm–egg fusion is also suggested by the fact that its polyclonal antibody inhibits sperm penetration of zona-free hamster eggs (Focarelli et al., 1998).
In the present study, we analysed the N-terminal of gp20, revealing a 100% identity with the N-terminal of CD52, a GPI-anchored antigen abundantly expressed on almost all human leukocytes (Hale et al., 1990). The presence of CD52 in the reproductive system was already known. Monoclonal antibodies against CD52, collectively called CAMPATH-1, are remarkably good effectors of complement-mediated lysis (Hale et al., 1983; Bindon et al., 1988) and have been widely used to treat lympho-proliferative disorders (Hale et al., 1988; Dyer et al., 1989) and to deplete lymphocytes in tissue transplants (Hale et al., 1983; Ettenger and Yudin, 1995). In a routine screening of the reactivity of these antibodies with other tissues, it was noted that they only reacted with the reproductive system (Hirsh et al., 1989). The antigen was associated with epididymal sperm maturation since it was found on epididymis, on seminal plasma and on the surface of mature but not testicular spermatozoa (Hale et al., 1993; Kirchhoff, 1996). This was confirmed by the finding of epididymal cDNA colinear with CD52 cDNA (Kirchhoff et al., 1993) and of CD52 transcripts in the epithelial cells of the distal epididymis and deferent ducts (Kirchhoff et al., 1993; Krull et al., 1993).
Our results, with spermatozoa instead of leukocytes, confirm that a CD52 homologue (gp20) is present on the sperm surface and is acquired during epididymal transit (Focarelli et al., 1998). The present data also show that gp20 behaves as a GPI-anchor protein in a phase separation test and that anti-gp20 also reacts with a leukocyte antigen which has the same mobility as the antigen found in spermatozoa and which remains in the detergent phase after phase separation. CAMPATH-1, used as a control on the same sperm sample treated with anti-gp20, confirmed that the bands reacting to our antibody and to the anti-CD52 monoclonal antibody have the same mobility. Although anti-gp20 and CAMPATH-1 react with the same antigen there are some intriguing differences. The CAMPATH-1 leukocyte antigen is reported to separate as a very broad band with an apparent molecular mass of 21–28 kDa (Xia et al., 1991) and the antigen recognized by the same antibody in the reproductive system runs as a broad smear between 18 and 25 kDa (Hale et al., 1993). In leukocyte and sperm immunoblots anti-gp20 gave rise to a well-defined band at 20 kDa; the only difference was that the band produced by sperm extract was wider than that produced by leukocytes, and in some cases it appeared as a doublet (Focarelli et al., 1998).
Although we have not yet any explanation for this difference, it is tempting to speculate that anti-gp20 and CAMPATH-1 recognize different epitopes. The structure of CD52 is unusual and well known. It consists of a short peptide (12 aminoacids) linked to a large sialylated, polylactosamine-containing, core-fucosylated tetra-antennary oligosaccharide and to a simple GPI membrane anchor (Xia et al., 1993; Treumann et al., 1995). The CAMPATH-1 antibodies only seem to recognize an epitope including the GPI-anchor plus a C-terminal tripeptide (Xia et al., 1993). This suggests that anti-gp20 prevalently interacts with a carbohydrate moiety, which could also explain the different localization of the antigen revealed by the two antibodies. In fact, on freshly ejaculated spermatozoa, CAMPATH-1 and anti-gp20 both revealed generic presence of the antigen on the whole sperm surface (Hale et al., 1993; Yeung et al., 1997; Focarelli et al., 1998), whereas on capacitated spermatozoa, anti-gp20 mainly reacted with components in the equatorial region of the head (Focarelli et al., 1998) and CAMPATH-1 was only positive on the tail. This difference could be related to the presence of differently glycosylated forms of the antigen on the tail and in the equatorial region of the head. However, since the antigen recognized by anti-gp20 on spermatozoa contains two reactive subcomponents, our antibody may recognize a different glycosylated form of CD52, specific to the reproductive system and localized prevalently on capacitated spermatozoa.
In line with the immunolocalization, the two antibodies affected the capacitated spermatozoa differently. Anti-gp20 did not agglutinate spermatozoa at dilutions over 1:10 (Focarelli et al., 1998) whereas CAMPATH-1 agglutinated spermatozoa, tail to tail (Hale et al., 1993) in a dose-dependent manner up to a dilution of 1:800. This agglutination effect makes it difficult to correctly evaluate the zona-free hamster egg inhibition test with CAMPATH-1. In fact, like anti-gp20, this antibody brings about a decrease in penetrated spermatozoa (Focarelli et al., 1998). However, since CAMPATH-1 agglutinates spermatozoa up to the dilution (1:800) at which minimum inhibition was obtained, it is impossible to evaluate to what extent agglutination influences the phenomenon. Work is now underway in our laboratory to explore the potential of our antibody in the immunological dilssection of gp20-CD52.
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Acknowledgments |
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We thank Professor Geoffrey Hale for helpful discussion and comments. We also thank Leonardo Gamberucci for assistance to artwork. This work was supported by a grant (60%) from Ministero della Università e della Ricerca Scientifica e Tecnologica.
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Notes |
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4 To whom correspondence should be addressed
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References |
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Submitted on July 10, 1998; accepted on October 6, 1998.
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