Molecular Human Reproduction, Vol. 6, No. 5, 429-434,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Purification of GP-83, a glycoprotein secreted by the human epididymis and conjugated to mature spermatozoa
1 Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, 2 Graduate Institute of Life Sciences, Department of Biology and Anatomy, National Defense Medical Center, Taipei, 3 Department of Biology and Anatomy, National Defense Medical Center, Taipei and 4 Graduate Institute of Medical Science, Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan 100, ROC
Abstract
Epididymal secretions are critical for mammalian spermatozoa to acquire both forward motility and an ability to recognize and penetrate oocytes. Previous studies identified two glycoproteins, GP-83 and GP-39, which were secreted by the human epididymis and may be related to maturation of sperm function. In this study, GP-83 was purified from human seminal fluid by DEAE–ion exchange, gel filtration chromatography and preparative gel elution. The isoelectric point (pI) of purified GP-83 was 6.57. Monospecific antiserum to GP-83 was induced in male New Zealand rabbits and confirmed on immunoblots. GP-83 was found in fluid, tissue and sperm extracts of corpus and cauda epididymis, but not in the caput. Immunohistochemical localization identified GP-83 in the luminal contents and in the supranuclear region and cell membrane of principal cells of the corpus and cauda epididymis. GP-83 was found on the anterior acrosome in ejaculated spermatozoa, and shifted to the equatorial region after capacitation and the acrosome reaction.
epididymis/glycoprotein/human/sperm maturation
Introduction
Sperm membrane molecules play an important role in the acrosome reaction (Yeung et al., 1997b
; Cooper and Yeung, 1998), sperm–zona binding (Gamzu et al., 1998) and sperm–oocyte fusion (Gabriele et al., 1998). The membrane properties of mammalian spermatozoa, including membrane potential (Abou-Halia and Fain-Maurel, 1984), antigenicity (Eddy et al., 1985; Sarkar and Chatterjee, 1997; Yoshinaga et al., 1998), protein composition (Hunnicutt et al., 1997) and lectin binding (Liu et al., 1991; Fourie et al., 1996), are modified during epididymal maturation (Schoysman and Bedford, 1986; Silber, 1989).
Proteins secreted by mammalian epididymis are found conjugated to spermatozoa and are related to the development of forward motility and fertility. In humans, two sialoproteins of 24 and 37 kDa appeared on the sperm surface during epididymal transit (Dacheux et al., 1987). CD52 is a glycosylphosphatidylinositol-anchored glycoprotein that conjugates to spermatozoa in epididymis, is found in 80% of viable spermatozoa and is closely related to motility, swimming velocity and viability (Yeung et al., 1997a). GP-20, a sialyglycoprotein of 20 kDa secreted by the human epididymis, may be involved in the early stages of fertilization (Focarelli et al., 1998). SOB3 appears on human spermatozoa in the corpus epididymis, and participates in secondary binding to the zona pellucida (Martin Ruiz et al., 1998). In addition to these molecules, previous studies have identified two sperm maturation-related glycoproteins, GP-83 and GP-39, in human epididymis (Liu et al., 2000). In this study, the purification and further characterization of GP-83 are reported.
Materials and methods
Purification of GP-83 from human seminal fluid
Since it was difficult to collect enough human epididymis to purify GP-83 from, and GP-83 was shown to be a major component of seminal fluid, in this study, we chose to purify GP-83 from human seminal fluid. Human semen of normal quality was collected from Tri-Service General Hospital, Taipei, Taiwan. Liquefied semen was diluted with 5 volumes of phosphate-buffered saline (PBS, pH 7.4), and centrifuged at 10 000 g for 10 min to recover the supernatant, i.e. seminal fluid. The seminal fluid was dialysed in 0.05 mol/l Tris–HCl, pH 7.4 for 48 h.
Dialysed seminal fluid (20 ml) was added to a 1.6x40 cm DEAE–Sephacryl column (Pharmacia, Uppsala, Sweden), washed and equilibrated with dialysing buffer. After washing with 200 ml of 0.05 mol/l Tris–HCl, pH 7.4, until no significant protein was detected at 280 nm, the column was eluted at 4°C in a stepwise fashion with 200 ml of 0.05 mol/l Tris–HCl containing 0.1 mol/l NaCl, 0.2 mol/l NaCl and 0.5 mol/l NaCl respectively. Fractions of 3 ml were collected. The protein concentrations of each fraction were determined using the Micro-Bradford protein assay (Bradford, 1976). The protein profile of each fraction was examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blots probed with wheat germ agglutinin (WGA) as described previously (Liu et al., 1991).
Fractions containing GP-83 were pooled, dialysed with 0.05 mol/l Tris–HCl, pH 7.4 and concentrated with freeze-drying. The concentrated GP-83 sample (6 ml) was applied to a 1.6x100 cm equilibrated S-300 Sephacryl gel filtration column (Pharmacia). The column was washed with 500 ml of 0.05 mol/l Tris–HCl, pH 7.4 at 4°C. Fractions of 3 ml were collected. The protein concentration and profile of each fraction was examined as described above.
Fractions containing GP-83 were pooled, dialysed and concentrated as stated above. The concentrated sample was further separated by preparative SDS–PAGE with 5% stacking and 10% separation gels. GP-83 was recovered from the gel by gel elutor (GE200; Hoefer, San Francisco, CA, USA) from 0.025 mol/l Tris–HCl buffer containing 0.192 mol/l glycine, 0.1% SDS, pH 8.3 to 0.1 mol/l Tris–HCl buffer containing 0.768 mol/l glycine, 0.4% SDS, pH 8.3.
The isoelectric point (pI) of the purified GP-83 was determined by isoelectric focusing (IEF) gel electrophoresis in 5% polyacrylamide gels containing 2% Bio-Lyte 3/10 ampholyte (Bio-Rad, Richmond, CA, USA) on a mini IEF cell (model 111; Bio-Rad).
Preparation of GP-83 specific antiserum in rabbits
Purified GP-83 was dialysed with 0.05 mol/l (NH4)HCO3 for 24 h. The dialysed GP-83 was concentrated to 2 mg/ml by freeze drying. GP-83-specific antisera were induced in male New Zealand rabbits (1.5–2 kg body weight). The rabbits were immunized first by intradermal injection of purified GP-83 mixed with complete Freund's adjuvant. At 4 weeks after the primary immunization, the rabbits were boosted with purified GP-83 mixed with incomplete Freund's adjuvant three times every 2 weeks. One week after each boost, titres of the antiserum were examined on immunoblots.
Immunohistochemical localization
Human epididymides removed from patients with prostate carcinoma who received orchidectomy prior to hormonal therapy in Tri-Service General Hospital were used in this study. For each pair of epididymides recovered, one was used for immunohistochemical observation and one for protein analysis. Epididymides (12 pairs) that were not invaded by the carcinoma from the surrounding prostate, and revealed apparently normal histology and spermatozoa in the cauda, were selected for this study. The patients were aged 55–75 years. The epididymides were trimmed, washed in PBS immediately after removal, and fixed in Bouin's solution containing 15 parts saturated picric acid aqueous solution, 5 parts formalin and 1 part acetic acid. After excess fixative was removed by washing in PBS, epididymides were dehydrated through a graded series of 50, 70, 80, 95 and 100% ethanol, cleared in xylene and embedded in paraffin. Paraffin sections (6–8 µm) were cut on a microtome.
Paraffin sections of epididymis were deparaffinized by xylene, and rehydrated through a graded series of 100, 95, 80, 70 and 50% ethanol. Sections were then washed three times in PBS, and in PBS containing 0.5% Tween and 3% skimmed milk to block non-specific binding for 1 h. The sections were then incubated with GP-83 antiserum for 1 h at room temperature. After three washes in PBS, the sections were incubated with goat anti-rabbit immunoglobulin G (IgG) conjugated with peroxidase (Cappel, Turnhout, Belgium) for 1 h. Immunoreactivities were revealed with 0.05 mol/l Tris–HCl buffer containing 0.05% diaminobenzidine–HCl (Sigma-Aldrich, St Louis, MO, USA) and 0.1% H2O2. The sections were counterstained with haematoxylin, dehydrated and mounted with Permount (Fisher, Fair Lawn, NJ, USA).
Immunoblots probed with GP-83 antiserum
Epididymal fluid, tissue extracts and sperm extracts were prepared as described previously (Liu et al., 1991). Briefly, human epididymides were trimmed and separated into caput, corpus and cauda by obvious anatomical landmarks in PBS, i.e. caput and corpus were separated at the neck, corpus and cauda were separated at the site where engorged tubules were first recognized. Epididymal tissues were washed in PBS briefly, and cut into pieces to release the luminal contents into PBS. The pieces of epididymal tissues were further washed in PBS to remove residual luminal content. The luminal contents (in PBS) were centrifuged at 1000 g for 10 min, and the supernatants saved as epididymal fluid. Spermatozoa in the sediments were further washed in PBS to remove epididymal fluid.
Epididymal tissues and spermatozoa from the caput, corpus and cauda were extracted separately in ~10x of tissue volume of an extraction buffer containing 0.15 mol/l NaCl, 10 mmol/l HEPES, 0.5% Triton-X 100, 1 mmol/l aprotonin (Boehringer Mannheim, Mannheim, Germany) and phenylmethylsulphonyl fluoride (Boehringer Mannheim) at 4°C for 30 min. The supernatant was recovered after centrifugation at 10 000 g for 20 min. Protein concentrations were measured by the Micro-Bradford protein assay.
Cell extracts, 25 µg protein per lane, were separated using SDS–PAGE, with 5% stacking and 10% separating gels. After electrophoresis, the proteins were electrotransferred to nitrocellulose paper with a semi-dry transfer unit (TE-70; Hoefer). After non-specific binding was blocked with PBS containing 0.5% Tween and 3% skimmed milk, the blots were then incubated with GP-83 antiserum for 1 h at room temperature. After three washes in PBS, the sections were incubated with goat anti-rabbit IgG conjugated with peroxidase (Cappel) for 1 h. Immunoreacted proteins were revealed with 0.05 mol/l Tris–HCl buffer containing 0.05% diaminobenzidine–HCl (Sigma) and 0.1% H2O2.
Immunofluorescent localization of GP-83 in ejaculated spermatozoa
After semen was liquefied, spermatozoa were washed in PBS three times, then fixed in PBS containing 2% formaldehyde and 0.5% Triton X-100 for 15 min. After excess fixative was removed with three washes in PBS, spermatozoa were washed in PBS containing 0.5% Tween-20 (Sigma) and 3% skimmed milk for 30 min to block non-specific binding. Spermatozoa were then incubated with GP-83 antiserum for 1 h at room temperature. After three washes in PBS, spermatozoa were incubated with goat anti-rabbit IgG antibody conjugated with fluorescein isothiocyanate (FITC; Cappel) for 1 h. After three washes in PBS, GP-83 localization was observed with a Epiphot microscope (Nikon, Tokyo, Japan) equipped with a vertical illuminator for epifluorescence.
Capacitation and acrosome reaction of ejaculated spermatozoa in vitro
After semen was liquefied, spermatozoa were collected by three washes in Biggers–Whitten–Whittingham medium (BWW) and resuspended in BWW at 1x107 spermatozoa/ml. Washed spermatozoa (0.5 ml) were added to the top of 0.5 ml BWW containing 10% human serum albumin in a test tube. After incubated at 37°C for 3 h, the swim-up spermatozoa in the upper 0.5 ml were recovered and recognized as `capacitated spermatozoa'. The capacitated spermatozoa were incubated with 30 nmol/l calcium ionophore A23187 at 37°C for 15 min to induce the acrosome reaction. Capacitation and acrosome reaction of spermatozoa were evaluated by trichromatic stain (Talbot and Chacon, 1981). The presence of Gp-83 on these spermatozoa was investigated by immunofluorescent stain.
Trichromatic stain of spermatozoa
The trichromatic stain used in this study was modified from a previously described method (Talbot and Chacon, 1981). After proper preparation, spermatozoa were stained with 0.5% Trypan Blue at 37°C for 15 min. After three washes in BWW, spermatozoa were fixed in 2% glutaraldehyde for 15 min. After two washes in deionized water, resuspended spermatozoa were smeared on precleaned slides. The slides were stained with 0.8% Bismark brown solution, pH 1.8 at 40°C for 5 min. After three washes in deionized water, the slides were stained with 0.8% rose bengal in 0.1 mol/l Tris–HCl buffer, pH 5.3 at 24°C for 30 min. After washing three times in deionized water, the slides were mounted with Kaiser's glycerol gelatin (Merck, Darmstadt, Germany).
Results
Purification of GP-83 from seminal fluid
Although secreted by the epididymis, GP-83 is a major component of seminal fluid. Since epididymis is difficult to collect, and seminal fluid of normal quality is easier to collect from the laboratory, in this study, we purified GP-83 from seminal fluid by DEAE ion exchange, S-300 gel filtration chromatography and preparative gel elution.
Seminal fluid applied to DEAE ion exchange chromatography was eluted subsequently by 0.05 mol/l Tris–HCl, pH 7.2 containing 0.1 mol/l, 0.2 mol/l and 0.5 mol/l NaCl (Figure 1a). SDS–PAGE revealed that GP-83 was a major component in the fractions eluted with 0.05 mol/l Tris–HCl without NaCl (Figure 1b), and was accompanied by five other proteins. Western blots probed with WGA confirmed that WGA-binding of GP-83 was retained after elution from the column (Figure 1c). In addition, GP-39 was separated from GP-83 during the elution.
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GP-83 containing eluent from DEAE ion exchange chromatography were pooled and further fractionated by Sephacryl S-300 gel filtration chromatography (Figure 2a
). SDS–PAGE revealed that GP-83 was eluted in the fractions of first peak, which already showed apparent homogeneity (Figure 2b). WGA blots confirmed GP-83 and no other glycoprotein in the fractions (Figure 2c).
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GP-83 containing eluent was further purified by preparative gel elution. The estimated yield calculated from the total protein in the purified sample divided by total protein loaded on the DEAE ion exchange column was 0.5%. IEF gel electrophoresis again revealed that GP-83 was purified to apparent homogeneity with single pI (Figure 3a
). The pI of GP-83 determined from the regression curve of relative mobilities of IEF standards was 6.57 (Figure 3b).
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The presence of GP-83 in human epididymis
GP-83 specific antisera were induced in male New Zealand rabbits. The specificity and titre were evaluated with immunoblotting. One week after the fourth boost, antiserum was found specific to GP-83 on immunoblots at dilution of 1:10 000 (Figure 4
). GP-83 was found in fluids, tissue and sperm extracts of the corpus and cauda, but not in the caput (Figure 4).
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Immunohistochemical localization with monospecific antiserum revealed the presence of GP-83 in the supranuclear region and stereocilia of principal cells, and luminal contents of the corpus and cauda epididymis, but not in the principal cells or luminal contents of the caput, nor in interstitial tissue of any regions (Figure 5
). White staining of GP-83 in the luminal contents of the corpus epididymis was not so obvious, the immunoblots (Figure 4
) demonstrating its presence in the fluid of the corpus. Furthermore, GP-83 specific antiserum did not react with human testis, seminal vesicle, prostate, mouse and rat epididymis (data not shown).
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The presence of GP-83 on spermatozoa after capacitation and the acrosome reaction
Ejaculated semen was capacitated in vitro by swim-up in 10% human serum protein, and the acrosome reaction was induced by calcium ionophore A23187. Trichromatic stain (Talbot and Chacon, 1981
) revealed that acrosomes of capacitated spermatozoa were pinkish in colour, as were those of ejaculated spermatozoa (Figure 6b), but were whitish after acrosome reaction (Figure 6d). Immunofluorescent stain showed that GP-83 was located on the surface of the anterior acrosome in ejaculated (Figure 6a) and capacitated spermatozoa (Figure 6c), and on the equatorial region in spermatozoa after the acrosome reaction (Figure 6e).
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Discussion
In this study, sperm maturation-related GP-83 was purified from human seminal fluid by DEAE ion exchange chromatography and then by Sephacryl S-300 gel filtration chromatography and preparative gel elution. Homogeneity was confirmed by SDS–PAGE, WGA blots and isoelectric focusing gel electrophoresis. GP-83 specific antisera induced in male New Zealand rabbits identified GP-83 in fluids, tissue and sperm extracts of the corpus and cauda, but not in the caput. These results are consistent with our previous findings that wheat germ agglutinin (WGA) can be used to identify GP-83 in mouse epididymis (Liu et al., 1991).
Immunohistochemical localization revealed GP-83 first in the supranuclear region and stereocilia of principal cells, and then in luminal contents of corpus and cauda epididymis; but not in the principal cells or luminal contents of caput (Figure 5). These results suggest that GP-83 may be synthesized and secreted by principal cells of the corpus and cauda, and conjugated to spermatozoa when they are passing by. GP-83, therefore, may be a human sperm maturation-related glycoprotein.
In addition to GP-83, GP-20 (Focarelli et al., 1998), SOB3 (Martin Ruiz et al., 1998), CD52 (Yeung et al., 1997a,b) and P34H (Boue et al., 1996) are reported as proteins secreted by epididymis and conjugated to spermatozoa during maturation. GP-20 is a sialyglycoprotein of 20 kDa found in epididymal epithelia, seminal fluid and sperm surface, but not germ cells in testis (Focarelli et al., 1995). SOB3 appears on human spermatozoa in corpus epididymis, and is not detected in testis (Martin Ruiz et al., 1998). CD52 is a glycosylphosphatidylinositol-anchored glycoprotein secreted by human epididymis, and conjugates to spermatozoa in epididymis (Yeung et al., 1997b). P34H is secreted by corpus epididymis (Legare et al., 1999) and appeared in the acrosome of spermatozoa during epididymal maturation (Boue et al., 1996). These results suggest that conjugation of epididymal secretions to spermatozoa is a common mechanism in epididymal maturation.
The overall yield of GP-83 from human seminal fluid by our purified procedure was 0.5%, which indicated that GP-83 secreted by epididymis is a major component of seminal fluid. These results also indicate that GP-83 not only conjugates to spermatozoa during their passage in epididymis, but also accompanies spermatozoa through the whole male reproductive tract. Therefore, besides its role in epididymal maturation, other physiological functions of GP-83 deserve further characterization.
Proteins secreted by human epididymis have been intensively investigated for their specific roles in maturation of sperm function and fertilization (Kirchhoff, 1998). Especially, monospecific antibodies are used to investigate functional roles of human sperm maturation-related antigens (Yeung et al., 1997b; Focarelli et al., 1998; Martin Ruiz et al., 1998). Motility, swimming velocity and viability of spermatozoa are hampered by CAMPATH-1G, a CD52-specific monoclonal antibody (Yeung et al., 1997a). The antiserum specific to GP-20 blocks sperm penetration of zona-free hamster oocytes (Focarelli et al., 1998). LB5, a SOB3-specific monoclonal antibody, inhibits sperm binding to human zona pellucida by 35.7% for ejaculated spermatozoa and 59.9% for acrosome-reacted spermatozoa (Martin Ruiz et al., 1998). These results indicate that the sperm maturation-related proteins secreted in the epididymis play an important role in the development of sperm motility and fertility. In this study, GP-83 was found in the equatorial region after the acrosome reaction. Since the equatorial region is where spermatozoa bind and fuse with the oocyte (Soupart and Strong, 1974), these results suggest that GP-83 may be involved in sperm–oocyte fertilization. Nevertheless, the role of GP-83 in fertilization needs to be confirmed by more specific investigation, e.g. inhibition of sperm–zona binding or IVF.
Sperm maturation-related molecules in the sperm membrane have been found to be redistributed during capacitation and acrosome reaction. GP-20 is shifted from the sperm head and midpiece to a sharp zone of the equatorial region during capacitation (Focarelli et al., 1998). In this study, GP-83 was shifted from the anterior acrosome to the equatorial region after the acrosome reaction. Although the mechanisms of redistribution of GP-20 and GP-83 have not been documented, the redistribution of fertilin during maturation has been studied extensively (Hunnicutt et al., 1997). Fertilin is a heterodimer of subunits 
and ß that mediate sperm–oocyte fusion. Fertilin is processed in the testis, and fertilin ß is processed in epididymis. Fertilin, therefore, migrates from the entire sperm head to the posterior head during epididymal maturation (Hunnicutt et al., 1997). Serine protease associated with testicular spermatozoa is shown to process fertilin ß in vitro in a manner that mimics epididymal maturation (Lum and Blobel, 1997). Whether protease activity of spermatozoa is involved in migration of GP-83 needs further verification.
In conclusion, GP-83, a glycoprotein secreted by epididymis, is found on ejaculated, capacitated and acrosome-reacted spermatozoa, and may be involved in sperm–oocyte fertilization.
Acknowledgments
The authors are grateful to Professor Gu-Gang Chang of Department of Biochemistry, National Defense Medical Center for his valuable advice in preparing this paper. This study was supported in part by a grant from the National Science Council, ROC (NSC 84–2331-B-016-091).
Notes
5 To whom correspondence should be addressed 
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Submitted on November 1, 1999; accepted on February 14, 2000.
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