Molecular Human Reproduction, Vol. 6, No. 5, 422-428,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation
1 Graduate Institute of Medical Science, Department of Biology and Anatomy, National Defense Medical Center, Taipei, 2 Graduate Institute of Life Sciences, Department of Biology and Anatomy, National Defense Medical Center, Taipei and 3 Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan 100, ROC
Abstract
Surface glycoconjugates of spermatozoa are modified during epididymal maturation, which is closely related to the development of sperm function. In addition, recognition of surface glycoconjugates is one of very critical events in sperm–oocyte interaction. The binding of carbohydrate-specific lectins to the human sperm surface during epididymal maturation has been investigated. However, the glycoproteins responsible for lectin binding in sperm maturation are not well documented. This study used wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (Con-A) to identify sperm maturation-related glycoproteins in human epididymis. Histochemical localization revealed that the binding sites of WGA, PNA and Con-A were mainly in the principal cells and luminal contents of the human epididymis, but not in the interstitial regions. Each lectin displayed a fairly distinct regional localization. On Western blots probed with WGA and Con-A, glycoproteins of 83 kDa (GP-83) and 39 kDa (GP-39) were identified in the sperm extracts, epididymal fluid and tissue extracts of the corpus and cauda epididymides, but not in the caput. PNA identified GP-83 in the same manner as WGA and Con-A, but did not recognize GP-39. These results suggest that lectin-binding glycoproteins GP-83 and GP-39 found on mature spermatozoa may be secreted by the principal cells of corpus and cauda epididymis, and conjugated to spermatozoa during their transit in human epididymis.
epididymis/glycoprotein/human/sperm maturation
Introduction
During epididymal transit, human spermatozoa have been shown to acquire forward motility and fertility potential (Cooper, 1990
). Although >90% of spermatozoa remain immotile in the epididymal caput, the percentage of motile spermatozoa increases abruptly in the corpus (Dacheux et al., 1987). The majority of epididymal spermatozoa acquire fertility potential after passing through the corpus and cauda epididymis (Moore, 1998). IVF studies showed that the fertilization rate is significantly higher in spermatozoa from the corpus than those from the caput (Mahadevan and Trounson, 1985; Pryor, 1987). Moreover, sperm motility and pregnancy rate were significant improved when the vas was surgically joined to the corpus in those patients who received `specific tubule' vasoepididymostomy for obstructive azoospermia (Fogdestam et al., 1986; Schoysman and Bedford, 1986; Silber, 1989).
Surface glycoconjugates of mammalian spermatozoa play important roles in epididymal maturation (Liu et al., 1991b) and sperm–oocyte binding (Kumar et al., 1990). Sialic acid (sial) and N-acetyl glucosamine (glcNAc) inhibit binding of spermatozoa to zona-free oocytes in mammals (Lambert and van Le, 1984; Ahuja, 1985). Wheat germ agglutinin (WGA), a lectin specific to sial and glcNAc (Nagata, 1974), has been found to inhibit the binding of hamster oocytes to spermatozoa (Oikawa et al., 1973). In addition, sperm binding to WGA-coated microbeads is correlated with the sperm morphology rating (Gabriel and Franken, 1997).
Lectin binding to the sperm surface during epididymal maturation has been investigated in the mouse (Liu et al., 1991b), rat (Kumar et al., 1990), dog (Bains et al., 1993) and monkey (Fourie et al., 1996; Navaneetham et al., 1996). In the human, WGA and concanavalin A (Con-A) binding was found in principal cells and luminal contents of the whole epididymis (Arenas et al., 1996). Peanut agglutinin (PNA) binding was detected in microvilli of principal cells and epididymal fluid of the caput and corpus, but not in the cauda epididymis. However, the glycoproteins responsible for lectin binding were not documented. Previous studies identified two WGA-binding glycoproteins, GP-49 and GP-83, which were secreted by the epididymis and conjugated to spermatozoa during maturation in BALB/c mice (Liu et al., 1991b). Since peanut agglutinin (PNA) and Con-A also bind specifically to the outer and inner acrosomal membranes of human spermatozoa (Mortimer et al., 1987; Holden et al., 1990; Carver-Ward et al., 1997), this study used WGA, PNA and Con-A to identify sperm maturation-related glycoproteins in human epididymis.
Materials and methods
Human epididymis
Epididymides were obtained from patients with prostate carcinoma who received orchidectomy before hormone therapy at the Division of Urology, Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan. Experiments were conducted immediately after the epididymides were removed. For each pair of epididymides recovered, one was used for immunohistochemical observation and one for protein analysis. Epididymides (12 pairs) from patients aged 55–75 years 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.
Histochemical localization of WGA, PNA and Con-A binding sites in human epididymis
After removed, the epididymides were trimmed, washed in phosphate-buffered saline (PBS, pH 7.4), fixed in Bouin's solution (15 parts of saturated picric acid aqueous solution, 5 parts of formalin and 1 part of acetic acid) and then washed in PBS to remove excessive fixative. After dehydrating through a graded series of ethanol (50, 70, 80, 95 and 100%), the epididymides were cleared in xylene and embedded in paraffin. Paraffin sections (6–8 µm) were cut using a microtome, deparaffinized by xylene and dehydrated through a graded series of ethanol (100, 95, 80, 70 and 50%). The sections were washed with PBS and then with PBS containing 0.5% Tween and 1% gelatin to block the non-specific binding. After incubating with 0.25 µg/ml WGA (Triticum vulgaris, 36 kDa, specific for sial and glcNAc; Sigma, St Louis, MO, USA), PNA (Arachis hypogaea, 120 kDa, specific for ß-galactose; Sigma) or Con-A (Canavalia ensiformis, 102 kDa, specific for 
-mannose and -glucose; Sigma) conjugated with peroxidase for 30 min. The WGA, PNA and Con-A binding sites were revealed with a solution containing 0.05% diaminobenzidine-4HCl (Sigma), 0.1% H2O2 and 0.05 mol/l Tris–HCl buffer, pH 7.6. The sections were then counterstained with haematoxylin, dehydrated and mounted with Permount.
WGA, PNA and Con-A binding proteins on Western blots
After removal, human epididymides were trimmed, placed in PBS and separated into caput, corpus and cauda by obvious anatomical landmarks, i.e. caput and corpus were separated at neck, corpus and cauda were separated at the site where engorged tubules were first recognized. Epididymal tissues were washed briefly in PBS, and cut into small pieces to release the luminal contents into PBS. The epididymal tissues were further washed in PBS to remove the residual luminal content. The luminal contents in PBS were centrifuged at 1000 g for 10 min, and the supernatants were saved as epididymal fluid. Spermatozoa in the sediments were further washed in PBS three times to remove epididymal fluid.
Epididymal tissues and spermatozoa from the caput, corpus and cauda were extracted separately in ~10x tissue volume of an extraction buffer containing 0.15 mol/l NaCl, 10 mmol/l HEPES, 0.5% Triton-X 100, 0.2 mmol/l aprotonin (Boehringer Mannheim, Mannheim, Germany) and 0.2 mmol/l 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 using the Micro-Bradford protein assay (Bradford, 1976).
Cell extracts (25 µg per lane) were separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) with 5% stacking and 10% separating gels. After electrophoresis at 15 mA for 1 h on a mini vertical slab gel unit (Bio-Rad, Richmond, CA, USA), the proteins were electrotransferred to nitrocellulose paper with a semi-dry transfer unit (TE-70; Hoefer, San Francisco, CA, USA). After blocking the non-specific binding with PBS containing 0.5% Tween and 1% gelatin, the blots were incubated with 0.25 µg/ml WGA, PNA or Con-A conjugated with peroxidase for 30 min. WGA, PNA and Con-A binding proteins were revealed in 0.05 mol/l Tris–HCl buffer containing 0.05% diaminobenzidine-4HCl (Sigma) and 0.1% H2O2, pH 7.6.
Con-A–agarose affinity chromatography
In order to confirm whether the 83 and 39 kDa glycoproteins recognized by WGA, PNA and Con-A represented identical molecules for each lectin, sequential lectin-binding studies were carried out. Con-A binding proteins were first recovered from a Con-A affinity column since Con-A recognized more proteins than WGA and PNA. Con-A–agarose (1 ml) was packed in a poly-prep column (Bio-Rad), and washed with 10 ml of starting buffer containing 1% Nonidet P-40, 10 mmol/l Tris–HCl, 0.15 mol/l NaCl, 2 mmol/l CaCl2 and 0.1% sodium azide. Epididymal fluid from the cauda was applied to the column and incubated for 30 min. After non-binding proteins were washed out with 10 ml of starting buffer, Con-A binding proteins were eluted in starting buffer containing 0.5 mol/l -methyl-D-mannoside (Sigma). The eluents were dialysed in a buffer containing 0.05 mol/l Tris–HCl and 0.1% sodium azide for 48 h. Finally, WGA-, PNA- and Con-A binding proteins in the eluents were identified on Western blots as described above.
Results
WGA, PNA and Con-A binding sites in human epididymis
The binding sites of WGA, PNA and Con-A in human epididymis were investigated by histochemical localization on paraffin sections. The binding sites of all these lectins were mainly in principal cells and luminal contents, but not in the interstitial region. WGA binding sites were located on stereocilia and in the apical region of principal cells in the caput and cauda, and extended through some principal cells in the corpus (Figure 1). PNA binding sites in principal cells were gradually increased from the caput to the cauda (Figure 2). Con-A binding sites were confluent in principal cells of the whole epididymis (Figure 3), and present only sparsely in interstitial regions of the corpus and cauda. The distribution of lectin binding sites was essentially consistent in all 12 epididymides used in this study.
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WGA, PNA and Con-A binding proteins in human epididymis
WGA, PNA and Con-A binding proteins in human epididymal spermatozoa (Figure 4
), fluid and tissue (Figure 5) were identified on Western blots probed with the lectins. Among the glycoproteins recognized by WGA, two major proteins with molecular weights of 83 kDa (GP-83) and 39 kDa (GP-39) were found in membrane extracts of spermatozoa from the corpus and cauda (Figure 4). Since spermatozoa from the corpus and cauda epididymis are functionally mature, GP-83 and GP-39 were considered as sperm maturation-related glycoproteins. These two proteins were also found in tissue homogenates and epididymal fluid of corpus and cauda (Figure 5b). Blots probed with PNA revealed GP-83 only in tissue homogenate and epididymal fluid of corpus and cauda (Figure 5c). Con-A blots revealed both GP-83 and GP-39 in tissue homogenates and epididymal fluids of corpus and cauda (Figure 5d). In addition to GP-83 and GP-39, other lectin-binding proteins were found. However, GP-83 and GP-39 were the only major proteins consistently present in the corpus and cauda, but not in the caput. The lectin-binding proteins present in the whole epididymis were not considered to be spermatozoa maturation-related. The patterns of lectin-binding proteins were essentially the same among all 12 epididymides used in this study.
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Con-A–agarose affinity chromatography
Since Con-A recognized more proteins than WGA and PNA, Con-A binding proteins were recovered from a Con-A affinity column (Figure 6a
) and probed with WGA (Figure 6b) and PNA (Figure 6c) to confirm whether GP-83 and GP-39 identified by WGA, PNA and Con-A were the same molecules. Among Con-A binding proteins, WGA identified both GP-83 and GP-39 (Figure 6b), whereas PNA identified only GP-83 (Figure 6c). These results revealed that GP-83 bound WGA, PNA and Con-A, but GP-39 bound WGA and Con-A only.
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Discussion
In this study, WGA, Con-A and PNA were found to bind to principal cells through the entire length of human epididymis. These findings are consistent with an earlier observation (Arenas et al., 1996) that regional differences in lectin binding were less pronounced in human epididymis than those reported in other mammals. However, the intensity of WGA and PNA binding in principal cells showed remarkable regionalization. WGA binding sites were restricted to stereocilia and the apical region of the principal cells in caput and cauda, but extended through some principal cells in the corpus. PNA binding sites in principal cells were gradually increased from caput to cauda. Since Western blots probed with WGA and Con-A revealed region-related protein profiles, the difference in intensities may be due to the number of proteins bound to lectins.
Although lectin binding in the epididymis has been extensively investigated in a number of mammals (Bains et al., 1993; Fourie et al., 1996; Navaneetham et al., 1996; Bendahmane and Abou-Halia, 1997; Parillo et al., 1997) including humans (Arenas et al., 1996, 1998), the biological functions of the lectin-binding proteins were not well characterized. A Con-A binding protein of 40 kDa, 436/10, is found in the acrosomal region of spermatozoa in human, horse, guinea pig, bull and ram (Runnebaum et al., 1995). However, this antigen is not related to epididymal maturation. Since spermatozoa become motile and fertile in the corpus and cauda epididymis, GP-83 and GP-39 identified in spermatozoa of the corpus and cauda in this study are probably sperm maturation-related glycoproteins. The consistent presence of GP-83 and GP-39 in tissue homogenates and epididymal fluid of the corpus and cauda further support our contention that they are sperm maturation-related.
In a series of studies, Tezon et al. investigated protein synthesized in human epididymal tissue in vitro (Tezon et al., 1985a; 1985b; 1987). Androgen might stimulate the secretion of five proteins of 38, 21, 69, 13.9 and 29 kDa in cultured human epididymal tubules (Tezon et al., 1985a). The antiserum raised against 0.6 mol/l NaCl extract of human ejaculated spermatozoa recognized three epididymal proteins of Ra (mobility relative to albumin) 0.3 (38 kDa), 0.43 (21 kDa) and 1.0 (29 kDa) (Tezon et al., 1985b). The antiserum generated from luminal content of the cauda epididymis identified a protein of 70 kDa purified from the proteins synthesized by human epididymis in vitro (Tezon et al., 1987). Although these molecules were identified by antisera of ejaculated spermatozoa or luminal content of the cauda epididymis, their functional roles were not documented. Among those proteins, the protein of 38 kDa was similar to GP-39 in molecular weight. However, the molecule was secreted by the caput, proximal and distal corpus, especially the caput (Tezon et al., 1985a). In this study, GP-39 was found in the corpus and cauda, but not in the caput. Hence, GP-83 and GP-39 are novel sperm maturation-related glycoproteins identified in human epididymis.
The presence of GP-83 and GP-39 in the spermatozoa of the corpus and cauda may result from de-novo synthesis by spermatozoa in the epididymis, or be secreted by principal cells of the corpus and cauda epididymis. The morphology of epididymal spermatozoa (including very condensed chromatin, sparse cytoplasm and organelles), does not support active protein synthesis. On the contrary, the well-developed endoplasmic reticulum and Golgi apparatus of principal cells indicate active protein synthesis. Some studies have shown that the principal cells of epididymis might incorporate radiolabelled amino acids and transport the radiolabelled molecules through the cell (Flickinger, 1979, 1981). In addition, our previous studies demonstrated that the WGA-binding glycoproteins, GP-83 and GP-49, found on mature spermatozoa (Liu et al., 1991b) were secreted by the epididymis and conjugated to spermatozoa during epididymal maturation in BALB/c mice (Liu et al., 1991a). Therefore, GP-83 and GP-39 identified in this study may be secreted by the epididymis and conjugated to spermatozoa as they pass through, in essentially the same manner as GP-83 and GP-49 in BALB/c mice (Liu et al., 1991a). However, whether human GP-83 is identical to mouse GP-83 remains to be determined.
Principal cells are actively involved in the physiological functions of the epididymis, including endocytosis (Hermo et al., 1998) and secretion (Legare et al., 1999). Proteins secreted by principal cells may interact with spermatozoa in the lumen of epididymal ducts, and enable spermatozoa to develop motility (Jaiswal and Majumder, 1998) and fertility (Smithwick and Young, 1999). Forward motility protein, a heat-stable protein in epididymal plasma, induces sperm motility in a dose-dependent manner (Jaiswal and Majumder, 1998). P34H, which appears on the acrosome of spermatozoa during epididymal maturation (Boue et al., 1996), is secreted by the corpus epididymis (Legare et al., 1999) and is involved in sperm–oocyte interaction (Boue et al., 1994). In this study, GP-83 and GP-39 were found on mature spermatozoa in the corpus and cauda, but not immature spermatozoa in the caput, indicating that GP-83 and GP-39 are sperm maturation-related. In addition, GP-83 and GP-39 were first found simultaneously in epididymal tissue, luminal fluid and spermatozoa of the corpus, but not in those of the caput. Since the corpus is the site where spermatozoa become motile and fertile (Cooper, 1990), these results further support our contention that GP-83 and GP-39 are indeed sperm maturation-related glycoproteins that may play important roles in sperm function.
Among Con-A binding proteins eluted from a Con-A affinity column, WGA identified both GP-83 and GP-39, and PNA identified GP-83. These results indicate that GP-83 is a glycoprotein composed of monosaccharides recognized by WGA, PNA and Con-A. GP-39 contains monosaccharides recognized by WGA and Con A. Therefore, GP-83 on mature spermatozoa contains sial, glcNAc, ß-galactose, -mannose and -glucose, whereas GP-39 contains sial, glcNAc, -mannose and -glucose. Since appropriate recognition of surface carbohydrates is a crucial event in sperm–oocyte fertilization (reviewed by Wassarman, 1990), WGA binding to human spermatozoa is closely related to sperm motility (Lassalle and Testart, 1994) and fertilization in vitro (Gabriel et al., 1995). WGA efficiently blocks human sperm–zona pellucida binding (Mori et al., 1993). These results strongly suggest that GP-83 and GP-39 may be involved in sperm–oocyte interaction.
In conclusion, WGA, PNA and Con-A identified glycoproteins GP-83 and GP-39 on mature spermatozoa in the corpus and cauda of human epididymis, but not in the caput epididymis. Both GP-83 and GP-39 may be secreted by the corpus and cauda epididymis and conjugated to spermatozoa as they pass through. These two glycoproteins may be involved in sperm–oocyte interaction.
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 83–0412-B-016-012).
Notes
4 To whom correspondence should be addressed 
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Submitted on November 1, 1999; accepted on February 14, 2000.
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T. C. McCauley, B. E. Kurth, E. J. Norton, K. L. Klotz, V. A. Westbrook, A. J. Rao, J. C. Herr, and A. B. Diekman Analysis of a Human Sperm CD52 Glycoform in Primates: Identification of an Animal Model for Immunocontraceptive Vaccine Development Biol Reprod, June 1, 2002; 66(6): 1681 - 1688. [Abstract] [Full Text] [PDF]
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Y.-C. Lin, G.-H. Sun, Y.-M. Lee, Y.-W. Guo, and H.-W. Liu Cloning and Characterization of a Complementary DNA Encoding a Human Epididymis-Associated Disintegrin and Metalloprotease 7 Protein Biol Reprod, September 1, 2001; 65(3): 944 - 950. [Abstract] [Full Text] [PDF]
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