Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print February 18, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.023259
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/6/1710    most recent
biolreprod.103.023259v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chiu, P.C.N.
Right arrow Articles by Yeung, W.S.B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiu, P.C.N.
Right arrow Articles by Yeung, W.S.B.
Agricola
Right arrow Articles by Chiu, P.C.N.
Right arrow Articles by Yeung, W.S.B.
BIOLOGY OF REPRODUCTION 70, 1710–1719 (2004)
DOI: 10.1095/biolreprod.103.023259
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

The Contribution of D-Mannose, L-Fucose, N-Acetylglucosamine, and Selectin Residues on the Binding of Glycodelin Isoforms to Human Spermatozoa1

P.C.N. Chiu3, H.Y. Tsang3, R. Koistinen4, H. Koistinen4, M. Seppala5, K.F. Lee3, and W.S.B. Yeung2,3

Departments of Obstetrics and Gynaecology,3 University of Hong Kong, Queen Mary Hospital, Hong Kong, China Departments of Obstetrics and Gynaecology4 Clinical Chemistry,5 University Central Hospital,00290 HUS Helsinki, Finland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.

fertilization, follicle, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycodelin, formerly known as placental protein 14 and progesterone-associated endometrial protein, was originally extracted from human term placenta [1]. It is also present in the amniotic fluid [2], endometrium [3], ovary [4], oviduct [5], maternal serum [6], and hematopoietic cells of the bone marrow [7]. The biological role of glycodelin-A is via its immunomodulatory activity in the maternal endometrium during implantation [8, 9] and its absence is characteristic of the fertile window [10].

Glycodelin-F, also known as zona binding inhibitory factor-1 [11], is a glycodelin isoform present in human follicular fluid [12]. Glycodelin-F has the same protein core as amniotic fluid glycodelin-A but with different oligosaccharide structure [12]. Although both glycodelin-F and -A inhibit sperm-zona pellucida binding [12, 13], the former forms three complexes while the latter produces only two complexes with the soluble extract of human sperm [14]. Interestingly, solubilized zona pellucida proteins reduce the binding of glycodelin-F to human sperm extract, suggesting that the glycodelin-F binding sites and the zona pellucida protein receptors on human sperm are closely related [14]. Similar data on glycodelin-A is not available. Therefore, the first objective of this investigation was to determine the effect of zona pellucida protein on the binding of glycodelin-A to sperm extract.

Sperm-zona pellucida interaction is a critical step to fertilization. Its failure during the fertilization process accounts for some cases of male infertility [15, 16]. It is believed that, in mammals, sperm-zona pellucida interaction is carbohydrate mediated [17–22], with sperm recognizing specific oligosaccharides of the zona pellucida glycoproteins. The involvement of carbohydrate moieties in sperm- zona pellucida binding is demonstrated by a decrease in the number of N-acetylglucosamine-, D-mannose-, or L-fucose- treated sperms bound to the zona pellucida in hemizona binding assay [23]. Putative sperm and oocyte receptors have been studied in several species, including humans. The best characterized sperm receptor for the zona pellucida protein, ZP3, is ß1,4-galactosyltransferase (GalT) that binds to the N-terminal N-acetylglucosamine residues on the oligosaccharide chains of ZP3. Oligosaccharides chains of ZP3 and synthetic polymers with N-acetylglucosamine terminal aggregate ß1,4-galactosyltransferase on mouse sperms [21, 24]. {alpha}-D-mannosidase in rat and human [25, 26] and ß-acetylglucosaminidase in human [27–29] have been suggested to be the sperm receptors for the mannosyl and N-acetylglucosamine residues, respectively, on the zona pellucida. Other potential zona pellucida receptors in mammalian sperm include fucosyltransferase [30], sp17 [31], PH-20 [32], and sp56 [33].

Selectins, including L-selectin (CD62L), E-selectin (CD62E), and P-selectin (CD62P), comprise a subfamily of Ca2+-dependent (C-type) animal lectins [34] and play important roles in cell-cell and cell-matrix interaction, such as during fertilization, implantation, embryogenesis, cell differentiation, and migration. They bind to various glycoconjugate ligands [35]. It has been suggested that the sialyl- Lewisx(a) and the unusual fucosylated and sialylated GalNAcß1, 4 GlcNAc (lacdiNAc) antennae of N-glycans in glycodelin-A with selectin ligand-like structure may participate in human sperm-zona pellucida interaction [36].

Immunohistochemical data show that glycodelin-A and -F bind to the acrosomal region of human sperm [12]. Acrosome reaction leads to the loss of the outer acrosome membrane and plasma membrane of sperm. If the receptors for glycodelin isoforms are present on these membranes, then the use of reagents inducing acrosome reaction, e.g., neoglycoproteins [37, 38] and solubilized zona pellucida protein [39], should affect their binding. Therefore, the second objective of this study was to determine the relationship between glycodelin-binding and acrosome reaction. The carbohydrate moieties of glycodelin-A and -F are important for their zona-binding inhibitory activity, and deglycosylated glycodelins have been shown to lose such activity in vitro [14]. Neoglycoproteins are synthesized by linkage of specific carbohydrate residues to a protein backbone. They have been used to investigate the impact of a given carbohydrate moiety in cell-to-cell interactions [40, 41]. The third objective of this study was to address the role of a single carbohydrate in neoglycoproteins and of selectins in the binding of glycodelin-A and -F to sperm. In this part, participation of the corresponding monosaccharide binding protein in sperm was demonstrated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The Ethics Committee of The University of Hong Kong approved the research protocol. Clinical samples were obtained from patients with informed consent.

Semen Samples

Semen samples with normal parameters [42] were from men visiting the subfertility clinics of the Queen Mary Hospital, University of Hong Kong. Sperm were separated by two-step Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation. A Hettich centrifuge (Universal II, Hettich, Tuttlingen, Germany) was used for Precoll sperm preparation (300 g) and cell washing (150 g). After capacitating in Earle balanced salt solution (EBSS; Flow Laboratories, Irvine, UK) supplemented with sodium pyruvate, penicillin-G, streptomycin sulfate, and 3% bovine serum albumin for 3 h, Percoll-processed sperm were resuspended in EBSS containing 0.3% BSA (EBSS/BSA).

Human Follicular Fluid

Three hundred human follicular fluid samples were collected during oocyte retrieval from women attending the assisted reproduction program of the Queen Mary Hospital. After downregulation of the pituitary with gonadotroph-releasing hormone agonist, human menopausal gonadotroph and human chorionic gonadotroph were used for ovarian stimulation. Only follicular fluid samples with no blood contamination were used. In this study, the samples were pooled, filtered through a 0.22-µm filter unit (Millipore, Bedford, MA) and stored at –20°C until used. Follicular fluid samples were thawed and diluted with EBSS/BSA to the desired concentration before use.

Purification of Glycodelin

The purification protocols of Chiu and coworkers [12] for glycodelin- A and -F were followed. Glycodelin-A was purified by mixing amniotic fluid with Triton X-100 (0.1%, v/v) and passing the mixture through a monoclonal anti-glycodelin antibody (clone F43–7F9) Sepharose column. The bound glycodelin-A was eluted with 0.1% trifluoroacetic acid. For glycodelin-F, follicular fluid was passed successively through Hi-Trap blue, protein-G, Con-A Sepharose columns (Pharmacia), Amicon-10 concentrator (Amicon Inc., Beverly, CA), Mono Q, and Superose columns. The concentrations of the purified glycodelins were determined by commercial assay kit (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA).

Radioiodination of Glycodelin

Fifty micrograms of glycodelin in 0.02 ml of 0.05 M PBS (pH 7.4) was mixed with 2 mCi of sodium 125I (20 µl; Amersham Biosciences, Buckinghamshire, UK) and freshly prepared chloramine T (100 g in 0.02 ml of 0.05 M PBS, pH 7.4). After vortexing for 60 sec, sodium metabisulphite (300 g in 0.05 ml of 0.05 M PBS, pH 7.4) was added to stop the reaction. A desalting column was used to remove free 125I. The first radioactive peak containing iodinated glycodelin was collected. Iodinated glycodelins had the same biological activity as their native form [14].

Glycodelin-A Binding to the Human Sperm Extract

Solubilized human zona pellucida was prepared by separating the zona pellucida from oocytes using glass micropipettes and by heat solubilizing the zona at 70°C for 90 min in distilled water with pH adjusted to 9 with Na2CO3 [14]. Soluble sperm extract was isolated as described [14] and divided into two identical portions (10 µg protein/ml). Each portion was incubated with 1 µg/ml of 125I-glycodelin-A in the presence of zona pellucida protein at concentrations equivalent to 0 or 0.2 zona pellucida/µl at 37°C for 3 h. After incubation, the mixtures were analyzed by native- gel electrophoresis and the radioactive band were visualized by exposing the gel to BIOMAX film (Kodak, Rochester, NY).

Determination of Acrosomal Status and Motility of Sperm

Fluorescein isothiocyanate labeled peanut (Pisum sativum) agglutinin (FITC-PSA; Sigma, St. Louis, MO) and Hoechst staining techniques were used to evaluate the acrosomal status of sperm as described [11]. The fluorescence patterns of 300 sperm in randomly selected fields were determined under a fluorescence microscope (Zeiss, Oberkochen, Germany) with 1000x magnification. Sperm without Hoechst staining and without FITC-PSA staining or with FITC-PSA staining confined to the equatorial segment only were considered as acrosome-reacted sperm. Hobson Sperm Tracker System (Hobson Tracking Systems Ltd., Sheffield, UK) was used to determine the motility of sperm. The set-up parameters of the system and the procedures were described elsewhere [14].

Effect of Acrosomal Status on Glycodelin Bindingto Sperm

Percoll-processed sperm from five normospermic men were used in the series of experiments described below. The experiments were divided into three groups. One group (control) was incubated in 300 pmol/ml of 125I- glycodelin-A or -F for 180 min at 37°C in an atmosphere of 5% CO2 in air. Sperm in the other two groups were treated with ionophore A23187 (Sigma) for 30 or 180 min at 37°C under 5% CO2 in air. A portion of the sperm from the two groups was incubated in EBSS alone in the corresponding conditions as control. After treatment, sperm were washed with fresh EBSS/BSA before being further incubated with 125I-glycodelin-A or -F (300 pmol/ml) for 180 min. Sperm from different groups were then washed with fresh EBSS/BSA and their cell-bound radioactivity was measured in a gamma counter (model 5500B; Beckman, Fullerton, CA). The specific binding of glycodelin was determined by subtracting the bound radioactivity in the presence of 100x concentration of unlabeled glycodelin from the bound radioactivity without unlabeled protein. The determination of total binding and nonspecific binding were done in triplicate. The acrosomal status was also determined as described above.

Glycodelin Binding on Fixed and Living Human Sperm

Results of the above experiment showed that ionophore-induced acrosome reaction reduced the binding of glycodelin to sperm (see below). To study the effect of neoglycoproteins on glycodelins binding, fixed sperm had to be used as neoglycoproteins might initiate acrosome reaction in live sperm. Fixation of sperm was performed by placing sperm briefly in 1% glutaraldehyde in EBSS for 5 min. To compare the glycodelin- binding ability of fixed and live sperm, sperm were washed thrice in fresh EBSS. Different concentrations of the sperm (0.01–10 x 106) were incubated with 125I-glycodelin-A or -F (300 pmol/ml) at 37°C in an atmosphere of 5% CO2 in air for 3 h. The treated sperm were then washed with fresh EBSS/BSA and the cell-bound radioactivity was measured as described above. Competition-binding analysis was also performed to compare the binding kinetics of labeled glycodelin-A and -F in the presence of increasing concentrations of the corresponding glycodelin to fixed and unfixed sperm using the protocol described below. The results were expressed as percentage of bound radioactivity on sperm without unlabeled glycodelin.

Competition Binding Assay of Glycodelinwith Neoglycoproteins

Competition binding assay was performed as described [14]. Briefly, the binding of labeled glycodelin (300 pmol/ml) to fixed human sperm (210 x 106/ml) was determined in the presence of increasing concentrations (0.3–30 000 pmol/ml based on monosaccharides) of neoglycoproteins, including bovine albumin p-aminophenyl-N-acetyl-ß-D-galactosaminide (BSA-GalNAc; Sigma), bovine albumin p-aminophenyl-N-acetyl- ß-D-glucosaminide (BSA-GlcNAc; Sigma), bovine albumin {alpha}-L-fucopyranosylphenyl isothiocyanate (BSA-Fuc; Sigma), bovine albumin p-aminophenyl-{alpha}-D-mannopyranoside (BSA-Man; Sigma) or bovine albumin galactosamide (BSA-Gal; Sigma) at 37°C for 3 h. After incubation, the cell-bound radioactivity was measured with a gamma counter as described above. Under this condition, the bound radioactivity did not change in the absence of competitor within the experimental period (data not shown). Each individual experiment was repeated thrice.

Effect of Glycosidases and Their Inhibitors on Glycodelin Binding to Sperm

The above experiments demonstrated the involvement of monosaccharide residues in the binding of glycodelin to sperm. Enzyme inhibitors were used to confirm that the corresponding glycosidases were present on sperm. Sperm were incubated in 0.1, 0.5, and 1 mol/ml of deoxyfuconojirimycin (DFJ; {alpha}-L-fucosidase inhibitor [43]), deoxygalactonojirimycin (DGJ; {alpha}-D-galactosidase inhibitor [44]). 3,4,5,6-tetrahydroxy-azepane (THA; ß-N-acetylglucosaminidase inhibitor [45]), deoxymannojirimycin (DMJ; {alpha}-D-mannosidase I and {alpha}-L-fucosidase inhibitor [46]), 1,4-dideoxy- 1,4-imino-D-mannitol (DIM; mannosidase I and II inhibitor [47]) (Industrial Research, Lower Hutt, New Zealand), uridine 5'diphosphogalactose disodium salt (UDP-Gal; galactosyltransferase inhibitor [48]) and alpha- lactalbumin (LA; galactosyltransferase inhibitor; Sigma [49]), or EBSS/ BSA (control) at 37°C under 5% CO2 in air for 3 h. After incubation, sperm were treated with 300 pmol/ml of iodinated glycodelin-A or -F for 3 h and the cell-bound radioactivity was counted as described above. In addition, the ability of ß1-2,3,4,6 linked N-acetyl-glucosaminidase (EC 3.2.1.52), {alpha}1-2,3,6-mannosidase (EC 3.2.1.24), {alpha}1-3,4-L-fucosidase (EC 3.2.1.51), and ß-galactosidase (EC 3.2.1.23; Calbiochem, San Diego, CA) at concentrations of 0.5, 1, 2, and 3 g/ml to inhibit the binding of labeled glycodelin (300 pmol/ml) to fixed human sperm (210 x 106/ml) was performed. The effect of glycosidases or glycosidase inhibitors on sperm motility, viability, and acrosomal status were also studied.

Effect of Selectins on Glycodelin Binding to Sperm

Two experiments were performed to determine the role of E-selectin, L-selectin, and P-selectin (Calbiochem) in the binding of glycodelin to human sperm. The first experiment addressed the kinetics of competition binding of glycodelin isoforms in the presence of different concentrations (0.3–30 000 pmol/ml) of selectins using the protocol described above. The second experiment was on the effect of antiselectin monoclonal antibodies on iodinated glycodelin binding. In this experiment, sperm from five men were incubated with increasing concentrations (1–10 000 ng/ml) of monoclonal antiselectin (E, L, and P) antibodies (anti-E- and anti-L-selectin from Sigma; anti-P-selectin from Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min at 37°C in an atmosphere of 5% CO2 in air. After incubation, the sperm were washed with fresh EBSS/BSA followed by incubation with iodinated glycodelin-A or -F (300 pmol/ml) for 3 h. The cell- bound radioactivity was then counted as described above. The effects of selectins and antiselectin antibodies on sperm motility, viability, and acrosomal status were studied.

Data Analysis

All the data were expressed as mean ± standard error of the mean (SEM). The data and effective concentration in competition binding assay (EC50) were analyzed using a Sigmastat statistical software (SigmaPlot 8.02, Ligand Binding Analysis Module and SigmaStat 2.03; Jandel Scientific, San Rafael, CA). For comparison of the effects of duration of ionophore treatment on the binding of glycodelin to sperm, the software determined that the data were normally distributed and repeated measure analysis of variance (ANOVA) was used followed by Tukey test as a post hoc test. For all other experiments, nonparametric ANOVA on rank was used for the multiple comparisons. Parametric Student t-test was subsequently used as a posttest and reported here because the statistical software confirmed that the data were normally distributed. Nonparametric Mann- Whitney U-test also were used as the posttest. The conclusions are identical to those using the parametric method (data not shown). A probability value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Solubilized Zonae Pellucidae on Bindingof Glycodelin-A

The effects of solubilized zonae pellucidae on the binding of glycodelin-A to sperm extracts are shown in Figure 1. Iodinated glycodelin-A (lane 1) showed a single radioactive band. Two additional radioactive bands appeared after incubation of glycodelin-A with the sperm extracts. All the additional bands had molecular sizes larger than that of iodinated glycodelin-A. Co-incubation of solubilized zona pellucida reduced the binding of glycodelin-A to one of the radioactive bands in the sperm extract (lane 3).



View larger version (121K):
[in this window]
[in a new window]
  		FIG. 1. Binding of radiolabeled glycodelin-A to the human sperm membrane fraction in the presence or absence of zona pellucida proteins. The binding was analysed by 8% native gel autoradiography. Lane 1: purified iodinated glycodelin-A; lane 2: iodinated glycodelin-A + human sperm extract; lane 3: iodinated glycodelin-A + human sperm extract + zona pellucida protein. The arrow depicts glycodelin-A-derived band with intensity reduced in the presence of zona pellucida protein

Effect of Acrosomal Status on the Binding of Glycodelin to Sperm

Figure 2 shows the effect of calcium ionophore A23187- induced acrosome reaction on the binding of glycodelin isoforms to human sperm. Compared with control without ionophore, preincubation with A23187 for 30 and 180 min significantly reduced the specific binding of iodinated glycodelin-A and -F. Thirty minutes of treatment significantly increased (P < 0.05) the percentage of acrosome-reacted sperm from 9.6% ± 1.5% to 34% ± 2.1% and decreased the specific binding of 125I-glycodelin-A by 62.0% ± 4.1% and glycodelin-F by 42.3% ± 6.2% compared with the control. Prolongation of the incubation (180 min) increased the percentage of acrosome-reacted sperm to 54.2% ± 2.0% and further reduced the 125I-glycodelin-A and -F binding to 18.9% ± 2.8% and 23.3% ± 1.1% of the control, respectively (P < 0.05 for both).



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 2. The effects of acrosome status of human sperm on the binding of 300 pmol/ml of 125I-glycodelin-A and -F. The values are mean ± SEM of five experiments. Each experiment used sperm from different donors. #, * P < 0.05 when compared with the corresponding control without ionophore treatment

Binding of Glycodelin to Live and Fixed Sperm

There was a linear relationship between the binding of 125I-glycodelin-A (r2 = 0.96) and glycodelin-F (r2 = 0.91) and the number of fixed and unfixed sperm in the incubation mixture (Fig. 3). The fractional binding (bound/free) was similar for the fixed and unfixed sperm. When the fixed sperm were incubated with saturating concentrations of 125I-glycodelin-A (Fig. 4A) or 125I-glycodelin-F (Fig. 4B) in the presence of increasing concentrations of the corresponding unlabeled glycodelin, there was an exponential decrease in the binding of labeled molecules. Comparable effect was found with unfixed sperm. The log EC50 of glycodelin-A on fixed and unfixed sperm were 2.19 ± 0.7 and 2.19 ± 0.07 pmol/ml, respectively. The corresponding values for glycodelin-F were 2.62 ± 0.08 and 2.54 ± 0.07 pmol/ml.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3. Competition binding to fixed and live sperm between saturated concentrations of 125I-glycodelin-A and 125I-glycodelin-F and increasing concentration (0.3–30 000 pmol/ml) of unlabeled glycodelins



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4. Competition binding to fixed and live sperm between saturated concentrations of 125I-glycodelin-A and 125I-glycodelin-F and increasing concentration (0.3–30 000 pmol/ml) of unlabeled glycodelins

Competition Binding Analysis of Glycodelinwith Neoglycoproteins

The effects of neoglycoproteins on the binding of 125I- glycodelin to fixed sperm are shown in Figure 5. As expected, unlabeled glycodelin was the best competitor for the corresponding iodinated glycodelin (log EC50: glycodelin-A, 2.17 ± 0.07 pmol/ml; glycodelin-F, 2.48 ± 0.08 pmol/ml). BSA alone had no effect. Among the neoglycoproteins, BSA-mannose and BSA-fucose competed with 125I-glycodelin-A for binding sites, with log EC50 of 3.69 ± 0.08 pmol/ml and 4.07 ± 0.07 pmol/ml, respectively, while the other neoglycoproteins did not affect the binding of labeled glycodelin-A except at high concentrations (log EC50 > 5 pmol/ml; Fig. 5A). Neoglycoprotein of N-acetylglucosamine, mannose, and fucose inhibited the binding of 125I-glycodelin-F to similar extent, with log EC50 of 4.38 ± 0.03, 4.47 ± 0.03, and 4.63 ± 0.04 pmol/ml, respectively (Fig. 5B). BSA-N-acetylgalactosamine and BSA-galactose were not effective in competing with glycodelin-F (log EC50 > 5 pmol/ml).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 5. Competition binding to sperm between saturated concentration of 125I-glycodelin-A or 125I-glycodelin-F and increasing concentration (0.3– 30 000 pmol/ml) of different neoglycoproteins. BSA-N-acetylgalactosamine, bovine albumin p-aminophenyl-N-acetyl-ß-D-galactosaminide; BSA-N-acetyl-glucosamine, bovine albumin p-aminophenyl-N-acetyl-ß- D-glucosaminide; BSA-Fuc, bovine albumin {alpha}-L-fucopyranosylphenyl isothiocyanate; BSA-Man, bovine albumin p-aminophenyl-{alpha}-D-mannopyranoside; BSA-Gal, bovine albumin galactosamide

Effect of Glycosidase Inhibitors on Glycodelin Binding

Figure 6 shows the effect of glycosidase inhibitors on glycodelin binding to human sperm. Among the inhibitors used, DIM had the greatest inhibitory effect on glycodelin- A binding. It dose-dependently inhibited the binding of glycodelin-A to human sperm (Fig. 6A). Significant inhibition (P < 0.05) was found at the concentrations greater than 0.5 µmol/ml. At 1 µmol/ml, it inhibited 48.2% ± 1.5% of the glycodelin-A binding. Glycodelin-A binding was also inhibited by DFJ and THA (P < 0.05) although with smaller magnitude, ranging from 10% to 20%. Deoxymannojirimycin (DMJ) inhibited glycodelin-A binding only at the concentration of 1 µmol/ml. The binding of glycodelin-A was not affected by DGJ, UDP-Gal, and LA.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 6. Effects of different concentrations (0.1, 0.5, and 1 µmol/ml) of glycosidase inhibitor on binding of iodinated glycodelin-A or glycodelin-F to human sperm. DFJ, Deoxyfuconojirimycin hydrochloride; DGJ, deoxygalactonojirimycin hydrochloride; THA, 3,4,5,6-getrahydroxyazepane; DMJ, deoxymannojirimycin; DIM, 1,4-dideoxy-1,4-imino-D-mannitol hydrochloride; UDP-Gal, uridine 5'diphosphogalactose disodium salt; and LA, alpha-lactalbumin. * P < 0.05 when compared with the control without inhibitor treatment

While no significant inhibition was observed for UDP- Gal, DGJ, and LA, DIM and THA dose-dependently inhibited glycodelin-F binding (Fig. 6B). Significant inhibition was found at the concentration of 0.5 µmol/ml. The inhibitions of glycodelin-F binding at a concentration of 1 µmol/ml of DIM and THA were 35.8% ± 2.1% and 30.4% ± 2.9%, respectively. At the same concentration, DFJ and DMJ only inhibited 10.6% ± 1.6% and 11.4% ± 1.3% of the binding, respectively. Their effects at lower concentrations were not different from the control without glycosidase inhibitor. The glycosidase inhibitors at the concentrations used did not affect sperm viability, acrosomal status, and motility (data not shown).

Effect of Glycosidases on Glycodelin Binding

Mannosidase and fucosidase significantly inhibited the binding of glycodelin-A at 0.5 µg/ml when compared with the control without glycosidase treatment (P < 0.05). At 3 g/ml, the inhibition was 54.7% ± 3.5% and 34.7% ± 3.2% of the maximal glycodelin-A binding, respectively (Fig. 7A). The inhibition was dose dependent within the concentrations tested. No significant inhibition was observed with galactosidase and acetylglucosaminidase in any of the concentrations used.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 7. Inhibition binding to sperm between saturated concentration (300 pmol/ml) of 125I-glycodelin-A or 125I-glycodelin-F and increasing concentration (0.5, 1, 2, and 3 µg/ml) of different enzymes; ß-N-acetylglucosaminidase, {alpha}-mannosidase, {alpha}-L-fucosidase, and ß-galactosidase. {dagger}, #, * P < 0.05 when compared with the control without glycosidase treatment

Similar to glycodelin-A, mannosidase and fucosidase had the greatest inhibitory effect on glycodelin-F binding, with significant inhibition at a concentration of 0.5 µg/ml (Fig. 7B). The corresponding inhibition at 3 µg/ml was 51.7% ± 2.0% and 22.3% ± 4.2%. Unlike that of glycodelin-A, acetylglucosaminidase inhibited glycodelin-F binding dose dependently; the inhibition was 43.7% ± 2.8% at 3 µg/ml. No inhibition was observed with galactosidase. At the concentrations employed, the glycosidases did not affect sperm viability, acrosomal status, and motility (data not shown).

Effects of Selectins E, L, and P on Glycodelin Bindingto the Human Sperm

At a concentration of 1000 pmol/ml, E-selectin significantly inhibited (P < 0.05) the binding of glycodelin-A to sperm (Fig. 8A). All other selectins (Fig. 8) and their monoclonal antibodies (data not shown) had no significant effects on the binding of glycodelin-A and -F to human sperm. The selectins and antiselectin antibodies used had no effect on sperm viability, acrosomal status, or motility (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 8. Inhibition of binding to sperm between saturated concentration (300 pmol/ml) of 125I-glycodelin-A or 125I-glycodelin-F and increasing concentration (0.3–30 000 pmol/ml) of selectins. * P < 0.05 when compared with the control without selectin treatment


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Solubilized zonae pellucidae protein dose-dependently reduces the binding of glycodelin-F to the sperm extract [14]. This report demonstrates that zona pellucida proteins also reduce the binding of glycodelin-A to sperm extract. Glycodelin-A and -F form two and three complexes, respectively, with sperm extract [14]. While the formation of two of the glycodelin-F complexes are inhibited by solubilized zona pellucida [14], only one of the glycodelin-A complexes is affected by the zona pellucida proteins. These observations are consistent with the binding kinetic experiments demonstrating that human sperm possess two receptors for glycodelin-F, one of which is common with glycodelin-A [14]. Chiu and coworkers [14] also suggest that glycodelins and zona pellucida proteins may bind to the same receptor(s) or that their receptor(s) are closely related so that the binding of one would affect binding of the other.

Sperm bind to zona pellucida with the acrosomal region, and glycodelin-A and -F bind to the acrosomal region of sperm [12]. The present study demonstrates that the glycodelin-binding sites are on the outer acrosomal membrane or in the sperm plasma membrane overlying the acrosome, as the binding of glycodelin isoforms to sperm was greatly reduced after ionophore-induced acrosome reaction. These observations lend support to the close relationship between glycodelin receptor(s) on sperm and zona pellucida proteins.

Neoglycoproteins are frequently used in studies on sperm-zona pellucida interactions, some of which mimic the action of ZP3 glycoprotein and induce acrosome reaction in capacitated mouse [28, 40] and human sperm [50]. The acrosome reaction-inducing ability of neoglycoprotein and the reduction of glycodelin binding after acrosome reaction requires the use of fixed, instead of live, sperm for characterization of the glycodelin-binding sites on sperm using neoglycoproteins; otherwise, equilibrium binding would have been impossible to achieve. The fixed cell model has been used successfully to characterize mouse sperm- zona pellucida interaction [51]. The suitability of this model in this study was demonstrated by sperm concentration-dependent binding of 125I-glycodelin to fixed sperm, and by the comparable binding kinetics of glycodelin isoforms with fixed and live sperm.

It has been proposed that sperm-zona pellucida recognition is mediated by a number of complementary proteins and carbohydrate structures on the surface of sperm and oocytes [17–22, 24]. Even though several carbohydrate- binding proteins have been proposed to be involved in sperm-oocyte interaction [22, 25, 26, 30, 52–54], the exact identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding remains unresolved.

The carbohydrate moieties of glycodelin-A and -F are critical for their zona-binding inhibitory activity as well as their binding on human sperm, and deglycosylated glycodelin loses such activities in vitro [14]. Glycodelin-F has the same protein core as glycodelin-A, but their oligosaccharide chains are different [12]. In the present study, we demonstrated that different sets of carbohydrate residues affected the binding of these glycodelin isoforms to sperm. While the binding of glycodelin-A appears to be affected by the mannose and fucose residues, that of glycodelin-F is influenced by N-acetylglucosamine in addition to fucose and mannose. This is in keeping with the observation that both glycodelin isoforms have similar lectin-binding spectrum except that glycodelin-F binds more strongly to wheat germ agglutinin and its succinylated form with specific affinity for N-acetylglucosamine residue [12].

Mannose and fucose neoglycoprotein suppress the binding of radiolabeled glycodelin-A to human sperm to a greater extent than that of glycodelin-F. One possible explanation is that glycodelin-F has an additional high-affinity binding site on sperm when compared with glycodelin-A [14]. This may make it more difficult for the neoglycoproteins to compete with glycodelin-F for the binding sites. It is also possible that the contribution of mannose and fucose residues in the binding of glycodelin-F to sperm is smaller than that in glycodelin-A sperm binding. The present data do not allow us to distinguish between these possibilities.

The involvement of mannose and N-acetylglucosamine residues in glycodelin binding is supported by the ability of mannosidase and acetylglucosaminidase and their inhibitors to inhibit glycodelin sperm binding. Both DIM and DMJ are mannosidase inhibitors. DIM inhibited but DMJ had only marginal effect on glycodelin binding to sperm. 1,4-dideoxy-1,4-imino-D-mannitol (DIM) is an inhibitor of mannosidase I and II [47], while DMJ is a mannosidase I and {alpha}-L-fucosidase inhibitor [46]. Mannosidase I acts specifically on {alpha}1-2-linked mannose residues and mannosidase II is specific for {alpha}1-3-linked and {alpha}1-6-linked mannose residues. Therefore, it is possible that the {alpha}1-3 and/or {alpha}1-6 mannose residue in the glycan chain of glycodelin participate in sperm binding.

Rat, mouse, hamster, and human sperm possess mannosidase activity [25, 55]. Mannosidase binds and hydrolyses terminal mannose of N-linked oligosaccharides. Alpha-mannosidase activity has been localized to the plasma membranes of rat [25], mouse [26], and human sperm [55]. However, the exact mannosidase isoform on sperm remains to be identified. The importance of mannosyl residues in fertilization is demonstrated by the observation that incubation of mouse sperm with D-mannose results in a dose- dependent decrease in the number of sperm bound to the oocyte [56]. Pretreatment of human sperm with D-mannose also inhibits sperm penetration through the zona [57].

Acetylglucosaminidase and its inhibitor affect the binding of glycodelin-F to human sperm but not that of glycodelin-A. Glycodelin-F has a high-affinity receptor that is not shared with glycodelin-A [14]. Thus, the binding of glycodelin-F to the high-affinity receptor may involve N- acetylglucosamine residue. The importance of N-acetylglucosamine residues in human sperm function has been reported [23, 27, 28, 38]. N-acetylglucosamine reduces the binding of capacitated human sperm to the zona pellucida [23]. This is supported by the observation that acetylglucosaminidase, which hydrolyses the terminal N-acetylglucosamine of the glycan, reduces the number of human sperm bound to the zona pellucida [27]. Although N-acetylglucosaminidase also hydrolyses N-acetylgalactosamine residue, this monosaccharide residue is unlikely to be involved in the binding of glycodelin to sperm, as its neoglycoprotein did not affect glycodelin binding in the present study. This is consistent with previous observations that neither this monosaccharide nor its neoglycoprotein affects sperm-zona pellucida binding [27] or acrosomal status of the human sperm in vitro [28].

In this study, the contribution of fucose residues in the binding of glycodelin varied depending on the assays performed. Fucose-neoglycoprotein and fucosidase suppressed but fucosidase inhibitor only marginally inhibited the binding of glycodelin-A and -F to sperm. Plasma membrane- associated {alpha}-L-fucosidase has been found on sperm of different species, including rat [58] and human [59]. L-fucosyl-binding sites have also been reported on intact [41] and acrosome-reacted [60] human sperm. Fucose-neoglycoprotein binds primarily to the sperm head, which could be inhibited by solubilized human zona pellucida proteins [41]. The reason for the lack of effect of fucosidase inhibitor on glycodelin binding is unknown. It could be that the fucosidase in sperm is different from that in the other cells or that the fucose-binding site in sperm is not due to fucosidase, thus making the inhibitor ineffective in suppressing the glycodelin binding to sperm.

Beta-1,4-galactosyltransferase is the best characterized potential sperm receptor for zona pellucida. It binds specifically to the N-terminal N-acetylglucosamine residues of ZP3 but not to the other zona pellucida proteins [24, 61]. The enzyme is present in the head region of all mammalian sperm studied, including human, consistent with a role in gamete recognition [24, 49, 62, 63]. Reagents blocking the activity of the enzyme, including UDP-Gal and alpha-lactalbumin, inhibit sperm-zona pellucida binding [24, 49, 64]. In this study, both UDP-Gal and alpha-lactalbumin did not affect the binding of glycodelin-A and -F to human sperm. These observations suggest that, although glycodelin-A and -F inhibit sperm-zona pellucida binding [12, 13], the binding of glycodelins to sperm differs from that of the zona pellucida in that the former does not involve galactosyltransferase activity.

Glycodelin-A contains N-glycans with selectin-like ligands, e.g., sialyl-Lewisx(a) and fucosylated and sialylated lacdiNAc-antennae [22, 36]. Recently, glycodelin-A has been reported to inhibit E-selectin-mediated cell adhesion [65]. Using purified selectins and antiselectin antibodies, we demonstrated that the binding of glycodelin-F was independent of selectins. On the other hand, E-selectin had slight inhibitory effect on the binding of glycodelin-A to sperm. These results are consistent with previous conclusions that glycodelin-F and glycodelin-A have different oligosaccharide chains [12]. However, the inability of antiselectin antibody to affect the binding of glycodelin-A to sperm suggests that sperm protein(s) with selectin-like activity, but not selectin, is involved in the sperm binding. A similar conclusion was reached for the binding of sperm to the zona pellucida [22].

The present data show that no single residue, even at high concentration, could produce a complete blockade of glycodelin binding to sperm, suggesting that the interaction of glycodelin with the sperm receptor is multivalent, involving several residues and possibly several receptor complexes. Such a multivalent binding would strengthen the protein-oligosaccharide interaction, even if the individual monosaccharide-protein interactions appeared weak [66]. Multivalent ligand binding exists between the L- and P- selectin proteins and sialyl Lewis-X [67]. This is in agreement with the suggestion that sperm-zona pellucida interaction is a complex event involving interaction of multiple sperm proteins with multiple sugar residues of the zona pellucida protein(s) [66].

In summary, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E-selectin-binding residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine. The reduction in glycodelin binding after acrosome reaction and the ability of the zona pellucida to displace glycodelin from sperm extract suggest a close relationship between glycodelin receptors and zona pellucida protein receptors. However, a difference in the requirement for the binding of glycodelin and zona pellucida to sperm is noted when comparing the present study with previous reports [24, 49, 64]. While the binding of zona pellucida proteins requires galactosyltransferase, the enzyme activity is not needed for glycodelin binding, indicating that the receptors for glycodelin and zona pellucida proteins are not identical. It has been suggested that the receptors of zona pellucida proteins in sperm are complexes comprised of a number of molecules [17, 19, 21, 68]. Whether the glycodelin isoforms exert their sperm-zona binding inhibitory activity by blocking part of the zona pellucida receptor complexes awaits further investigation.


   ACKNOWLEDGMENTS
 
The authors thank the laboratory staff in the IVF team for their skillful technical assistance.


   FOOTNOTES
 
1 Supported by grants from the Research Grant Council, Hong Kong (HKU7188/99M and HKU7261/01M), CRCG, University of Hong Kong, Helsinki University Central Hospital Research Funds, Federation of the Finnish Life and Pension Insurance Companies, the Cancer Society of Finland, the Academy of Finland, and University of Helsinki. Back

2 Correspondence: W.S.B. Yeung, Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong. FAX: 852 2855 0947; wsbyeung{at}hkucc.hku.hk Back

Received: 15 September 2003.

First decision: 13 October 2003.

Accepted: 22 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bohn H, Kraus W, Winckler W. New soluble placental tissue proteins: their isolation, characterization, localization and quantification. Placenta Suppl 1982 4:67-81[Medline]
  2. Joshi SG, Smith RA, Stokes DK. A progestogen-dependent endometrial protein in human amniotic fluid. J Reprod Fertil 1980 60:317-321[Abstract/Free Full Text]
  3. Julkunen M, Wahlstrom T, Seppala M. Human fallopian tube contains placental protein 14. Am J Obstet Gynecol 1986 154:1076-1079[Medline]
  4. Critchley HO, Chard T, Olajide F. Role of the ovary in the synthesis of placental protein-14. J Clin Endocrinol Metab 1992 75:97-100[Abstract]
  5. Julkunen M, Koistinen R, Sjoberg J, Rutanen EM, Wahlstrom T, Seppala M. Secretory endometrium synthesizes placental protein 14. Endocrinology 1986 118:1782-1786[Abstract]
  6. Bolton AE, Chapman MG, Stoker RJ, Andrew CE, Wass D, Bohn H. The radioimmunoassay of human placental protein 14 (PP14). Clin Chim Acta 1983 135:283-291[CrossRef][Medline]
  7. Kamarainen M, Riittinen L, Seppala M, Palotie A, Andersson LC. Progesterone-associated endometrial protein—a constitutive marker of human erythroid precursors. Blood 1994 84:467-473[Abstract/Free Full Text]
  8. Pockley AG, Mowles EA, Stoker RJ, Westwood OM, Chapman MG, Bolton AE. Suppression of in vitro lymphocyte reactivity to phytohemagglutinin by placental protein 14. J Reprod Immunol 1988 13:31-39[CrossRef][Medline]
  9. Okamoto N, Uchida A, Takakura K. Suppression by human placental protein 14 of natural killer cell activity. Am J Reprod Immunol 1991 26:137-142
  10. Seppala M, Taylor RN, Koistinen H, Koistinen R, Milgrom E. Glycodelin: a major lipocalin protein of the reproductive axis with diverse actions in cell recognition and differentiation. Endocr Rev 2002 23:401-430[Abstract/Free Full Text]
  11. Chiu PC, Ho PC, Ng EH, Yeung WS. Comparative study of the biological activity of spermatozoa-zona pellucida binding inhibitory factors from human follicular fluid on various sperm function parameters. Mol Reprod Dev 2002 61:205-212[CrossRef][Medline]
  12. Chiu PC, Koistinen R, Koistinen H, Seppala M, Lee KF, Yeung WS. Zona-binding inhibitory factor-1 from human follicular fluid is an isoform of glycodelin. Biol Reprod 2003 69:365-372[Abstract/Free Full Text]
  13. Oehninger S, Coddington CC, Hodgen GD, Seppala M. Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida. Fertil Steril 1995 63:377-383[Medline]
  14. Chiu PC, Koistinen R, Koistinen H, Seppala M, Lee KF, Yeung WS. Binding of zona binding inhibitory factor-1 (ZIF-1) from human follicular fluid on spermatozoa. J Biol Chem 2003 278:13570-13577[Abstract/Free Full Text]
  15. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW. Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 1980 33:534-542[Medline]
  16. Lih CH, Grunfeld L, Sandler B, Drews MR, Navot D, Gordon JW. Infertile couples with normal counts who require subzonal sperm insertion possess a fertility defect that affects zona pellucida penetration. Hum Reprod 1994 9:2335-2338[Abstract/Free Full Text]
  17. Wassarman PM. Toward molecular mechanisms for gamete adhesion and fusion during mammalian fertilization. Curr Opin Cell Biol 1995 7:658-664[CrossRef][Medline]
  18. Miller DJ, Ax RL. Carbohydrates and fertilization in animals. Mol Reprod Dev 1990 26:184-198[CrossRef][Medline]
  19. Tulsiani DR, Yoshida-Komiya H, Araki Y. Mammalian fertilization: a carbohydrate-mediated event. Biol Reprod 1997 57:487-494[CrossRef][Medline]
  20. Benoff S. Carbohydrates and fertilization: an overview. Mol Hum Reprod 1997 3:599-637[Abstract/Free Full Text]
  21. Shur BD. Is sperm galactosyltransferase a signaling subunit of a multimeric gamete receptor?. Biochem Biophys Res Commun 1998 250:537-543[CrossRef][Medline]
  22. Oehninger S. Molecular basis of human sperm-zona pellucida interaction. Cells Tissues Organs 2001 168:58-64[CrossRef][Medline]
  23. Miranda PV, Gonzalez-Echeverria F, Marin-Briggiler CI, Brandelli A, Blaquier JA, Tezon JG. Glycosidic residues involved in human sperm- zona pellucida binding in vitro. Mol Hum Reprod 1997 3:399-404[Abstract/Free Full Text]
  24. Nixon B, Lu Q, Wassler MJ, Foote CI, Ensslin MA, Shur BD. Galactosyltransferase function during mammalian fertilization. Cells Tissues Organs 2001 168:46-57[CrossRef][Medline]
  25. Tulsiani DR, Skudlarek MD, Orgebin-Crist MC. Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions. J Cell Biol 1989 109:1257-1267[Abstract/Free Full Text]
  26. Cornwall GA, Tulsiani DR, Orgebin-Crist MC. Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro. Biol Reprod 1991 44:913-921[Abstract]
  27. Miranda PV, Gonzalez-Echeverria F, Blaquier JA, Mahuran DJ, Tezon JG. Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro. Mol Hum Reprod 2000 6:699-706[Abstract/Free Full Text]
  28. Brandelli A, Miranda PV, Tezon JG. Participation of glycosylated residues in the human sperm acrosome reaction: possible role of N-acetylglucosaminidase. Biochim Biophys Acta 1994 1220:299-304[Medline]
  29. Miller DJ, Gong X, Shur BD. Sperm require beta-N-acetylglucosaminidase to penetrate through the egg zona pellucida. Development 1993 118:1279-1289[Abstract]
  30. Ram PA, Cardullo RA, Millette CF. Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse. Gamete Res 1989 22:321-332[CrossRef][Medline]
  31. Richardson RT, Yamasaki N, O'Rand MG. Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reaction. Dev Biol 1994 165:688-701[CrossRef][Medline]
  32. Primakoff P, Hyatt H, Myles DG. A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. J Cell Biol 1985 101:2239-2244[Abstract/Free Full Text]
  33. Cheng A, Le T, Palacios M. Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3. J Cell Biol 1994 125:867-878[Abstract/Free Full Text]
  34. Drickamer K. Recognition of complex carbohydrates by Ca(2+)-dependent animal lectins. Biochem Soc Trans 1993 21:456-459[Medline]
  35. Crockett-Torabi E. Selectins and mechanisms of signal transduction. J Leukoc Biol 1998 63:1-14[Abstract]
  36. Dell A, Morris HR, Easton RL, Panico M, Patankar M, Oehninger S, Koistinen R, Koistinen H, Seppala M, Clark GF. Structural analysis of the oligosaccharides derived from glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J Biol Chem 1995 270:24116-24126[Abstract/Free Full Text]
  37. Blackmore PF, Eisoldt S. The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa. Mol Hum Reprod 1999 5:498-506[Abstract/Free Full Text]
  38. Brandelli A, Miranda PV, Anon-Vazquez MG, Marin-Briggiler CI, Sanjurjo C, Gonzalez-Echeverria F, Blaquier JA, Tezon JG. A new predictive test for in vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein. Hum Reprod 1995 10:1751-1756[Abstract/Free Full Text]
  39. Schuffner AA, Bastiaan HS, Duran HE, Lin ZY, Morshedi M, Franken DR, Oehninger S. Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive G(i) protein and extracellular calcium, and priming effect of progesterone and follicular fluid. Mol Hum Reprod 2002 8:722-727[Abstract/Free Full Text]
  40. Loeser CR, Lynch C, Tulsiani DR. Characterization of the pharmacological-sensitivity profile of neoglycoprotein-induced acrosome reaction in mouse spermatozoa. Biol Reprod 1999 61:629-634[Abstract/Free Full Text]
  41. Tesarik J, Mendoza C, Ramirez JP, Moos J. Solubilized human zona pellucida competes with a fucosylated neoglycoprotein for binding sites on the human sperm surface. Fertil Steril 1993 60:344-350[Medline]
  42. World Health Organization. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge, UK: Cambridge University Press; 1998
  43. Winchester B, Barker C, Baines S, Jacob GS, Namgoong SK, Fleet G. Inhibition of alpha-L-fucosidase by derivatives of deoxyfuconojirimycin and deoxymannojirimycin. Biochem J 1990 265:277-282[Medline]
  44. Legler G, Pohl S. Synthesis of 5-amino-5-deoxy-D-galactopyranose and 1,5-dideoxy-1,5-imino-D-galactitol, and their inhibition of alpha- and beta-D-galactosidases. Carbohydr Res 1986 155:119-129[CrossRef][Medline]
  45. Qian X, Moris-Varas F, Fitzgerald MC, Wong CH. C2-symmetrical tetrahydroxyazepanes as inhibitors of glycosidases and HIV/FIV proteases. Bioorg Med Chem 1996 4:2055-2069[CrossRef][Medline]
  46. Fuhrmann U, Bause E, Legler G, Ploegh H. Novel mannosidase inhibitor blocking conversion of high mannose to complex oligosaccharides. Nature 1984 307:755-758[CrossRef][Medline]
  47. Palamarczyk G, Mitchell M, Smith PW, Fleet GW, Elbein AD. 1,4-Dideoxy-1,4-imino-D-mannitol inhibits glycoprotein processing and mannosidase. Arch Biochem Biophys 1985 243:35-45[CrossRef][Medline]
  48. Takayama S, Chung SJ, Igarashi Y, Ichikawa Y, Sepp A, Lechler RI, Wu J, Hayashi T, Siuzdak G, Wong CH. Selective inhibition of beta- 1,4- and alpha-1,3-galactosyltransferases: donor sugar-nucleotide based approach. Bioorg Med Chem 1999 7:401-409[CrossRef][Medline]
  49. Shur BD, Hall NG. A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida. J Cell Biol 1982 95:574-579[Abstract/Free Full Text]
  50. Brandelli A, Miranda PV, Tezon JG. Voltage-dependent calcium channels and Gi regulatory protein mediate the human sperm acrosomal exocytosis induced by N-acetylglucosaminyl/mannosyl neoglycoproteins. J Androl 1996 17:522-529[Abstract/Free Full Text]
  51. Thaler CD, Cardullo RA. The initial molecular interaction between mouse sperm and the zona pellucida is a complex binding event. J Biol Chem 1996 271:23289-23297[Abstract/Free Full Text]
  52. Miller DJ, Macek MB, Shur BD. Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding. Nature 1992 357:589-593[CrossRef][Medline]
  53. Durr R, Shur B, Roth S. Sperm-associated sialyltransferase activity. Nature 1977 265:547-548[CrossRef][Medline]
  54. Godknecht A, Honegger TG. Isolation, characterization, and localization of a sperm-bound N-acetylglucosaminidase that is indispensable for fertilization in the ascidian, Phallusia mammillata. Dev Biol 1991 143:398-407[CrossRef][Medline]
  55. Tulsiani DR, Skudlarek MD, Orgebin-Crist MC. Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity. Biol Reprod 1990 42:843-858[Abstract]
  56. Yoshida-Komiya H, Tulsiani DR, Hirayama T, Araki Y. Mannose- binding molecules of rat spermatozoa and sperm-egg interaction. Zygote 1999 7:335-346[CrossRef][Medline]
  57. Mori K, Daitoh T, Irahara M, Kamada M, Aono T. Significance of D- mannose as a sperm receptor site on the zona pellucida in human fertilization. Am J Obstet Gynecol 1989 161:207-211[Medline]
  58. Aviles M, Abascal I, Martinez-Menarguez JA, Castells MT, Skalaban SR, Ballesta J, Alhadeff JA. Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum alpha-L-fucosidase from rat testis and epididymal spermatozoa. Biochem J 1996 318:821-831
  59. Khunsook S, Bean BS, McGowan SR, Alhadeff JA. Purification and characterization of plasma membrane-associated human sperm alpha- L-fucosidase. Biol Reprod 2003 68:709-716[Abstract/Free Full Text]
  60. Gabriele A, D'Andrea G, Cordeschi G, et al Carbohydrate binding activity in human spermatozoa: localization, specificity, and involvement in sperm-egg fusion. Mol Hum Reprod 1998 4:543-553[Abstract/Free Full Text]
  61. Gong X, Dubois DH, Miller DJ, Shur BD. Activation of a G protein complex by aggregation of beta-1,4-galactosyltransferase on the surface of sperm. Science 1995 269:1718-1721[Abstract/Free Full Text]
  62. Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios. Biol Reprod 1997 56:1020-1024[Abstract]
  63. Larson JL, Miller DJ. Sperm from a variety of mammalian species express beta 1,4-galactosyltransferase on their surface. Biol Reprod 1997 57:442-453[Abstract]
  64. Macek MB, Lopez LC, Shur BD. Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction. Dev Biol 1991 147:440-444[CrossRef][Medline]
  65. Jeschke U, Wang X, Briese V, Friese K, Stahn R. Glycodelin and amniotic fluid transferrin as inhibitors of E-selectin-mediated cell adhesion. Histochem Cell Biol 2003 119:345-354[Medline]
  66. Kiessling LL, Pohl NL. Strength in numbers: non-natural polyvalent carbohydrate derivatives. Chem Biol 1996 3:71-77[CrossRef][Medline]
  67. Rosen SD, Bertozzi CR. Two selectins converge on sulphate. Leukocyte adhesion. Curr Biol 1996 6:261-264[CrossRef][Medline]
  68. Wassarman PM. Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 1999 96:175-183[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
M. Seppala, H. Koistinen, R. Koistinen, P.C.N. Chiu, and W.S.B. Yeung
Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations
Hum. Reprod. Update, May 1, 2007; 13(3): 275 - 287.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. C. N. Chiu, M.-K. Chung, R. Koistinen, H. Koistinen, M. Seppala, P.-C. Ho, E. H. Y. Ng, K.-F. Lee, and W. S. B. Yeung
Cumulus Oophorus-associated Glycodelin-C Displaces Sperm-bound Glycodelin-A and -F and Stimulates Spermatozoa-Zona Pellucida Binding
J. Biol. Chem., February 23, 2007; 282(8): 5378 - 5388.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
P. C. N. Chiu, M.-K. Chung, R. Koistinen, H. Koistinen, M. Seppala, P.-C. Ho, E. H. Y. Ng, K.-F. Lee, and W. S. B. Yeung
Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding
J. Cell Sci., January 1, 2007; 120(1): 33 - 44.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
K. Lapid and N. Sharon
Meet the multifunctional and sexy glycoforms of glycodelin
Glycobiology, March 1, 2006; 16(3): 39R - 45R.
[Abstract] [Full Text] [PDF]

This Article