Binding of zona binding inhibitory factor-1 (ZIF-1) from human follicular fluid on spermatozoa.

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Human follicular fluid from women undergoing controlled ovarian stimulation (1) and natural cycle (2) in vitro fertilization and embryo transfer treatment inhibits the binding of spermatozoa to the zona pellucida. The number of spermatozoa bound to the zona decreases significantly after exposing the spermatozoa to human follicular fluid. The inhibitory effect occurred in all the follicular fluid samples tested (1)(2)(3). Zona binding inhibitory factor-1 (ZIF-1), 1 a glycoprotein isolated from human follicular fluid, is partly responsible for the zona binding inhibitory activity of follicular fluid (4).
ZIF-1 is similar to glycodelin-A in a number of aspects. They have similar molecular size: 28 kDa for glycodelin-A (8, 12) and 32 kDa for ZIF-1 (4). Both are found in follicular fluid of women undergoing assisted reproduction treatment (2,4,8,12) and inhibit the binding of human spermatozoa to zona pellucida (4,9) in a dose-dependent manner. Neither of the glycoproteins affects the motility and spontaneous acrosome reaction of spermatozoa in vitro (2,4,9). ZIF-1 is also immunologically similar to glycodelin (13). Our observations using N-terminal sequencing and protease-digested peptide mapping to be published elsewhere show that ZIF-1 has the same protein core as glycodelin-A. However, they are different in their oligosaccharide chains as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin binding ability, and isoelectric focusing. 2 These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin.
It is generally believed that follicular fluid stimulates fertilization. The physiological role of the paradoxical activity of ZIF-1 on spermatozoa zona pellucida binding inhibition is unclear. The presence of specific receptor/binding sites is a prerequisite for most physiological activities. Our previous data showed the presence of specific binding of iodinated ZIF-1 on human spermatozoa (2). To study further the binding and biological properties of these ZIF-1 binding sites, we perform binding kinetics experiment of ZIF-1 on human spermatozoa. We confirm these findings by investigating the ability of ZIF-1 binding to soluble extract of spermatozoa.
Spermatozoa zona pellucida binding in mammals involves carbohydrate-mediated events (14 -16). It has been shown that sperm carbohydrate-binding proteins bind to glycoconjugates of the zona pellucida in different species and mediate gamete recognition (17)(18)(19). We hypothesize that ZIF-1 exerts its zona binding inhibitory activity via its carbohydrate moieties. In the second part of this report, we study the effects of deglycosylation on the biological activity of ZIF-1 and demonstrate that the oligosaccharide chains derived from ZIF-1 as well as monosaccharides can compete with ZIF-1 for the binding sites on human spermatozoa.

EXPERIMENTAL PROCEDURES
Semen Samples-The Ethics Committee of the University of Hong Kong approved the study protocol. Semen samples with normal sperm parameters (20) from men visiting the subfertility clinics of the Queen Mary Hospital, University of Hong Kong were used in this study. Spermatozoa were separated by Percoll (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation as described previously (21). The resulting pellet was resuspended in Earle's balanced salt solution (EBSS, Flow Laboratories, Irvine, UK) supplemented with sodium pyruvate, penicillin-G, streptomycin sulfate, and 3% bovine serum albumin. The spermatozoa were washed and resuspended in EBSS containing 0.3% BSA (EBSS/BSA) before experimentation.
Human Follicular Fluid-Fifteen batches of human follicular fluid samples (20 samples per batch) were collected during oocyte retrieval from women undergoing assisted reproduction treatment in the Queen Mary Hospital, Hong Kong. Human menopausal gonadotropins after down-regulation with gonadotropin releasing hormone agonist and human chorionic gonadotropin were used for ovarian stimulation in these women. Only human follicular fluid samples without blood contamination and from follicles with a retrieved oocyte were used in this study. The cell debris in human follicular fluid was removed by centrifugation at 300 ϫ g for 10 min. The samples were sterilized by filtration with a 0.22-m filter unit (Millipore, Bedford), pooled, and stored at Ϫ20°C until used. Before experimentation, human follicular fluid was thawed and diluted with EBSS/BSA to the desired concentration.
Purification of ZIF-1-The purification of ZIF-1 was performed as described previously (2). Briefly, human follicular fluid was passed successively through a Hi-Trap blue and a protein-G column (Amersham Biosciences). The flow-through fraction from these two columns was applied to a concanavalin A-Sepharose column (Amersham Biosciences). The fraction that bound to the concanavalin A column was eluted with 0.3 M ␣-D-methylglucoside and was separated into two parts using Amicon-10 concentrator (Amicon Inc., Beverly, CA) according to their molecular size. The part with molecular size Ͼ10 kDa was further purified with Mono Q and Superose columns as described (4). The concentration of purified ZIF-1 was measured with a protein assay kit (Bio-Rad, Hercules, CA). The yield of ZIF-1 obtained was 30 -100 g/liter.
Purification of Glycodelin-Glycodelin-A and glycodelin-S were purified from amniotic fluid and seminal plasma, respectively. Seminal plasma was diluted 1:4 (v/v) with 50 mM Tris-HCl-buffered saline containing 9 g/liter NaCl, pH 7.7 (TBS), before purification. Triton X-100 (0.1%, v/v) was mixed with amniotic fluid or diluted seminal plasma and passed through a monoclonal anti-glycodelin antibody (clone F43-7F9)-Sepharose column as described previously (22). The column was then washed successively with TBS, 1 M NaCl, containing 1% isopropanol and 10 mM ammonium acetate before the bound glycodelin was eluted with 0.1% trifluoroacetic acid. The purified protein was dialyzed against 100 mM sodium phosphate, pH 7.2, and its concentration was measured. Glycodelin-S was further purified using anion exchange column as described (13).
Purification of Deglycosylated Glycodelin and ZIF-1-A kit, N-Glycosidase F Deglycosylation (Bio-Rad), was used to deglycosylate ZIF-1 or glycodelin-A. Fifty microliters of ZIF-1 or glycodelin-A (1 g/l in PBS) was denatured in 1 l of 5% SDS and 1.5 l of 1:10 dilution of ␤-mercaptoethanol at 95°C for 5 min. Four microliters of 10% Nonidet P-40 was added to entrap excess SDS after the reaction mixture was cooled to room temperature. The denatured protein was incubated in an equal volume of releasing buffer supplied with the kit containing 2 l of N-glycosidase F for 24 h at 37°C. Deglycosylated protein was obtained after three successive protein precipitations, each with 3 volumes of cold 100% ethanol followed by centrifugation for 5 min at 5000 ϫ g. The deglycosylated ZIF-1 (deglyco-ZIF-1) or glycodelin-A (deglyco-GdA) obtained was dried in a vacuum concentrator (Virtis, New York), redissolved in 20 l of PBS and further purified by gel filtration using Superdex-75 column in a SMART system (Amersham Biosciences). The purity of the deglycosylated proteins was checked by SDS-PAGE, and their concentrations were determined.
Purification of Oligosaccharide Chains from ZIF-1 and Glycodelin-A-Oligosaccharide chains from ZIF-1 and glycodelin-A were isolated as reported (23). In brief, the glycoproteins were denatured as described above except that no Nonidet P-40 was added. The denatured protein was precipitated by the addition of 4 volumes of 80% acetone at Ϫ20°C, incubation of the mixture at Ϫ20°C for 30 min and centrifugation at 13,000 ϫ g at 4°C for 20 min. The pellet obtained was redissolved and deglycosylated using the N-Glycosidase F Deglycosylation kit (Bio-Rad). Deglycosylated protein and oligosaccharide chains were precipitated in 80% acetone at Ϫ20°C for 1 h, and the supernatant was discarded. The pellet was first triturated (23) in 50 l of ice-cold 60% aqueous methanol and subsequently dispersed in 1 ml of the same solution. The supernatant was obtained by centrifugation at 13,000 ϫ g at 4°C for 20 min. This extraction process was repeated twice, and the methanol supernatant containing the peptide N-glycosidase F-released oligosaccharide chain (ZIF-1-glyco/GdA-glyco) was pooled, dried in a vacuum concentrator (Virtis, New York), and redissolved in 20 l of water. The percentage recovery of oligosaccharide chains using this method was greater than 90% (23). Therefore, the amount of oligosaccharide chains used in each experiment was expressed as the amount of native glycoprotein required for producing that quantity of oligosaccharide chains. Fifty microliters of PBS was also processed through all the above procedures and served as control (PBS control).
Hemizona Binding Assay-The hemizona binding assay was performed as described (24). Unfertilized oocytes from our intracytoplasmic sperm injection program were bisected into two identical hemizonae by a micromanipulator. In the assay, each hemizona was incubated with 2 ϫ 10 5 spermatozoa/ml in a 100-l droplet of EBSS/BSA under mineral oil for 3 h at 37°C in an atmosphere of 5% CO 2 in air. We used a sperm concentration higher than those in a typical HZA because more spermatozoa would bind to the zona pellucida in this condition and, therefore, the variation of the assay would be smaller. Loosely attached spermatozoa were removed by pipetting the hemizonae through a micropipette of internal diameter 200 m. The numbers of tightly bound spermatozoa on the outer surface of the hemizonae were counted. The hemizona binding index (HZI) was defined as, HZI ϭ number of spermatozoa bound in test droplet number of spermatozoa bound in control droplet ϫ 100 (Eq. 1) Radioiodination of ZIF-1/Glycodelin-A-Fifty micrograms of ZIF-1/ gylcodelin-A in 0.02 ml of 0.05 M PBS (pH 7.4) was mixed with 2 mCi of carrier-free Na 125 I (20 l, Amersham Biosciences, England) in a small conical vial. Freshly prepared chloramine T (100 g in 0.02 ml of 0.05 M PBS, pH 7.4) was then added and mixed thoroughly. After 60 s, sodium metabisulfite (300 g in 0.05 ml of 0.05 M PBS, pH 7.4) was used to stop the reaction. Free 125 I was removed by passing the mixture through a 10-ml disposable desalting column. The first radioactive peak containing iodinated ZIF-1/glycodelin-A was collected.
Equilibrium Binding of 125 I-ZIF-1 to Spermatozoa-Two million spermatozoa in 1 ml of EBSS/BSA were incubated with different concentrations (from 0.03 to 3000 pmol/ml) of 125 I-ZIF-1 at 37°C for 3 h. The binding was terminated by the addition of 15 ml of ice-cold PBS followed by centrifugation at 150 ϫ g for 10 min. The spermatozoa were further washed twice with fresh EBSS. A gamma counter (Model 5500B, Beckman) was used to count the radioactivity associated with the spermatozoa. Specific binding of ZIF-1 was determined by subtracting the counts bound in the presence of a 100-fold concentration of unlabeled ZIF-1 from the counts bound in the absence of unlabeled protein. The determinations of total binding and nonspecific binding were done in triplicate.
Association Kinetics of 125 I-ZIF-1 on Human Spermatozoa-After equilibration of 2 ϫ 10 6 spermatozoa (n ϭ 3) to the desired temperature (4, 25, or 37°C), 125 I-ZIF-1 (300 pmol/ml) was added at the appropriate temperature and mixed rapidly. The mixture was incubated for 0, 5, 10, 20, 30, 60, 90, 120, 150, 180, or 210 min. A large volume of ice-cold buffer was added to terminate the binding. Nonspecific binding was determined by the inclusion of 30 nmol/ml cold ZIF-1. Bound and free ligands were separated by centrifugation at 150 ϫ g for 10 min. The radioactivity associated with the treated spermatozoa after washing twice in EBSS/BSA was measured.
Specificity of ZIF-1 Binding to Human Spermatozoa-Competition binding analysis was used to investigate the affinity of ZIF-1 binding sites to glycodelin and other lipocalins. The binding of labeled ZIF-1 (300 pmol/ml) to human spermatozoa (2 ϫ 10 6 /ml) was determined in the presence of an increasing concentration (from 0.3 pmol/ml to 30 nmol/ml) of unlabeled ZIF-1, lipocalins, or EBSS/BSA at 37°C for 3 h. The lipocalins used included glycodelin-A, glycodelin-S, bovine ␤-lactoglobulin A (Sigma), and human retinol-binding protein (Sigma). Another zona binding inhibitory factor that we purified from the human follicular fluid, ZIF-2 (4), was also used in this competition assay. Cell-bound radioactivity was determined after washing the treated spermatozoa twice with EBSS/BSA. Each individual experiment was repeated three times.
Determination of Acrosomal Status of Spermatozoa-The acrosomal status of the spermatozoa was evaluated by fluorescein isothiocyanate-Pisum sativum agglutinin (Sigma) as described (2). Hoechst staining was used to determine the viability of the spermatozoa. The acrosomal status and viability of 300 spermatozoa were determined in randomly selected fields under a fluorescence microscope (Zeiss, Germany) with ϫ1000 magnification. The filter set used for Hoechst staining consisted of an excitation filter G365, a chromatic beam splitter FT395, and a barrier filter LP420, whereas that for fluorescein isothiocyanate-Pisum sativum agglutinin staining consisted of an excitation filter BP 450 -490, a chromatic beam splitter FT510, and a barrier filter LP520. The acrosomal status of spermatozoa was classified into: (i) intact acrosome: complete staining of the acrosome; (ii) reacting acrosome: Hoechstnegative and partial or patchy staining of the acrosome; and (iii) reacted acrosome: Hoechst-negative and complete staining of the equatorial segment only or no staining of the whole sperm head. The proportions of spermatozoa with reacted acrosomes were determined.

125
I-ZIF-1/Glycodelin-A Binding to the Human Sperm Extracts-Sperm extracts was isolated as described (25). Spermatozoa were separated from the seminal plasma by centrifugation at 5000 rpm for 5 min, washed three times with 0.01 M PBS, pH 7.4, and resuspended in 0.01 M Tris-HCl buffer containing 0.5 M KCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5% glycine, pH 8.3. Triton X-100 (1% v/v) was then added to the sperm suspension and stirred at 4°C overnight. The insoluble material was discarded after centrifugation at 15,000 rpm for 40 min. The supernatant containing mainly proteins of the plasma membrane and acrosome (25) was dialyzed against 100 mM sodium phosphate, pH 7.2. The protein concentration in the solution was determined by using a commercial kit (Bio-Rad). Human zona pellucida was separated from the oocytes using glass micropipettes under a microscope (26) and heat-solubilized at 70°C for 90 min in distilled water with pH adjusted to 9 with Na 2 CO 3 (27).
The purified sperm extracts was divided into six identical portions (10 g/ml). Each portion was incubated with 1 g/ml 125 I-ZIF-1 in the presence of zona pellucida protein at concentrations of 0, 0.01, 0.05, 0.1, or 0.2 zona pellucida/l, or with 1 g/ml 125 I-glycodelin-A at 37°C for 3 h. After incubation, the mixtures were analyzed by native-gel electrophoresis, and the radioactive bands were visualized by exposing the gel to BIO-MAX film (Kodak, New York).
Effects of ZIF-1/Glycodelin-A on Progesterone-induced Acrosome Reaction of Human Spermatozoa-Spermatozoa (n ϭ 5) were incubated in 0.03, 3, and 30 pmol/ml ZIF-1, glycodelin-A, or EBSS/BSA (as control) at 37°C under 5% CO 2 in air for 3 h. After washing with fresh EBSS/BSA, the treated spermatozoa were incubated with 1 g/ml progesterone or EBSS/BSA (as control) for 30 min. The acrosomal status of the spermatozoa was then evaluated as described above.
Data Analysis-All the data were expressed as means Ϯ S.E. The data were analyzed by SigmaStat statistical software (SigmaPlot 2000 & Enzyme Kinetics Analysis Module 1.0, Jandel Scientific, San Rafael, CA). A paired Student's t test was used to compare the number of spermatozoa bound to zona pellucida between matching hemizona.

RESULTS
Purification of ZIF-1/Glycodelin-A and Deglycosylated ZIF-1/Glcyodelin-A-After glycosidase digestion and purification by gel filtration, both ZIF-1 and glycodelin-A showed a single band of size 19 kDa (Fig. 1) corresponding to the size of deglycosylated glycodelin-A. Our unpublished data showed that the sequence for the first 25 amino acids from the N-terminal of deglycosylated ZIF-1 was identical to that reported for glycodelin-A (11). The identity of this band was further confirmed by its immunoreactivity to monoclonal anti-glycodelin antibody (F43-7F9) in enzyme-linked immunosorbent assay (data not shown). Equilibrium Studies-The mean number of spermatozoa bound to the control hemizona was 164.7 Ϯ 1.6. 125 I-ZIF-1 and 125 I-glycodelin-A had the same zona binding inhibitory activity as their native form (Fig. 2). The HZI decreased in a dose-dependent manner as the concentration of 125 I-ZIF-1 or cold ZIF-1 increased. There is a discrepancy in the efficiency of spermatozoa zona pellucida binding inhibition of glycodelin-A between this report and two previous studies (5,11). Several possibilities might account for this. First, the condition of HZA was different among the studies. Spermatozoa incubated in EBSS/BSA, medium used in the present study, have a higher zona binding capacity than those treated with Ham's F-10 medium used in previous studies (24). Spermatozoa prepared by swim-up technique were used in the previous studies, whereas Percoll density gradient-processed spermatozoa were used in the present study. Sperm preparation methods had been shown to affect sperm binding to the zona pellucida in hemizona assay (28). Second, unpublished data 3 show that glycodelin-A isolated from different amniotic fluid samples differs slightly in glycosylation as well as spermatozoa zona binding inhibitory activities. Third, the purification protocol was different among studies, which might preferentially isolate certain isoforms of glycodelin. Fig. 3 shows the specific binding of 125 I-ZIF-1 to the spermatozoa after incubation with different concentrations of ZIF-1. This binding increases with the amount of ZIF-1 used up to 300 pmol/ml, after which no further increase in binding was observed. This result indicated that the binding of ZIF-1 to spermatozoa was saturable. Analysis of the saturation data revealed a curvilinear plot. Scatchard plots best fitted by nonlinear regression analysis (R 2 Ͼ 0.99) suggested the presence of two specific binding sites for ZIF-1 (Fig. 3A). The low affinity binding site (K D , 24.98 Ϯ 2.36 pmol/ml; B max , 6.73 Ϯ 0.11 pmol/2 ϫ 10 6 spermatozoa) was more abundant than the higher affinity site (K D , 3.94 Ϯ 0.08 pmol/ml; B max , 2.72 Ϯ 0.06 pmol/2 ϫ 10 6 spermatozoa). Analysis of the binding data by the Hill equation analysis yielded a Hill coefficient of less than unity (0.731 Ϯ 0.041) (Fig. 3B), raising the possibility of binding heterogeneity.
Binding Kinetics- Fig. 4 shows the kinetics of ZIF-1 (300 pmol/ml) binding to spermatozoa at different temperatures. The specific binding increased rapidly for the first 60 min reaching equilibrium after about 150 min at 37°C. Specific binding of ZIF-1 at 4 and 25°C also increased with increasing time but did not reach equilibrium even after 210 min. In this study, the value of maximum equilibrium binding (B eq ) was highest at 37°C and lowest at 4°C.
At 37°C, the association kinetic data were best fitted to a double-exponential equation, indicating the presence of two populations of binding sites with different observed association rate constants (K obs1 and K obs2 , Table I). The major population (75.4% of the total) had a slow association rate constant of 0.043 Ϯ 0.0075 per min (K obs1 ) compared with the minor population (24.5% of the total) with an association rate constant of 0.1735 Ϯ 0.024 (K obs2 ).
In agreement with the association data, dissociation experiments showed that the kinetics of dissociation at 37°C could also be best described by two exponential functions (Fig. 5), suggesting the presence of two populations of binding sites. These binding sites had similar dissociation constants (K off1 and K off2 , Table I). The larger population (60.2% of the total) had a dissociation rate constant of 0.0038 Ϯ 0.0004 (K off1 ), which corresponded to a half-life of dissociation of 182 min (obtained from ln 2/K off ). The smaller population (38.9% of the total) had a dissociation rate constant of 0.0028 Ϯ 0.0001 (K off2 ) and a half-life of 247 min.
These data indicate that the binding of ZIF-1 is reversible. The true association rate constant (K on ) was calculated using the equation, (K obs Ϫ K off )/L, where L is the concentration of radio-ligand used in the kinetic binding studies (300 pmol/ml). The K D values derived from the rate constants according to the relationship: K D ϭ K off /K on were 4.92 and 29.08 pmol/ml. These values closely agreed with that obtained in the above equilibrium study, which were 3.94 and 24.98 pmol/ml, respectively.
Specificity of ZIF-1 Binding to Human Spermatozoa- Fig. 6 shows the results of the competition binding study of 125 I-ZIF-1 binding to human spermatozoa by ZIF-2 (another spermatozoa zona binding inhibitory factor from human follicular fluid) and different lipocalin family members. Glycodelin-A competitively inhibited the binding of ZIF-1 to human spermatozoa in a dose-dependent manner. The half-maximal inhibition (IC 50 ) of 300 pmol/ml ZIF-1 was 271.82 pmol/ml. Glycodelin-A could not further inhibit the binding of 125 I-ZIF-1 at concentrations greater than 2.82 nmol/ml. Furthermore, glycodelin-A could not completely inhibit ZIF-1 binding even when its concentration was 100-fold greater than that of 125 I-ZIF-1 and had a maximum inhibitory effect of about 70% only. ␤-Lactoglobulin A and ZIF-2 inhibited the binding of 125 I-ZIF-1 only at very high concentrations (IC 50 Ͼ 30 nmol/ml), whereas glycodelin-S and retinol-binding protein had no effect on 125 I-ZIF-1 binding to the spermatozoa at all. Scatchard analysis of the stoichiometric data of ZIF-1 gave a curvilinear plot with upward concavity that could be resolved into two straight lines (R 2 Ͼ 0.99). These data supported the results from the above binding kinetic study that there were two classes of ZIF-1 binding sites on the human spermatozoa. The Hill equation analysis also yielded a Hill coefficient that was much less than unity (0.462 Ϯ 0.030), thus providing further evidence for the presence of multiple binding sites for ZIF-1 on spermatozoa.
For glycodelin-A, the Hill coefficient was only slightly lower than unity (0.845 Ϯ 0.078) (Fig. 6A), and a two-sites fit was not significantly better than a one-site fit in Scatchard analysis of the competition results (data not shown). This result suggested glycodelin-A shared one common binding site with ZIF-1 on spermatozoa. The K D of glycodelin-A from Scatchard analysis was 20.76 Ϯ 2.15 pmol/ml, which was close to the value of ZIF-1 low affinity binding site (24.98 Ϯ 2.36 pmol/ml).

I-ZIF-1/Glycodelin-A Binding to the Human Sperm
Extracts-Previous study using the same protocol for preparation of human sperm extracts suggested that the isolated proteins contained mainly membrane proteins (25). The binding of 125 I-ZIF-1 and 125 I-glycodelin-A to the sperm extracts and the effects of solubilized zona pellucida on the binding of ZIF-1 to the sperm extracts are shown in Fig. 7. Iodinated ZIF-1 (lane 7) and glycodelin-A (lane 1) showed a single radioactive band at similar position. Two and three additional radioactive bands appeared after incubation of glycodelin-A (lane 2) and ZIF-1 (lane 8), respectively, with the sperm extracts. All the additional bands had their molecular sizes larger than that of iodinated ZIF-1 or glycodelin-A. The two bands from glycodelin-A had similar mobility as the upper two bands from ZIF-1. Coincubation of solubilized zona pellucida reduced the binding of ZIF-1 to the two lower radioactive bands in a manner that was inversely proportional to the amount of zona pellucida protein added (lane 3-6).
Effect of ZIF-1/Glycodelin-A on Progesterone-induced Acrosome Reaction of Human Spermatozoa-ZIF-1 or glycodelin-A at concentration of 0.3, 3, and 30 pmol/ml did not affect the spontaneous acrosome reaction of human spermatozoa (col- umns 1, 3, 5, and 7, Fig. 8), consistent with previous observation (2,8). Progesterone induced acrosome reaction in spermatozoa incubated in EBSS/BSA only (Fig. 8). Although preincubation with glycodelin-A at the concentrations tested did not affect progesterone-induced acrosome reaction, ZIF-1 at all the concentrations tested significantly suppressed the progesterone-induced acrosome reaction. This suppressive effect of ZIF-1 was dose-dependent, and the incidences of acrosome reaction after 30 pmol/ml ZIF-1 treatment with and without subsequent progesterone incubation were similar.

Effects of Glycosylation of ZIF-1 and Glycodelin-A on Spermatozoa zona Pellucida
Binding Capacity-The mean number of spermatozoa bound to the control hemizona was 157.1 Ϯ 2.1 in this experiment. The changes in HZI with different concentrations of ZIF-1, deglycosylated ZIF-1, or ZIF-1-glyco treatment are shown in Fig. 9A. ZIF-1 treatment decreased the HZI in a linear dose-dependent manner. Although ZIF-1-glyco inhibited spermatozoa zona pellucida binding, the magnitude of the inhibition was much lower than that of the native protein.
A similar pattern was found for the glycodelin-A (Fig. 9B),  although the inhibitory effect of glycodelin-A and GdA-glyco were significantly less than their ZIF-1 counterpart at concentration greater than 1 g/ml (p Ͻ 0.05). Both the deglycosylated form of ZIF-1 and that of glycodelin-A did not affect the HZI. Deglyco-ZIF-1/deglyco-GdA and ZIF-1-glyco/GdA-glyco did not affect sperm viability, acrosomal status, and motility (data not shown).
Effect of Oligosaccharide Chains on the ZIF-1 Binding to the Human Spermatozoa- Fig. 10 shows the results of the competition binding study of 125 I-ZIF-1 binding to human spermatozoa with the deglycosylated form or carbohydrate moieties of the glycoproteins. The oligosaccharide chains derived from glycodelin-A and ZIF-1 competed with 125 I-ZIF-1 for binding to spermatozoa. At concentrations greater than 30 pmol/ml, the glycans of ZIF-1 suppressed the binding of 125 I-ZIF-1 significantly more than their glycodelin-A counterparts (p Ͻ 0.05). The deglycosylated form of ZIF-1 and glycodelin-A at the concentrations tested did not compete with 125 I-ZIF-1 for binding sites on spermatozoa. All the glycans at the concentration used in this study did not affect sperm motility, viability, and acrosomal status (data not shown).

DISCUSSION
Our previous study showed that ZIF-1 binds specifically to human spermatozoa (2). The present study is the first report on the sperm binding sites for ZIF-1. Binding kinetic analysis shows that the binding of ZIF-1 to human spermatozoa is timeand temperature-dependent. There are two binding sites for ZIF-1 on spermatozoa, which are of low and a high affinities. Unlike ZIF-1, glycodelin-A shares only one binding site on human spermatozoa with K D value in competition binding assay close to the low affinity binding site of ZIF-1. Here, the low affinity sites of ZIF-1 occupy about 70% of the total binding sites. These data are consistent with the observation that gly- codelin-A cannot inhibit 30% of 125 I-ZIF-1 binding in the competitive binding assay. This profile likely represents the interaction between 125 I-ZIF-1 and the high affinity binding sites. The alternative interpretation of the present binding data is that different glycoforms of ZIF-1 interact with a single set of binding site with different affinities. The data presented in this report cannot distinguish between these two possibilities. The exact binding mechanism of ZIF-1 and glycodelin on human spermatozoa is being investigated in our laboratory.
The present study shows that ZIF-1 and glycodelin-A form three and two complexes, respectively, with the sperm extracts. Interestingly, the binding of ZIF-1 to two of the complexes is reduced in the presence of zona pellucida proteins in a dosedependent manner. Coincidentally, one of the glycodelin-Asperm protein complexes has the same mobility as the one of the ZIF-1 binding complexes. This result is consistent with the binding kinetic experiment demonstrating the presence of two receptors for ZIF-1 on human spermatozoa and that glycodelin-A shares one binding site with ZIF-1. The two ZIF-1 binding complexes may represent the receptor complexes of ZIF-1. We are uncertain whether ZIF-1 binds to a sperm receptor responsible for the binding to the zona pellucida or to one or more molecules that regulate spermatozoa zona pellucida binding. The ability of zona pellucida proteins to displace the binding of glycodelin from soluble sperm extract suggests that the ZIF-1 binding sites are closely associated with the putative zona pellucida protein binding sites even if they were regulatory proteins. The identities of the sperm components involved in these complexes are currently under investigation.
At the time of ovulation, follicular fluid together with the oocyte cumulus mass are transported to the oviduct where fertilization occurs. Using immunoassay with antibody raised against glycodelin-A and cross-reacted with ZIF-1, the concentration of ZIF-1 in the follicular fluid is estimated to be about 12 pmol/ml. This result together with the present kinetic data suggest that ZIF-1 in the follicular fluid could affect sperm function via its high affinity receptor. Whether the low affinity receptor of ZIF-1 is physiologically relevant is uncertain, because its K D is much higher to the concentration of ZIF-1 in the follicular fluid. The receptor may complement the function of the high affinity receptor and serve to fine-tune the action of ZIF-1 on sperm function. It is also possible that there is a local high concentration of ZIF-1 in the cumulus matrix, where the low affinity receptor will be occupied and exert its biological activity.
There is experimental evidence showing that lipocalins bind to specific cell surface receptors like retinol-binding protein (29) and ␤-lactoglobulin (30). Recombinant glycodelin from Escherichia coli binds to the CD14 ϩ monocyte lineage cells but not to CD20 ϩ (B cell lineage) or CD3 ϩ (T cell lineage) cells (31). Using monocytes (CD14 ϩ ), Scatchard analysis has demonstrated a single class of receptors for the recombinant glycodelin with an estimated K D of about 10 pmol/ml. However, the K D of glycodelin purified from decidual cytosol on monocyte-like U937 cells was reported to be higher, 48 pmol/ml (32), suggesting that glycosylation may be important for the binding of glycodelin to monocytes. Our finding, that the spermatozoa zona pellucida binding inhibitory activity of glycodelins is also affected by glycosylation, further supports the importance of glycosylation. Yet, the K D values of glycodelin reported for U937 cells (32) are higher than those of the binding sites of glycodelin-A obtained in this study, suggesting that the binding sites for glycodelin-A in different cell types are different.
Glycodelin, retinol-binding protein, and ␤-lactoglobulin, all are members of the lipocalin family (33). ZIF-1 shares amino acid sequence and structural similarity with glycodelin-A. Hence, it was of interest to study the specificity of the ZIF-1 sperm binding sites with respect to other lipocalin family members. From our data, retinol-binding protein and ␤-lactoglobulin do not effectively compete for binding of 125 I-ZIF-1 to human spermatozoa. Thus, lipocalins do not promiscuously bind to human spermatozoa. A similar conclusion was made for the glycodelin receptor on human monocytes (31). Another form of glycodelin, GdS, also had no effect on the binding of ZIF-1 to spermatozoa, indicating that specific oligosaccharide recognition is required for ZIF-1 binding on human sperm.
Fertilization is a complex process during which the spermatozoa undergo a cascade of events that lead to the eventual fusion between the spermatozoa and the oocyte. The recognition and interaction between complementary molecules present on the spermatozoa and the zona pellucida is the first step in this cascade. There is compelling evidence that carbohydratebinding proteins on the sperm surface mediate gamete recognition of different species by binding with high affinity and specificity to complex glycoconjugates on the zona pellucida (14 -16, 19, 34), including human (16,19,35,36). In the past two decades, the presence of several carbohydrate-binding proteins, such as galactosyltransferase (37), fucosyltransferase (38), ␣-mannosidase (39,40), fucose-binding protein (41), selectin-like molecules (42), ␤-hexosaminidase (43), FA-1 (44), and others, on the sperm plasma membrane and their complementary sugar molecules on the zona pellucida have been suggested to be involved in sperm-egg interaction.
In the present study, we showed that the carbohydrate moieties of ZIF-1 and glycodelin-A were important for their zona binding inhibitory activity and the deglycosylated glycoproteins lost such inhibitory activity in vitro. The glycans of ZIF-1 and glycodelin-A also reduce the sperm binding to the zona pellucida but with reduced magnitude. This result is further supported by the ability of ZIF-1 and glycodelin-A glycans to compete with iodinated ZIF-1 for binding sites on the spermatozoa. Interestingly, the ability of ZIF-1-derived glycans to inhibit spermatozoa zona pellucida binding and to compete with iodinated ZIF-1 is higher than those from glycodelin-A. These observations are consistent with our unpublished results 2 that there are differences in the glycosylation between the two glycoproteins and with present observations that ZIF-1 has an additional high affinity binding site on spermatozoa.
The reason for the lower spermatozoa zona pellucida binding inhibitory activity of the glycans when compared with the native glycoprotein is unknown. It is possible that the inhibi- tion requires the simultaneous binding of more than one site on spermatozoa. The theoretical three-dimensional structure of glycodelin (45) suggests that the carbohydrate chains are located in a way that might allow formation of "clustered saccharide patch" (46). In the intact glycoprotein, the protein backbone might be needed to bring the sugars from different glycosylation sites in the close proximity to each other to form a high affinity ligand for ZIF-1 receptor. This situation is less likely to occur when the glycans are dispersed randomly in solution. Therefore, the binding and the inhibition would be stronger for the native molecules. Such multivalent ligand binding has been suggested for the binding event between the L-and P-selectin proteins and sialyl Lewis-X (47). This result may well explain that high concentration of single species of glycan is required to compete with the native molecule for binding sites in this study.
The exact physiological role of ZIF-1 is still under investigation in our laboratory. The spermatozoa zona pellucida binding inhibitory effect ZIF-1 may serve to reduce the incidence of polyspermic fertilization or to select spermatozoa with stronger zona pellucida binding capacity for fertilization. It is also possible that ZIF-1 has other effects on sperm functions in the cumulus mass where local high concentration of the molecule may exist. The present data showed that ZIF-1 suppressed progesterone-induced acrosome reaction of human spermatozoa. The concentration of ZIF-1 required to elicit this biological activity is well below the K D value of the ZIF-1 receptors and within the concentration range of ZIF-1 found in follicular fluid, indicating that such activity may have physiological relevance. Previous work had demonstrated the presence of sperm plasma membrane progesterone receptors and a receptor-activated increase in intracellular calcium of human spermatozoa (48). Thus, progesterone, which is found in the cumulus matrix and follicular fluid, may stimulate acrosome reaction of human spermatozoa (49). This action of progesterone may not be beneficial to fertilization, because acrosome-reacted spermatozoa have a reduced zona pellucida binding capacity (50). ZIF-1 in the follicular fluid and in the cumulus matrix may protect the spermatozoa from premature progesterone-induced acrosome reaction when they are traversing through the cumulus mass.
In addition, cumulus cells take up and modify glycodelin-A obtained from the surrounding environment (13). 2 Because ZIF-1 is highly similar to glycodelin-A, it is possible that the cumulus cells also modified ZIF-1 to another form with different biological activity during fertilization in vivo. We have recently identified another glycodelin-like molecule from the extracellular matrix of cumulus mass of human that stimulates spermatozoa zona pellucida binding. 2 Taking these data together, the other possible function of ZIF-1 is to serve as a substrate for the production of other glycodelin-like molecules that enhance the zona pellucida binding capacity of spermatozoa traversing the cumulus mass. This hypothesis is currently being tested in this laboratory. The exact physiological function of different glycosylated forms of the glycodelin remains to be elucidated.