From the Department of Obstetrics and Gynaecology,
University of Hong Kong, Queen Mary Hospital, Hong Kong, Special
Administrative Region China and the § Department of
Obstetrics and Gynecology, University Central Hospital, Helsinki
FIN-00290, Finland
Received for publication, November 27, 2002, and in revised form, February 5, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 125I-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 125I-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 (1-3). 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).
Glycodelin, previously known as placental protein 14 (PP14), is a
member of the lipocalin superfamily. It is a glycoprotein consisting of
180 amino acids with three putative N-glycosylation sites
(Asn-28, Asn-63, and Asn-85). There are two known isoforms of
glycodelin, amniotic fluid glycodelin (glycodelin-A, GdA) and seminal
plasma glycodelin (glycodelin-S, GdS). The two isoforms have identical
protein backbones but have different glycosylation processes (5-8).
Only glycodelin-A has spermatozoa zona pellucida binding inhibitory
activity (9), whereas glycodelin-S does not affect spermatozoa zona
binding (5, 10). These data suggest that glycosylation determines the
biological activities of these two isoforms.
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 (17-19).
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.
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 × g for 10 min. The samples were sterilized by
filtration with a 0.22-µm filter unit (Millipore, Bedford), pooled,
and stored at 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 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 µg/µl in PBS) was denatured in 1 µl of 5% SDS and 1.5 µl of 1:10 dilution of 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 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 × 105 spermatozoa/ml in a
100-µl droplet of EBSS/BSA under mineral oil for 3 h at 37 °C
in an atmosphere of 5% CO2 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 µm. The numbers of tightly bound spermatozoa on the outer surface of the hemizonae were counted. The hemizona binding index (HZI) was defined as,
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 Na125I (20 µl, Amersham
Biosciences, England) in a small conical vial. Freshly prepared
chloramine T (100 µg in 0.02 ml of 0.05 M PBS, pH 7.4)
was then added and mixed thoroughly. After 60 s, sodium metabisulfite (300 µg in 0.05 ml of 0.05 M PBS, pH 7.4)
was used to stop the reaction. Free 125I 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.
Biological Activity of 125I-Labeled
ZIF-1/Glycodelin-A--
Percoll-processed spermatozoa
(n = 5) were divided into eight portions. They were
incubated with 0, 0.01, 0.1, 1, 10, 25, or 50 µg/ml
125I-labeled ZIF-1/glycodelin-A in a 1-ml volume or in
unlabeled ZIF-1/glycodelin-A at 37 °C in an atmosphere of 5%
CO2 for 3 h. After incubation, the spermatozoa were
washed with fresh EBSS/BSA. HZA were performed on these treated spermatozoa.
Equilibrium Binding of 125I-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 125I-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 × 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 125I-ZIF-1 on Human
Spermatozoa--
After equilibration of 2 × 106
spermatozoa (n = 3) to the desired temperature (4, 25, or 37 °C), 125I-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 × g for 10 min. The radioactivity associated with the treated
spermatozoa after washing twice in EBSS/BSA was measured.
Dissociation Kinetics of 125I-ZIF from Human
Spermatozoa--
Two million human spermatozoa in 1 ml of EBSS/BSA
were incubated with 125I-ZIF-1 (300 pmol/ml) at 37 °C
for 150 min. A 100-fold excess of unlabeled ZIF-1 (30 nmol/ml) was then
added. After a further incubation for 0, 5, 10, 20, 30, 60, 90, 120, 180, 210, 240, 270, 300, or 360 min, 15 ml of ice-cold buffer was added
to stop the dissociation. The spermatozoa were then washed and their
associated radioactivity was determined.
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 × 106/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 Effects of Glycosylation of ZIF-1 and Glycodelin-A on Spermatozoa
zona Pellucida Binding--
Percoll-processed spermatozoa
(n = 5) were divided into eleven portions. Each portion
was incubated in 0, 0.01, 0.1, 1, or 10 µg/ml ZIF-1, deglycosylated
ZIF-1 (deglyco-ZIF-1), glycans of ZIF-1 (ZIF-1-glyco), glycodelin-A
(GdA), deglycosylated glycodelin-A (deglyco-GdA), glycans of
glycodelin-A (GdA-glyco), or EBSS/BSA (control) at 37 °C in an
atmosphere of 5% CO2 in air for 3 h. After
incubation, the spermatozoa were washed with fresh EBSS/BSA. HZA were
performed on the treated spermatozoa. To confirm that the effects of
oligosaccharide chains or deglycosylated protein cores from ZIF-1 and
glycodelin-A on spermatozoa zona pellucida binding are not due to their
effects on sperm motility, viability, and acrosomal status, we also
studied these parameters of the spermatozoa treated above.
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 ×1000 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:
Hoechst-negative 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.
Determination of Sperm Motility--
Hobson Sperm Tracker System
(HST, Hobson Tracking Systems Ltd., Sheffield, UK) was used to
determine sperm motility. The system consisted of a phase-contrast
microscope, closed circuit television video camera, a
microcomputer, a tracking screen, and HST software (Hobson Tracking
Systems Ltd.). The set-up parameters of the system were: (i)
framing rate, 50 Hz; (ii) aspect ratio, 1.39; (iii) calibration,
×10.0; (iv) refresh time, 5 s; (v) thresholds, +20/ Competition Binding Analysis of ZIF-1 with Oligosaccharide
Chains--
The binding of 125I-ZIF-1 (300 pmol/ml) to
human spermatozoa (2 × 106/ml) was measured in the
presence of increasing concentration (from 0.3 pmol/ml to 30 nmol/ml) of deglyco-ZIF-1, ZIF-1-glyco, deglyco-GdA, GdA-glyco,
or EBSS/BSA (control). After incubation at 37 °C for 3 h, the
treated spermatozoa were washed with fresh EBSS/BSA and the cell-bound
radioactivity was measured. Each experiment was repeated three times.
125I-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 Na2CO3 (27).
The purified sperm extracts was divided into six identical portions (10 µg/ml). Each portion was incubated with 1 µg/ml
125I-ZIF-1 in the presence of zona pellucida protein at
concentrations of 0, 0.01, 0.05, 0.1, or 0.2 zona pellucida/µl, or
with 1 µg/ml 125I-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 = 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%
CO2 in air for 3 h. After washing with fresh EBSS/BSA,
the treated spermatozoa were incubated with 1 µg/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 ± 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.
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 ± 1.6. 125I-ZIF-1 and
125I-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 125I-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
data3 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
125I-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 (R2 > 0.99) suggested the
presence of two specific binding sites for ZIF-1 (Fig. 3A).
The low affinity binding site (KD, 24.98 ± 2.36 pmol/ml; Bmax, 6.73 ± 0.11 pmol/2 × 106 spermatozoa) was more abundant than the
higher affinity site (KD, 3.94 ± 0.08 pmol/ml;
Bmax, 2.72 ± 0.06 pmol/2 × 106 spermatozoa). Analysis of the binding data by the Hill
equation analysis yielded a Hill coefficient of less than unity
(0.731 ± 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 (Beq) 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 (Kobs1 and Kobs2, Table
I). The major population (75.4% of the total) had a slow association rate constant of 0.043 ± 0.0075 per
min (Kobs1) compared with the minor population
(24.5% of the total) with an association rate constant of 0.1735 ± 0.024 (Kobs2).
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 (Koff1 and
Koff2, Table I). The larger population
(60.2% of the total) had a dissociation rate constant of 0.0038 ± 0.0004 (Koff1), which corresponded to a
half-life of dissociation of 182 min (obtained from ln
2/Koff). The smaller population (38.9% of the
total) had a dissociation rate constant of 0.0028 ± 0.0001 (Koff2) and a half-life of 247 min.
These data indicate that the binding of ZIF-1 is reversible. The true
association rate constant (Kon) was calculated
using the equation, (Kobs Specificity of ZIF-1 Binding to Human Spermatozoa--
Fig.
6 shows the results of the competition
binding study of 125I-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
(IC50) of 300 pmol/ml ZIF-1 was 271.82 pmol/ml.
Glycodelin-A could not further inhibit the binding of
125I-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
125I-ZIF-1 and had a maximum inhibitory effect of about
70% only.
Scatchard analysis of the stoichiometric data of ZIF-1 gave a
curvilinear plot with upward concavity that could be resolved into two
straight lines (R2 > 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 ± 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 ± 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 KD of glycodelin-A from Scatchard analysis was
20.76 ± 2.15 pmol/ml, which was close to the value of ZIF-1 low
affinity binding site (24.98 ± 2.36 pmol/ml).
125I-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
125I-ZIF-1 and 125I-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 (columns 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 ± 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 µg/ml (p < 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 125I-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
125I-ZIF-1 for binding to spermatozoa. At concentrations
greater than 30 pmol/ml, the glycans of ZIF-1 suppressed the binding of 125I-ZIF-1 significantly more than their glycodelin-A
counterparts (p < 0.05). The deglycosylated form of
ZIF-1 and glycodelin-A at the concentrations tested did not compete
with 125I-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).
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 time- and
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 KD 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 glycodelin-A cannot inhibit 30%
of 125I-ZIF-1 binding in the competitive binding assay.
This profile likely represents the interaction between
125I-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 dose-dependent manner. Coincidentally, one of the glycodelin-A-sperm 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 KD 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
Glycodelin, retinol-binding protein, and 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 carbohydrate-binding 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),
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 results2
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 inhibition 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 KD 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until used. Before experimentation, human
follicular fluid was thawed and diluted with EBSS/BSA to the desired concentration.
-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 >10 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 µg/liter.
-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 µl 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 × 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 µl 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.
20 °C, incubation of the mixture at
20 °C for 30 min and centrifugation at 13,000 × 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
20 °C for 1 h, and the supernatant was
discarded. The pellet was first triturated (23) in 50 µl 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 × 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 µl 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).
(Eq. 1)
-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.
20; (vi)
filter weights, 1 and
1, 2 and 0, 3 and 0, 4 and 0; (vii) minimum
chamber depth, 20 µm; (viii) minimum trail point, 25; (ix) maximum
trail point, 250; and (x) trail draw time, 3 s. The motility of
spermatozoa was analyzed on a warmed microscope stage at 37 °C with
a Cell-VU disposable semen analysis chamber (Fertility Technologies,
Inc., Natick, MA). Five hundred spermatozoa per specimen in randomly
selected fields were evaluated to determine 1) curvilinear velocity
(VCL, µm/s), 2) mean straight line velocity (VSL, µm/s), 3) average path velocity (VAP,
µm/s), 4) mean linearity (LIN, VSL/VCL), 5)
amplitude of lateral head displacement (ALH, µm), 6) head
beat cross-frequency (BCF, Hz), and 7) percentage of motile
sperm (MOT).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (88K):
[in a new window]
Fig. 1.
Detection of purified or deglycosylated ZIF-1
and glycodelin-A in 12% SDS-PAGE gel. Lane 1, ZIF-1;
lane 2, glycodelin-A; lane 3, molecular weight
marker; lane 4, deglycosylated ZIF-1 purified by gel
filtration; lane 5, deglycosylated GdA purified by gel
filtration.
View larger version (16K):
[in a new window]
Fig. 2.
Effects of different concentrations of
125I-ZIF-1/GdA and cold ZIF-1/GdA on hemizona binding index
(HZI) (n = 5).
View larger version (21K):
[in a new window]
Fig. 3.
Saturation of 125I-ZIF-1 binding
to human spermatozoa. Each point represents the
mean ± S.E. of three experiments performed in triplicate.
A, Scatchard plot of ZIF-1 from the saturation curve.
B, Hill plot of ZIF-1 from the saturation curve.
View larger version (17K):
[in a new window]
Fig. 4.
Time courses of 125I-ZIF-1
binding to human spermatozoa at different temperature. Each
point represents the mean ± S.E. of three experiments
performed in triplicate.
Association and dissociation constants of ZIF-1 on sperm binding
sites
View larger version (11K):
[in a new window]
Fig. 5.
Dissociation of 125I -ZIF-1 from
human spermatozoa at 37 °C. Each point represents
the mean ± S.E. of three experiments performed in
triplicate.
Koff)/L, where L is the
concentration of radio-ligand used in the kinetic binding studies (300 pmol/ml). The KD values derived from the rate
constants according to the relationship: KD = Koff/Kon 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.
-Lactoglobulin A and ZIF-2 inhibited the binding of
125I-ZIF-1 only at very high concentrations
(IC50 > 30 nmol/ml), whereas glycodelin-S and
retinol-binding protein had no effect on 125I-ZIF-1 binding
to the spermatozoa at all.
View larger version (29K):
[in a new window]
Fig. 6.
Competition of binding to spermatozoa between
saturated concentration of 125I -ZIF-1 and increasing
concentration of native ZIF-1, glycodelin-A, glycodelin-S,
-lactoglobulin A, retinol-binding protein, and
native ZIF-2. Each point represents the
mean ± S.E. of three experiments performed in triplicate.
A, Hill plot of glycodelin-A using data from the competition
binding assay
View larger version (68K):
[in a new window]
Fig. 7.
Binding of iodinated ZIF-1/glycodelin-A
binding to the human sperm membrane fraction (MF) in
the presence or absence of zona pellucida proteins. The amount of
zona pellucida protein used is expressed as the number of zona
pellucida solubilized per microliter of the final incubation medium
(ZP/µl). The binding was analyzed by 8% native gel
autoradiography. Lane 1, purified iodinated GdA; lane
2, iodinated GdA plus MF; lane 3, iodinated ZIF-1 plus
MF plus 0.2 ZP/µl; lane 4, iodinated ZIF-1 plus MF plus
0.1 ZP/µl; lane 5, iodinated ZIF-1 plus MF plus 0.05 ZP/µl, lane 6, iodinated ZIF-1 plus MF plus 0.01 ZP/µl;
lane 7, purified iodinated ZIF-1; lane 8,
iodinated ZIF-1 plus MF. The arrows represent the
ZIF-1-derived bands with intensity reduced in the presence of ZP.
View larger version (40K):
[in a new window]
Fig. 8.
Effect of different concentrations of ZIF-1
or glycodelin-A on the acrosomal status of human spermatozoa.
Spermatozoa were incubated either with ZIF-1/glycodelin-A (0.3, 3, or
30 pmol/ml) or EBSS/BSA, for 3 h followed by 30-min treatment with
progesterone (1 µg/ml) or EBSS/BSA. Each point represents
the mean of results from five different sperm samples. *,
p < 0.05 when compared with column 2. #,
p < 0.05 when compared with the corresponding control
without progesterone treatment.
View larger version (17K):
[in a new window]
Fig. 9.
A, effects of different concentration of
ZIF-1, deglycosylated ZIF-1 (deglyco-ZIF-1), or a mixture of
ZIF-1 oligosaccharides (ZIF-1-glyco) on the HZI of human
spermatozoa. B, effects of different concentration of GdA,
deglycosylated GdA (deglyco-GdA), or a mixture of GdA
oligosaccharides (GdA-glyco) on the HZI of human
spermatozoa.
View larger version (18K):
[in a new window]
Fig. 10.
Competition of binding to spermatozoa
between saturated concentrations of 125I -ZIF-1 and
increasing concentrations of deglycosylated ZIF-1
(deglyco-ZIF-1), ZIF-1 oligosaccharides
(ZIF-1-glyco), deglycosylated GdA
(deglyco-GdA), or GdA oligosaccharides
(GdA-glyco).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactoglobulin (30). Recombinant glycodelin from Escherichia
coli binds to the CD14+ monocyte lineage cells but not
to CD20+ (B cell lineage) or CD3+ (T cell
lineage) cells (31). Using monocytes (CD14+), Scatchard
analysis has demonstrated a single class of receptors for the
recombinant glycodelin with an estimated KD of about
10 pmol/ml. However, the KD 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 KD 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.
-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
125I-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.
-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.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the laboratory staff in the in vitro fertilization team for their skillful technical assistance and Dr. A. Poon, Department of Physiology, University of Hong Kong for advice on the binding kinetics study.
![]() |
FOOTNOTES |
---|
* This work was supported by the Research Grant Council, Hong Kong (Grants HKU7188/99 M and HKU7261/01M), 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.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam Rd., Hong Kong, SAR China. Tel.: 852-285-53405; Fax: 852-281-75374; E-mail: wsbyeung@hkucc.hku.hk.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M212086200
2 P. C. N. Chiu, R. Koistinen, H. Koistinen, M. Seppala, K. F. Lee, and W. S. B. Yeung, unpublished data.
3 H. Koistinen, R. L. Easton, P. C. N. Chiu, M. Halttunen, A. Dell, H. R. Morris, W. S. B. Yeung, M. Seppala, and R. Koistinen, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ZIF-1, zona binding inhibitory factor-1; EBSS, Earle's balanced salt solution; GdA, glycodelin-A; GdS, glycodelin-S; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HZA, hemizona binding assay; HZI, hemizona binding index; ZP, zona pellucida; deglyco-ZIF-1, deglycosylated ZIF-1; ZIF-1-glyco, glycans of ZIF-1; deglyco-GdA, deglycosylated GdA; GdA-glyco, glycans of glycodelin-A.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Yao, Y. Q., Yeung, W. S. B., and Ho, P. C. (1996) Hum. Reprod. 11, 2674-2680[Abstract] |
2. | Chiu, P. C. N., Ho, P. C., Ng, E. H. Y., and Yeung, W. S. B. (2002) Mol. Reprod. Dev. 61, 205-212[CrossRef][Medline] [Order article via Infotrieve] |
3. | Qiao, J., Yeung, W. S. B., Yao, Y. Q., and Ho, P. C. (1998) Hum. Reprod. 13, 128-131[Abstract] |
4. | Yao, Y. Q., Chiu, P. C. N., Ip, S. M., Ho, P. C., and Yeung, W. S. B. (1998) Hum. Reprod. 13, 2541-2547[Abstract] |
5. |
Morris, H. R.,
Dell, A.,
Easton, R. L.,
Panico, M.,
Koistinen, H.,
Koistinen, R.,
Oehninger, S.,
Patankar, M. S.,
Seppala, M.,
and Clark, G. F.
(1996)
J. Biol. Chem.
271,
32159-32167 |
6. | Koistinen, H., Koistinen, R., Dell, A., Morris, H. R., Easton, R. L., Patankar, M. S., Oehninger, S., Clark, G. F., and Seppala, M. (1996) Mol. Hum. Reprod. 2, 759-765[Abstract] |
7. |
Dell, A.,
Morris, H. R.,
Easton, R. L.,
Panico, M.,
Patankar, M.,
Oehninger, S.,
Koistinen, R.,
Koistinen, H.,
Seppala, M.,
and Clark, G. F.
(1995)
J. Biol. Chem.
270,
24116-24126 |
8. |
Seppala, M.,
Taylor, R. N.,
Koistinen, H.,
Koistinen, R.,
and Milgrom, E.
(2002)
Endocr. Rev.
23,
401-430 |
9. | Oehninger, S., Coddington, C. C., Hodgen, G. D., and Seppala, M. (1995) Fertil. Steril. 63, 377-383[Medline] [Order article via Infotrieve] |
10. | Koistinen, H., Koistinen, R., Kamarainen, M., Salo, J., and Seppala, M. (1997) Lab. Invest. 76, 683-690[Medline] [Order article via Infotrieve] |
11. | Julkunen, M., Seppala, M., and Janne, O. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8845-8849[Abstract] |
12. | Chryssikopoulos, A., Mantzavinos, T., Kanakas, N., Karagouni, E., Dotsika, E., and Zourlas, P. A. (1996) Fertil. Steril. 66, 599-603[Medline] [Order article via Infotrieve] |
13. |
Tse, J. Y. M.,
Chiu, P. C. N.,
Lee, K. F.,
Seppala, M.,
Koistinen, H.,
Koistinen, R.,
Yao, Y. Q.,
and Yeung, W. S. B.
(2002)
Mol. Hum. Reprod.
8,
142-148 |
14. | Wassarman, P. M. (1999) Cell 96, 175-183[Medline] [Order article via Infotrieve] |
15. | Tulsiani, D. R., Yoshida-Komiya, H., and Araki, Y. (1997) Biol. Reprod. 57, 487-494[Medline] [Order article via Infotrieve] |
16. | Benoff, S. (1997) Mol. Hum. Reprod. 3, 599-637[Abstract] |
17. | Macek, M. B., and Shur, B. D. (1988) Gamete Res. 20, 93-109[Medline] [Order article via Infotrieve] |
18. | O'Rand, M. G. (1988) Gamete Res. 19, 315-328[Medline] [Order article via Infotrieve] |
19. | Chapman, N. R., and Barratt, C. L. (1996) Mol. Hum. Reprod. 2, 767-774[Medline] [Order article via Infotrieve] |
20. | World Health Organization. (1998) Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction , Cambridge University Press, Cambridge, UK |
21. | Yeung, W. S. B., Ng, V. K. H., Lau, E. Y. L., and Ho, P. C. (1994) Hum. Reprod. 9, 656-660[Abstract] |
22. | Riittinen, L., Narvanen, O., Virtanen, I., and Seppala, M. (1991) J. Immunol. Methods 136, 85-90[CrossRef][Medline] [Order article via Infotrieve] |
23. | Verostek, M. F., Lubowski, C., and Trimble, R. B. (2000) Anal. Biochem. 278, 111-122[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Yao, Y. Q.,
Yeung, W. S. B.,
and Ho, P. C.
(1996)
Hum. Reprod.
11,
1516-1519 |
25. | Shi, Y. L., and Ma, X. H. (1998) Mol. Reprod. Dev. 50, 354-360[CrossRef][Medline] [Order article via Infotrieve] |
26. | Franken, D. R., Kruger, T. F., and Oehninger, S. C. (1994) Andrologia 26, 277-281[Medline] [Order article via Infotrieve] |
27. | Dunbar, B. S., Wardrip, N. J., and Hedrick, J. L. (1980) Biochemistry 19, 356-365[Medline] [Order article via Infotrieve] |
28. | Yogev, L., Homonnai, Z. T., Gamzu, R., Amit, A., Lessing, J. B., Paz, G., and Yavetz, H. (1995) Hum. Reprod. 10, 851-854[Abstract] |
29. |
Sundaram, M.,
Sivaprasadarao, A.,
DeSousa, M. M.,
and Findlay, J. B.
(1998)
J. Biol. Chem.
273,
3336-3342 |
30. | Mansouri, A., Gueant, J. L., Capiaumont, J., Pelosi, P., Nabet, P., and Haertle, T. (1998) Biofactors 7, 287-298[Medline] [Order article via Infotrieve] |
31. | Miller, R. E., Fayen, J. D., Chakraborty, S., Weber, M. C., and Tykocinski, M. L. (1998) FEBS Lett. 436, 455-460[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Vigne, J. L.,
Hornung, D.,
Mueller, M. D.,
and Taylor, R. N.
(2001)
J. Biol. Chem.
276,
17101-17105 |
33. | Flower, D. R., North, A. C., and Sansom, C. E. (2000) Biochim. Biophys. Acta 1482, 9-24[Medline] [Order article via Infotrieve] |
34. | Sinowatz, F., Plendl, J., and Kolle, S. (1998) Acta Anat. (Basel) 161, 196-205[CrossRef][Medline] [Order article via Infotrieve] |
35. | Ozgur, K., Patankar, M. S., Oehninger, S., and Clark, G. F. (1998) Mol. Hum. Reprod. 4, 318-324[Abstract] |
36. | Oehninger, S., Patankar, M., Seppala, M., and Clark, G. F. (1998) Andrologia 30, 269-274[Medline] [Order article via Infotrieve] |
37. | Miller, D. J., Macek, M. B., and Shur, D. (1992) Nature 357, 589-593[CrossRef][Medline] [Order article via Infotrieve] |
38. | Ram, P. A., Cardullo, R. A., and Millette, C. F. (1989) Gamete Res. 22, 321-332[Medline] [Order article via Infotrieve] |
39. | Tulsiani, D. R., Skudlarek, M. D., and Orgebin-Crist, M. C. (1990) Biol. Reprod. 42, 843-858[Abstract] |
40. | Cornwall, G. A., Tulsiani, D. R., and Orgebin-Crist, M. C. (1991) Biol. Reprod 44, 913-921[Abstract] |
41. | Topfer-Petersen, E., Friess, A. E., Nguyen, H., and Schill, W. B. (1985) Histochemistry 83, 139-145[Medline] [Order article via Infotrieve] |
42. | Oehninger, S. (2001) Cells Tissues Organs 168, 58-64[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Miranda, P. V.,
Gonzalez-Echeverria, F.,
Blaquier, J. A.,
Mahuran, D. J.,
and Tezon, J. G.
(2000)
Mol. Hum. Reprod.
6,
699-706 |
44. | Kadam, A. L., Fateh, M., and Naz, R. K. (1995) J. Reprod. Immunol. 29, 19-30[CrossRef][Medline] [Order article via Infotrieve] |
45. | Koistinen, H., Koistinen, R., Seppala, M., Burova, T. V., Choiset, Y., and Haertle, T. (1999) FEBS Lett. 450, 158-162[CrossRef][Medline] [Order article via Infotrieve] |
46. | Varki, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7390-7397[Abstract] |
47. | Rosen, S. D., and Bertozzi, C. R. (1996) Curr. Biol. 6, 261-264[Medline] [Order article via Infotrieve] |
48. |
Blackmore, P. F.,
Neulen, J.,
Lattanzio, F.,
and Beebe, S. J.
(1991)
J. Biol. Chem.
266,
18655-18659 |
49. | Meizel, S., Pillai, M. C., Diaz-Perez, E., and Thomas, P. (1990) Serono Symp. 16, 205-222 |
50. | Liu, D. Y., and Baker, H. W. (1990) J. Reprod. Fertil. 89, 127-134[Abstract] |