* Cell Biology and Inflammation Research, Drug Delivery Research, § Biochemistry and
Molecular Biology Research,
Pharmacia and Upjohn, Inc., Kalamazoo, Michigan 49001
The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm- egg interactions.
Cell-cell and cell-matrix interactions play central
roles in biological development, such as during
fertilization, implantation, placenta formation,
embryogenesis, cell differentiation, migration, and organ
formation. These types of interactions are also essential for a variety of physiological and pathological processes,
such as lymphocyte trafficking, immune defense, hemostasis, wound healing, cancer cell invasion, and metastasis.
Cell-cell and cell-matrix interactions are precisely controlled and regulated by cell and matrix adhesion molecules with specificities appropriate for their particular
functions (Gumbiner, 1992 Recruitment of leukocytes, from the flowing blood
stream across the endothelial cells of postcapillary venules
into the tissue at sites of inflammation or injury, is a multistep paradigm requiring at least three sequential steps.
Three major families of cell adhesion molecules are involved in this process: selectins, integrins, and adhesion
molecules of the immunoglobulin superfamily (Butcher, 1991 All three selectins recognize a sialoglycoprotein ligand,
P-selectin glycoprotein ligand-1 (PSGL-1)1 (Moore et al.,
1992 All three selectins have been reported to bind to the
tetrasaccharide structure NeuNAc The involvement of oligosaccharide structures in mammalian gamete interactions is well documented (Florman
and Wassarman, 1985 Materials
Rabbit, mouse, and human IgGs, saponin, L-cysteine, and Hoechst 33258 (H258) were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse
IgM was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA).
Calcium ionophore A23187 was purchased from Boehringer Mannheim
Biochemicals (Indianapolis, IN). Tissue necrosis factor- Proteins and Antibodies
P-selectin was purified from outdated human platelets as published (Ma et al.,
1994 Monoclonal IgG antibodies against P-selectin, P7 and P23, and monoclonal IgM antibodies against PSGL-1, PL5, and against SLex, CSLEX,
were prepared and characterized as reported (Ma et al., 1994 A polyclonal IgG antibody against PSGL-1 was prepared as follows. A
peptide corresponding to residues 41-55 (QATEYEYLDYDFLPEGGC) of the amino acid sequence of PSGL-1 (Sako et al., 1993 For the immunostaining experiments, the protein G-purified IgG fraction (C3633) was further purified by affinity chromatography on the immobilized antigen peptide as follows. The peptide (2.2 mg) was coupled
directly onto ~3 ml of SulfoLink Coupling Gel (Pierce Chemical Co.,
Rockford, IL); nonspecific binding sites on the gel were blocked with 50 mM
L-cysteine, according to the manufacturer's protocol. The IgG fraction
was incubated with the immobilized peptide beads at 4°C overnight with
end-to-end rotation. After washing with 100 ml of PBS, the bound antibody was eluted with 0.1 M glycine-HCl, pH 2.7. The preparation was
then concentrated by Mono-Q chromatography as described above. This
procedure yielded ~2 mg of the affinity-isolated antibody protein from
~100 mg of the protein G-purified IgG fraction.
Preparation of Porcine Oocytes and Zona Pellucida
Porcine oocytes and zona pellucida were prepared from frozen or fresh
ovaries essentially as described (Dunbar et al., 1980 Isolation of Porcine Sperm Cells
Fresh porcine sperm were collected at a local farm and kept at 37°C until
use. No obvious contamination of blood cells could be found in the white
milky suspension. The sperm was kept still for at least 15 min at 37°C in
the presence of 5% CO2, and "swim-up" sperm cells were carefully collected on the top layer of the sperm suspension. This was done to avoid
the possible contaminating blood cells and to eliminate less viable sperm
cells. No obvious contamination by leukocytes could be observed by microscopy.
Preparation of Porcine Umbilical Vein
Endothelial Cells
Porcine umbilical vein endothelial cells (PUVEC) were prepared from the
freshly collected porcine umbilical cords exactly as previously described
for human umbilical vein endothelial cells (Geng et al., 1990 CHO Cell Line Expressing E-Selectin
A stable CHO-K1 cell line expressing human full-length E-selectin was established by cotransfection of E-selectin cDNA in CDM8 vector (10 µg/ml)
with pcDNA1/Neo (1 µg/ml) using a LipofectinTM (GIBCO BRL) method
according to manufacturer's protocol. 3 d later, CHO cells were selected
and maintained in DME (high glucose) in the presence of 10% FCS (vol/
vol) and 0.4 mg/ml of active GeneticinTM (wt/vol; GIBCO BRL).
SDS-PAGE and Silver Staining
Aliquots of total zona pellucida proteins (~38 µg per lane) and sperm
cells (5 × 106 cells per lane; washed three times with ice-cold PBS) were
mixed with SDS sample buffer in the presence or absence of 5% Ligand Blotting
Aliquots of total zona pellucida proteins (~115 µg per lane) and porcine
leukocyte membrane extracts (~500 µg per lane) were mixed with SDS
sample buffer in the presence or absence of 5% Immunoblotting
Aliquots of total zona pellucida proteins (~115 µg per lane), sperm cells
(1 × 106 cells per lane), and TNF- Flow Cytometric Analysis
Porcine sperm cells were used either unwashed or washed three times
with HBSS/FCS (1% heat-inactivated FCS in HBSS, vol/vol; 3,000 rpm for
10 min). More than 90% of the washed sperm cells were mechanically capacitated, as determined using Coomassie brilliant blue (Aarons et al.,
1991 Immunoelectron Microscopy
Porcine oocytes were washed with HBSS/FCS and incubated with purified
platelet P-selectin (10 µg/ml) at 22°C for 1 h. As controls, P-selectin was
either omitted or the oocytes were incubated with P-selectin in calcium
and magnesium-free HBSS/FCS containing 2 mM EDTA. After washing,
the oocytes were incubated with rabbit P-selectin antibody (25 µg/ml) for
30 min. For antibody staining, porcine oocytes were first incubated in 10%
(vol/vol) normal goat serum in PBS for 1 h at 22°C. After this step, the oocytes were incubated with 1 µg/ml of PSGL-1 peptide antibody or preimmune IgG for 1 h at 22°C. After washing, samples were incubated with 50 µg/ml of affinity-purified goat anti-rabbit IgG conjugated with HRP (Accurate Chemical Co., Westbury, NY) for 1 h at 22°C. The oocytes were
rinsed and fixed with 2.5% glutaraldehyde (vol/vol) and 2% paraformaldehyde (wt/vol) in 0.1 M sodium cacodylate buffer, pH 7.4, containing
0.5 mM CaCl2 at 4°C for 30 min. They were then processed for peroxidase
cytochemistry and EM as previously described (Raub et al., 1990 Rabbit P- and E-selectin antibodies were conjugated with gold particles
as previously described (Raub and Kuentzel, 1984 Sperm-Egg Binding Assay
A sperm-egg binding assay was set up according to a published procedure
(Almeida et al., 1995 For the inhibition studies, sperm cells were preincubated with mouse
IgG, P23 mAb, or P7 mAb (all at 30 µg/ml) in the presence of 5 µM
A23187 at 22°C for 30 min. Oocytes were preincubated with mouse IgM,
CSLEX mAb, PL5 mAb, or human IgG, E-selectin Rg, or P-selectin Rg
(all at 30 µg/ml) at 22°C for 30 min. The cells were then mixed with sperm
cells without washing for the binding assay, as described above.
Sperm Cell-HL-60 Cell Binding Assay
Freshly collected sperm (0.3 ml) were resuspended into 30 ml of HBSS/
BSA and loaded with 2 µM BCECF-AM at 37°C for 30 min (Ma et al.,
1994 The labeled sperm cells (1 × 106 cells in a 0.5-ml aliquot) were mixed
with either Ramos cells or HL-60 cells (2 × 105 cells in a 0.1-ml aliquot) in
the presence or absence of 5 µM A23187 at 22°C for 1 h, with end-to-end
rotating. The unbound sperm cells were removed on a FCS cushion (0.5 ml of FCS per tube, centrifuged at 700 rpm on a table-top centrifuge for 2 min). The cell pellets were fixed with 2% paraformaldehyde (wt/vol; 0.5 ml
per tube) for flow cytometric analysis (FACScan®). The binding of the fluorescence-labeled sperm cells to the Ramos or HL-60 cells was measured
as the mean fluorescence intensity from >100,000 cells in the gated windows for Ramos or HL-60 cells.
For the inhibition studies, the labeled sperm cells were preincubated
with mouse IgG, P23 mAb, or P7 mAb (all at 30 µg/ml) in the presence of
5 µM A23187 at 22°C for 30 min. HL-60 cells were preincubated with either mouse IgM, CSLEX mAb, PL5 mAb, or human IgG, E-selectin Rg,
or P-selectin Rg (all at 30 µg/ml) at 22°C for 30 min. The cells were subsequently mixed with oocytes without washing for the binding assay.
Expression of a P-selectin Ligand in Zona Pellucida of
Porcine Oocytes
To investigate whether the zona pellucida of porcine oocytes contains a specific ligand for P-selectin, we took advantage of the cross-reactivity of human platelet P-selectin
with porcine neutrophils, i.e., the fact that human P-selectin supports adhesion of porcine neutrophils (Geng, J.-G.,
unpublished observations). As shown in Fig. 1 A, porcine
zona pellucida contains many proteins as visualized by silver staining. Based on densitometric measurements, >50%
of the proteins were recovered in the 40-70-kD ranges, under reducing conditions. This is consistent with published
data (Dunbar et al., 1980
In a parallel experiment, the zona pellucida proteins,
separated by 7% SDS-PAGE and transferred to blotting
membranes, were probed with human platelet P-selectin.
Surprisingly, P-selectin specifically bound to a single protein with a molecular mass of ~210 kD under nonreducing
conditions (Fig. 1 B, lane 1) and ~80 kD under reducing
conditions (Fig. 1 C, lane 1). The binding was Ca2+ dependent as it was completely abolished by performing the
P-selectin incubation in the presence of 2 mM EDTA (Fig.
1, B and C, lanes 2). P-selectin also specifically recognized
a dimeric molecule from porcine leukocyte membrane extracts, with molecular masses identical to those of the zona
pellucida protein under both nonreducing and reducing
conditions (Fig. 1 D).
Three additional weaker bands (at ~210, ~180, and
~140 kD under reducing conditions) were also recognized
by P-selectin (Fig. 1 C, lane 1). The presence of the ~210kD band may be due to incomplete reduction of the ~210kD dimer, a phenomenon frequently observed for human
leukocyte PSGL-1 (Moore et al., 1992
To corroborate the above findings, the porcine zona pellucida proteins were also probed with an antibody against
a synthetic peptide encoding residues 41-55 of the amino
acid sequence of PSGL-1. Fig. 2 shows that this antibody,
but not preimmune IgG, bound to the ~210-kD protein
under nonreducing conditions (A, arrow) and the ~80-kD
protein under reducing conditions (B, arrow). Preincubation of the antibody with the synthetic peptide abrogated this binding (C, arrow). The protein bands at ~50-60 kD
were most likely due to nonspecific binding, since (a) they
existed in the blots probed with both preimmune IgG and
PSGL-1 peptide antibody (A and B), and (b) they were
not inhibited with the respective peptide antigen (C).
Using EM, the distribution of the P-selectin ligand in the
zona pellucida of porcine oocytes was examined after labeling either with P-selectin followed by P-selectin antibody or with PSGL-1 peptide antibody. The experiments
demonstrated that the P-selectin ligand was associated with
membrane fragments and vesicles embedded throughout
the matrix of the zona pellucida (Figs. 3 and 4). The specificities of these approaches were confirmed by the absence, or marked reduction, of peroxidase reaction product in control oocytes, where either P-selectin (Fig. 3 C) or
P-selectin antibody (data not shown) was omitted, where
P-selectin was incubated in the presence of 2 mM EDTA
(Fig. 3 D), or where preimmune IgG was used (Fig. 4 B).
P-selectin Expression on Acrosomal Membrane of
Porcine Sperm Cells
The expression of P-selectin on porcine sperm cells was
first established by FACS® analysis, using two different
P-selectin antibodies, an FITC-conjugated rabbit P-selectin antibody and an FITC-conjugated P7 mAb. Both antibodies were raised against human platelet P-selectin and
both reacted with porcine platelet P-selectin (see Fig. 7 C).
As shown in Fig. 5, rabbit P-selectin antibody (A) and P7
mAb (B) bound to repeatedly washed sperm cells whose
plasma membranes were no longer intact (see Fig. 8). By
contrast, FITC-conjugated rabbit E-selectin antibody did
not bind to the sperm cells (Fig. 5 C), although it clearly reacted with the TNF-
To corroborate the finding of P-selectin expression on
porcine sperm cells, an immunoblotting experiment was
carried out. Intact sperm cells were washed with ice-cold
PBS and lysed in SDS sample buffer. The total sperm cell
proteins were separated by SDS-PAGE and stained by silver staining. This resulted in numerous protein bands with
various molecular masses, under reducing and nonreducing conditions (Fig. 7 A). The separated proteins were also
transferred to blotting membranes and probed with either rabbit P- or E-selectin antibody or with P7 mAb. Both
P-selectin antibodies bound to ~120-kD proteins under
nonreducing conditions (Fig. 7 B, arrow). The observed
molecular mass is identical to the molecular mass for human platelet P-selectin (Ma et al., 1994 Further studies, using immunoelectron microscopy and
a gold-conjugated rabbit P-selectin antibody, revealed that
the localization of P-selectin on the porcine sperm cells
was confined to the region of the sperm head, containing
the dense nucleus, covered only by the exposed acrosome
(Fig. 8 A, 1), including the acrosomal cap (Fig. 8 A, arrow).
The gold-conjugated P-selectin antibody did not label the
lower one-third of the sperm head (Fig. 8 A, 2), the neck
(Fig. 8 A, 3), or the tail (Fig. 8 A, 4). Consistent with the
results from the flow cytometric studies (Fig. 6), label was not observed on sperm cells with the plasma membrane still intact (Fig. 8 B). Incubation of sperm cells with
the gold-conjugated P-selectin antibody in the presence of
the unconjugated P-selectin antibody abolished the binding (Fig. 8 C). Again, gold-conjugated rabbit E-selectin antibody did not label the sperm cells (Fig. 8 D). Neither the
broken plasma membrane (Fig. 8 D, arrow) nor the
acrosome was labeled, although it labeled the surface of a CHO cell line expressing human E-selectin (Fig. 8 E).
Thus, these results confirm the expression of P-selectin,
but not E-selectin, on the acrosomal membranes, but not
on the plasma membrane, of porcine sperm cells. Furthermore, the detection of P-selectin after acrosomal reaction
suggests the possible localization of the molecule on the
inner acrosomal membrane.
Function of the Oocyte P-selectin Ligand and the Sperm
Cell P-selectin
To evaluate the function of the P-selectin ligand in the
zona pellucida of porcine oocytes and P-selectin on the
acrosomal membrane of porcine sperm cells, an in vitro
sperm-oocyte binding assay was carried out, essentially
according to the published procedure (Almeida et al., 1995 Table I.
Adhesion of Sperm Cells to Oocytes
; Hynes and Lander, 1992
; Cross et al., 1994
; Wassarman, 1995
; Snell and White, 1996
).
; Lasky, 1992
; Springer, 1994
). The selectins comprise
a subfamily of Ca2+-dependent (C-type) animal lectins
(Drickamer, 1988
, 1993
) that are mainly responsible for the
initial leukocyte tethering to, and rolling on, the activated
endothelial cells. Three members of this family have been
described to date (Butcher, 1991
; Lasky, 1992
; Springer,
1994
). L-selectin (CD62L) is a constitutively expressed homing receptor on a majority of leukocytes for lymphatic and
vascular endothelial cells. E-selectin (CD62E) is a cytokine-inducible cell surface receptor on vascular endothelial
cells, and P-selectin (CD62P) is a rapidly inducible receptor on vascular endothelial cells and platelets. E- and P-selectins function as cell surface receptors for neutrophils,
monocytes, T lymphocyte subsets, eosinophils, and basophils.
; Sako et al., 1993
; Lenter et al., 1994
; Ma et al., 1994
;
Asa et al., 1995
; Spertini et al., 1996
; Tu et al., 1996
).
PSGL-1 is a disulfide-linked dimeric sialomucin expressed on the microvilli of human leukocytes. The recognition of
PSGL-1 by the three selectins is Ca2+ dependent and sialidase sensitive, characteristics of selectin-mediated leukocyte adhesion (Rosen et al., 1985
; Yednock and Rosen,
1989
; Bevilacqua et al., 1987
, 1989; Larsen et al., 1989
;
Geng et al., 1990
).
2-3Gal
1-4(Fuc
1-3)
GlcNAc (called sialyl Lewis x or SLex) and its isomer,
NeuNAc
2-3Gal
1-3(Fuc
1-4)GlcNAc (called sialyl Lewis
a or SLea); these structures may constitute the minimal
recognition motif for the three selectins (Brandley et al.,
1990
; Varki, 1994
). In a previous investigation, we isolated
PSGL-1 from [3H]glucosamine-labeled HL-60 cells, a human promyeloid cell line, by P- and E-selectin affinity
chromatography. The desialylated O-linked oligosaccharides released from this molecule were separated into five
well-defined peaks having elution volumes corresponding to glucose oligomers composed of 2.5, 3.5, 6.3, 9.8, and 12.8 glucose units, respectively (Asa et al., 1995
). Sequencing
of the carbohydrate structures in these peaks resulted in
the identification of a set of oligosaccharides (Aeed, P.,
J.-G. Geng, D. Asa, L. Raycroft, L. Ma, and Å. Elhammer,
manuscript submitted for publication), which had considerable similarities to structures previously identified on
glycoproteins isolated from the porcine zona pellucida
(Hirano et al., 1993
).
). In mouse, for instance, O-linked
oligosaccharides have been specifically implicated in the
sperm-egg binding (Wassarman, 1988
; Litscher et al.,
1995
). To determine whether porcine gametes carry cell
adhesion molecules similar to those described for the interaction of leukocytes with vascular endothelial cells, i.e., selectins and sialomucin-type ligand molecules (Butcher,
1991
; Lasky, 1992
; Springer, 1994
), we investigated whether a
P-selectin ligand and P-selectin are expressed on porcine
oocytes and sperm cells, respectively.
Materials and Methods
(TNF-
) was purchased from Genzyme (Boston, MA). 2
,7
-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from
Molecular Probes (Eugene, OR). Paraformaldehyde was purchased from
Mallinckrodt Inc. (St. Louis, MO). Glass microscope slides were purchased from Baxter Healthcare Corp. (McGaw Park, IL). Frozen porcine
ovaries were obtained from PelFreeze Biologicals (Rogers, AR); fresh
porcine ovaries were obtained from a local slaughter house; and fresh porcine umbilical cords were collected from a local farm. The membrane extracts of porcine leukocytes were prepared as previously described (Ma et
al., 1994
; Asa et al., 1995
). HL-60 cells (CCL 240) and Ramos cells (CRL
1596) were obtained from American Tissue Culture Collection (Rockville,
MD) and cultured as recommended. All other chemicals were purchased
from previously described sources (Ma et al., 1994
; Asa et al., 1995
).
). E- and P-selectin receptor-globulins (Rg) were prepared as described (Asa et al., 1995
).
; Asa et al.,
1995
). Rabbit polyclonal antibodies raised against human platelet P-selectin and E-selectin Rg were prepared as previously described (Toombs et al.,
1995
).
) was synthesized on a 430A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA).
Two glycine residues (underlined) were added before the carboxyl-terminal cysteine to provide a spacer such that flexibility and availability of the
peptide would be retained after conjugation. The peptide was coupled to
maleimide-activated keyhole limpet hemocyanin via the carboxyl-terminal cysteine (underlined). Two New Zealand white rabbits (C3633 and
C3365; Caltag Laboratories, San Francisco, CA) were immunized with the
peptide conjugate. Rabbit IgG fractions were isolated from preimmune
sera and antisera using mAb Trap G (Pharmacia Biotech, Piscataway, NJ).
The IgG fractions were dialyzed against 27.3 mM Tris-phosphoric acid, pH 6.3, and concentrated on a Mono-Q ion-exchange column (Pharmacia Biotech) by elution with the same buffer containing 2 M NaCl. The purified proteins were then dialyzed against PBS, pH 7.4, and stored at 4°C.
). No obvious contamination by leukocytes could be observed by microscopy. The concentration of total zona pellucida proteins was determined by amino acid compositional analysis.
; Ma et al.,
1994
). For induction of E-selectin expression, confluent monolayers of
PUVEC (third passage) were treated with 300 U/ml of TNF-
at 37°C for
4 h. After washing once with PBS, the cells were harvested by mechanical
detachment with a cell scraper (Nunc, Naperville, IL) in the presence of VersineTM (GIBCO BRL, Gaithersburg, MD).
-mercaptoethanol (vol/vol). After boiling for 5 min, samples were subjected to 7% SDS-PAGE. After electrophoresis, proteins were silver stained (Bio
Rad Laboratories, Hercules, CA).
-mercaptoethanol (vol/
vol). Samples were boiled for 5 min and subjected to 7% SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride
membranes (Immobilon-P; Millipore Corp., Bedford, MA). The membranes were probed with P-selectin followed by biotinylated P23 mAb
(1 µg/ml). The membranes were subsequently incubated with a streptavidin-peroxidase complex (Vectostain ABC kit; Vector Laboratories, Burlingame, CA). A chemiluminescent detection system (Amersham Corp.,
Arlington Heights, IL) was used for detection (Ma et al., 1994
; Asa et al.,
1995
). All incubation and washing buffers contained either 1 mM CaCl2 or
2 mM EDTA as indicated.
-treated PUVECs (confluent monolayer of cells from a 35-mm dish per lane) were subjected to 7% SDSPAGE. The separated proteins were transferred to the blotting membranes as described above. For the zona pellucida proteins, the blotting membranes were probed with preimmune IgG or PSGL-1 peptide antibody (both at 1 µg/ml). They were then incubated with biotinylated goat
antibodies against rabbit IgG (5 µg/ml). For sperm cells and PUVECs, the
blotting membranes were probed separately with either biotinylated rabbit P- or E-selectin antibody or biotinylated P7 mAb (all at 1 µg/ml). The
membranes were subsequently incubated with a streptavidin-peroxidase
complex followed by a chemiluminescent detection system as described
above.
). The sperm cells were resuspended in HBSS/FCS (2 × 106 cells per
ml) and incubated separately with either an FITC-conjugated rabbit preimmune IgG, an FITC-conjugated rabbit polyclonal antibody against P- or
E-selectin, an FITC-conjugated mouse preimmune IgG, or an FITC-conjugated P7 mAb (all at 10 µg/ml) at 22°C for 1 h. For staining of PUVECs,
mechanically detached cells (confluent monolayer of cells from a 35-mm
dish per aliquot) were resuspended in HBSS/FCS and incubated with an
FITC-conjugated rabbit preimmune IgG or an FITC-conjugated rabbit
E-selectin antibody, as outlined above. After incubation, the cells were
sedimented by centrifugation at 1,500 rpm for 5 min and washed twice with
HBSS/FCS. The cells were then resuspended in HBSS/FCS for flow cytometric analysis (FACScan®; Becton Dickinson & Co., Mountain View, CA).
).
). Porcine sperm cells
and CHO cells expressing E-selectin were rinsed with HBSS/FCS and incubated with a sixfold dilution of the gold-conjugated rabbit P- or E-selectin antibody in HBSS/FCS at 22°C for 1 h. For inhibition experiments as
control, sperm cells were incubated with the gold-conjugated P-selectin
antibody in the presence of 200 µg/ml of the unconjugated P-selectin antibody. The labeled cells were fixed with 2% paraformaldehyde (wt/vol) in
PBS at 4°C for 15 min followed by 0.1 M ammonium chloride for 5 min.
The cells were processed for EM as previously described (Raub and Kuentzel, 1984
). Semithick (0.25 µm), en face sections through the cells
were viewed with a JEM-1200EX electron microscope (JEOL, Inc., Peabody, MA) operated at 100-120 kV.
). Porcine sperm cells and oocytes were gently
washed once (1,000 rpm for 10 min) with HBSS/BSA (1% BSA in HBSS;
wt/vol) except in controls where calcium and magnesium-free HBSS/BSA
containing 2 mM EDTA was used throughout the entire assay. The sperm
(0.1-ml aliquots of 2 × 106 cells per ml) were mixed with oocytes (0.1-ml
aliquots of ~500 oocytes per ml), in the presence or absence of 5 µM
A23187, at 22°C for 1 h. The cell mixtures were carefully layered on the top of 100% dialyzed and heat-inactivated FCS (1 ml per tube) and spun
at 500 rpm on a table-top centrifuge for 2 min (FCS "cushion"). Under
these washing conditions, <10% of the free sperm cells were mechanically
capacitated as determined using Coomassie brilliant blue (Aarons et al.,
1991
), and more than 90% of the free sperm cells were viable according to
their ability to exclude Hoechst 33258 (Tao et al., 1993
). The supernatants
were discarded and the cell pellets were fixed with 0.2 ml per tube of 2%
paraformaldehyde (freshly prepared in PBS, wt/vol). The preparations
were transferred onto glass microscope slides and examined under a microscope (Nikon Phase Contrast-2, EL WD 0.3; Tokyo, Japan), equipped
with a screen monitor (VOCON Industries, Inc., New York) and a VC2400
video camera (VICON Industries, Inc., New York). The microscopic images were printed using a color video printer (UP-5200MD; Sony, Park Ridge, NJ).
; Asa et al., 1995
). The labeled sperm cells as well as HL-60 and Ramos cells were washed three times with PBS (1,000 rpm for 10 min). The
cells were resuspended in HBSS/BSA (2 × 106 cells per ml), except in controls where calcium and magnesium-free HBSS/BSA containing 2 mM
EDTA was used, throughout the assay. Under these washing conditions,
~30-50% of the sperm cells were mechanically capacitated as determined
using Coomassie brilliant blue (Aarons et al., 1991
), and >80% of the free
sperm cells were viable based on their ability to exclude Hoechst 33258 (Tao et al., 1993
).
Results
).
Fig. 1.
Ca2+-dependent
binding of P-selectin to a
dimeric molecule from porcine zona pellucida. (A) Silver staining of total zona pellucida proteins (~38 µg per
lane) separated on 7% SDSPAGE in the presence (R) or
absence (N) of 5% -mercaptoethanol (vol/vol). (B)
P-selectin ligand blotting of
zona pellucida proteins
(~115 µg per lane), fractionated on 7% SDS-PAGE under nonreducing conditions.
After electrophoresis, the
proteins were transferred
onto a blotting membrane
and probed with human
platelet P-selectin followed
by a biotinylated P23 mAb in
the presence of 1 mM CaCl2
or 2 mM EDTA, as indicated. The blots were visualized with a chemiluminescence detection system (Amersham Corp.). (C) P-selectin
ligand blotting of the zona pellucida proteins under reducing conditions. (D) P-selectin ligand blotting of the membrane extracts of porcine leukocytes (~0.5 mg per lane) under reducing (R) and nonreducing (N) conditions. Blotting procedures were exactly as described for B.
[View Larger Version of this Image (39K GIF file)]
; Sako et al., 1993
; Lenter et al., 1994
). The nature of ~180- and ~140-kD bands
is unclear; they were not recognized by P-selectin (Fig. 1 B,
lane 1) and PSGL-1 peptide antibody (see Fig. 3 A) when
proteins were separated under nonreducing conditions.
Plus, they were not recognized by PSGL-1 peptide antibody (Fig. 3, B and C), under reducing conditions. In addition, there was some background staining spots on the blots, which were not similar to the protein bands (Fig. 1 B, lane 2).
Fig. 3.
Distribution of a P-selectin ligand in porcine zona pellucida. Porcine oocytes were washed and incubated sequentially with platelet P-selectin, rabbit P-selectin antibody, and an HRP-conjugated secondary antibody. P-selectin binding was visualized by the
electron-dense reaction product. P-selectin is bound to the membrane vesicles and fragments embedded within the zona pellucida at or
near the (A) inner surface (arrows) of the zona pellucida, which is adjacent to the oocyte removed during the staining procedures, and
(B) outer surface. (C) Zona pellucida stained in the absence of P-selectin. (D) Zona pellucida stained with P-selectin in the presence of EDTA. Bar, 500 nm.
[View Larger Version of this Image (115K GIF file)]
Fig. 2.
Immunoblotting of
zona pellucida proteins with
PSGL-1 peptide antibody.
Porcine zona pellucida proteins, fractionated by 7% SDS-PAGE under nonreducing (A) and reducing (B
and C) conditions and transferred to blotting membranes,
were probed with rabbit preimmune IgG or PSGL-1 peptide antibody followed by biotinylated antibody to rabbit
IgG. Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.
[View Larger Version of this Image (44K GIF file)]
Fig. 4.
Staining of porcine
zona pellucida with PSGL-1
peptide antibody. Zona pellucida of porcine oocytes
were stained with either (A)
PSGL-1 peptide antibody or
(B) preimmune IgG followed
by indirect immunoperoxidase EM as described above.
The outer surfaces of the
zona are shown. Bar, 2 µm.
[View Larger Version of this Image (73K GIF file)]
-treated PUVEC (D). Interestingly,
P-selectin polyclonal antibody did not bind to the unwashed sperm cells (Fig. 6 A) unless they were treated
with A23187 (a calcium ionophore known to induce the
acrosomal reaction; Fig. 6 B), repeated washing (causing
disruption of the plasma membranes, as demonstrated in
Fig. 8; Fig. 6 C), or saponin (a detergent that selectively
permeabilizes the plasma membrane; Fig. 6 D). Together,
these results suggest that porcine sperm cells express P-selectin, but not E-selectin, and that P-selectin is expressed on
the acrosomal membrane of the sperm cells, but not on the
plasma membrane.
Fig. 7.
Immunoblotting of
porcine sperm cells. Porcine
sperm cells (5 × 105 cells per
lane for silver staining and
1 × 106 cells per lane for
immunoblotting), porcine
platelets (1 × 105 cells per
lane), and PUVEC (confluent monolayer of cells from a
35-mm dish per lane) were
mixed with SDS sample
buffer and boiled for 5 min.
After electrophoresis under
reducing (A, R) and nonreducing conditions (A, N; B-D),
proteins were either silver
stained (A) or transferred to
blotting membranes and
probed with 1 µg/ml of biotinylated rabbit P-selectin antibody, biotinylated P7 mAb
against P-selectin (B for
sperm cells and C for platelets), or biotinylated rabbit
E-selectin antibody (D). Immunoreactive proteins were
visualized as outlined in the
legend to Fig. 1.
[View Larger Version of this Image (46K GIF file)]
Fig. 5.
P-selectin antibody binding to sperm cells. Repeatedly
washed porcine sperm cells (2 × 106 cells per ml) were incubated
separately with FITC-labeled rabbit IgG, an FITC-labeled rabbit P-selectin antibody (A), FITC-labeled mouse IgG, an FITClabeled P7 mAb against P-selectin (B), or an FITC-labeled rabbit
E-selectin antibody (C), respectively, at 22°C for 1 h (all at 10 µg/
ml). Similarly, TNF--treated PUVECs were incubated separately with FITC-labeled rabbit IgG or an FITC-labeled rabbit
E-selectin antibody (D). After washing, the sperm and endothelial cells were analyzed by flow cytometry (FACScan®). Results
were presented as histograms of log fluorescence intensities over
cell numbers from 10,000 cells.
[View Larger Version of this Image (30K GIF file)]
Fig. 8.
Localization of P-selectin on sperm cells. Washed sperm cells were incubated with a gold-conjugated P- or E-selectin antibody and examined by EM. (A) The staining of P-selectin antibody was confined to the region of the sperm head (1), which contained the
dense nucleus, covered only by the acrosome including the acrosomal cap (arrow). P-selectin was not expressed on the lower third of the
head region (2), the neck (3), or the tail (4). (B) The gold-conjugated P-selectin antibody did not label sperm cells with the plasma membrane (arrow) still intact. (C) Control sperm cell was incubated with the gold-conjugated P-selectin antibody in the presence of 200 µg/
ml of the unconjugated P-selectin antibody. (D) The gold-conjugated E-selectin antibody did not label the sperm cell. Neither the broken plasma membrane (arrow) nor the acrosome were labeled. (E) The gold-conjugated E-selectin antibody labeled the cell surface of a
CHO cell expressing human E-selectin. Bar, 0.5 µm.
[View Larger Version of this Image (102K GIF file)]
Fig. 6.
P-selectin antibody binding to sperm cells after acrosomal reaction or permeablization. Porcine sperm cells (2 × 106
cells per ml) without prior washing (A), without prior washing in
the presence of 5 µM A23187 (B), with prior repeated washing (C), and without prior washing in the presence of 0.01% saponin (D) were incubated with either FITC-labeled rabbit IgG or
FITC-labeled rabbit P-selectin antibody at 22°C for 1 h (all at 10 µg/ml). After washing, the cells were analyzed by flow cytometry
as above.
[View Larger Version of this Image (29K GIF file)]
) and porcine platelet P-selectin (Fig. 7 C) (Toombs et al., 1995
). The identities of the additional protein bands, with lower molecular masses, observed on the blots are not known. They may
represent proteolytic fragments of P-selectin generated
during the lysis of the sperm cells, since (a) sperm cells
were known to contain a variety of proteases (Eddy, 1988
),
and (b) the antibodies used in this experiment specifically
bound to P-selectin among all the proteins present in the
platelet lysates (Fig. 7 C). The relatively sharper ~120-kD
bands from sperm cells (Fig. 7 B, arrow), as compared with
the relatively broad bands from platelets (Fig. 7 C), could also result from the proteolytic cleavage of some forms of
the sperm P-selectin. Differential protease accessibility of
heterogeneous glycosylated platelet P-selectin has been
demonstrated previously (Johnston et al., 1989
). E-selectin
antibody did not recognize any protein in the porcine
sperm cells, although it bound avidly to a ~90-kD protein
from TNF-
-treated PUVEC (Fig. 7 D).
).
In this assay, acrosome-reacted sperm cells bound to oocytes in numbers usually exceeding 15 sperm cells per oocyte (referring to the number of bound sperm cells viewed
in the single optical plane used; the total number of sperm cells bound to the entire oocyte was considerably larger;
Table I). The binding was Ca2+ and Mg2+ dependent, since
it could be dramatically reduced (to less than three sperm
cells per oocyte) by treatment with EDTA. The requirement for divalent cations for sperm-egg binding is consistent with previous observations (Yanagimachi, 1988
).
Sperm-egg binding was also significantly reduced (to less than five sperm cells per oocyte) by preincubation of the sperm cells with P7 (a leukocyte adhesion blocking mAb against P-selectin), or by preincubation of the oocytes with PL5 (a leukocyte adhesion blocking mAb against PSGL-1), purified platelet P-selectin, or recombinant P-selectin Rg (Table I). By contrast, the sperm-egg binding was not affected by preincubation of the oocytes with mouse IgM, CSLEX (an mAb against SLex), mouse IgG, P23 (a leukocyte adhesion nonblocking mAb against P-selectin), or E-selectin Rg (Table I).
These results strongly suggest that both the P-selectin
ligand in the zona pellucida of porcine oocytes and P-selectin
on the acrosome-reacted sperm cells are biologically functional. To corroborate this finding, we investigated whether
porcine sperm cells could bind to HL-60 cells, a human
promyeloid cell line that expresses the functional PSGL-1
(Moore et al., 1992; Sako et al., 1993
; Lenter et al., 1994
;
Ma et al., 1994
; Asa et al., 1995
; Spertini et al., 1996
; Tu et
al., 1996
). Fig. 9 shows that, in the presence of Ca2+, Mg2+,
and A23187, fluorescently labeled sperm cells avidly
bound to HL-60 cells (A4), but not to Ramos cells (A1
and A2), a human lymphoblast cell line that does not express the functional PSGL-1 (Vachino et al., 1995
). This
binding was reduced when the experiment was carried out
in the absence of A23187 (A3), a calcium ionophore that
induces the sperm acrosomal reaction (Aarons et al., 1991
; Tao et al., 1993
). The partial binding observed in the absence of A23187 (A3) was likely attributed to the broken
cytoplasmic membrane on some sperm cells, caused by the
repeated washing procedures used in their preparation
(Fig. 8). The requirement for A23187 for full binding activity is consistent with the expression of P-selectin on the
acrosomal membrane of sperm cells (Figs. 6 and 8).
As expected, the binding activity was blocked by preincubation of the sperm cells with P7 (a leukocyte adhesion blocking mAb against P-selectin; B3), but not with mouse IgG (B1) or P23 (a leukocyte adhesion nonblocking mAb against P-selectin; B2). Preincubation of HL-60 cells with PL5 (a leukocyte adhesion blocking mAb against PSGL-1; C3) or P-selectin Rg (D3) also neutralized the binding, but mouse IgM (C1), human IgG (D1), or E-selectin Rg (D2) did not. Taken together, the results provide independent and convergent evidence for the biological function of the zona pellucida P-selectin ligand and the sperm P-selectin.
It should be mentioned that the anti-SLex mAb, CSLEX,
partially inhibited the interaction in this assay. This partial
inhibition, by CSLEX mAb, of the binding of sperm cells
to HL-60 cells (Fig. 9; C2), but not on the binding of sperm
cells to the oocytes (Table I), may be due to subtle differences in assay formats. Alternatively, the difference in cell
type (different oligosaccharide structures on porcine oocytes vs human HL-60 cells) and/or the particulars of the
CSLEX mAb specificity (recognizes other carbohydrate
epitopes besides SLex; Stroud et al., 1996a,b) may contribute to this partial inhibition. In addition, the lack of inhibition by E-selectin Rg, particularly in sperm cell binding to
HL-60 cells (Fig. 9; D2), raises several possibilities, such as
(a) there is an insufficient amount of E-selectin Rg in the
assay; (b) the binding determinant(s) on PSGL-1 for P-
and E-selectin is quite distinct and separated; (c) there is
sufficient other ligand(s) for E-selectin; or (d) PSGL-1 is
not a functional ligand for E-selectin. Obviously, further experiments are required to clarify these issues.
Attachment of the sperm cell to the oocyte is the first step
in mammalian fertilization. This process involves a cascade of cell-cell and cell-matrix interactions with at least
six consecutive steps (Wassarman, 1995; Snell and White,
1996
). Sperm cells first bind to the zona pellucida, a large
extracellular matrix surrounding the oocyte. This binding
triggers the acrosomal reaction to facilitate the penetration of the sperm cell through the zona pellucida. After
penetration, the sperm cell binds to and fuses with the plasma membrane of the oocyte. The fertilized egg finally
undergoes implantation, placenta formation, and embryonic development.
Several adhesion molecules have been implicated to
play important roles in gamete binding. The sperm binding
to the zona pellucida in mouse is reportedly mediated by
ZP3 glycoprotein (Bleil and Wassarman, 1980), a reaction
that induces the acrosomal reaction on the mouse sperm
cells (Bleil and Wassarman, 1983
). Interestingly, this interaction appears to be mediated by O-linked carbohydrates on ZP3 molecule (Florman and Wassarman, 1985
; Litscher et al., 1995
). On the other hand, the binding of sperm
cells to the plasma membrane of the oocyte is mediated by
fertilin, a mouse sperm surface protein, and its counterligand,
6
1 integrin, on the oocyte surface (Blobel et al.,
1992
; Almeida et al., 1995
).
The specific distribution pattern of the P-selectin ligand
in the zona pellucida matrix argues against the possibility
that the detection of the ligand in this tissue represents an
artifact. An erroneous signal caused by contaminating leukocytes is unlikely for the following reasons. First, as discussed above, our microscopic studies show that both
P-selectin and PSGL-1 peptide antibody specifically bind
to membranous structures within the zona pellucida. Second,
large amounts of leukocytes (~0.5 mg proteins of membrane extract from ~100 million leukocytes per lane) are
typically required for positive detection with the P-selectin
blotting method used in this report (Fig. 1 D; Ma et al.,
1994). Hence, if the molecule detected in the zona pellucida represents the leukocyte ligand, truly major leukocyte
contaminations of the zona pellucida preparations would
be required to produce the signal observed on the blots. However, light and EM examination of these preparations
failed to show such leukocyte presence. Third, nonspecific
binding of P-selectin and PSGL-1 peptide antibody to a
contaminating or otherwise unrelated polysaccharide and/
or protein structure(s) on the surface of the membranous
structures in the zona pellucida is unlikely since our blotting experiments clearly show that both P-selectin and
PSGL-1 peptide antibody primarily recognize one protein
among the considerable numbers of proteins present in
the zona preparation; in addition, this protein is one of the
minor constituents of the zona pellucida (Fig. 1, compare
A, B, and C). Finally, the expression of a P-selectin ligand
in the zona pellucida is consistent with the detection of
PSGL-1 mRNA in the mouse ovary, as demonstrated by
Northern analysis (Yang et al., 1996
).
The P-selectin ligand from porcine zona pellucida and
leukocytes described in this report shares many characteristics with human leukocyte PSGL-1 (Moore et al., 1992;
Sako et al., 1993
; Lenter et al., 1994
; Ma et al., 1994
; Asa
et al., 1995
). These include biochemical properties (disulfide-linked dimeric protein), functional properties (recognition by P-selectin in a Ca2+-dependent manner), and
polypeptide properties (recognition by PSGL-1 peptide
antibody). However, there is a difference in molecular masses between the human leukocyte PSGL-1 (~240 kD
under nonreducing conditions and ~100 kD under reducing conditions) (Ma et al., 1994
) and the porcine oocyte
and leukocyte P-selectin ligands (~210 kD under nonreducing conditions and ~80 kD under reducing conditions)
(Figs. 1 and 2). Therefore, since amino acid sequencing information is not available for the porcine oocyte P-selectin ligand, the question as to whether this molecule is identical to PSGL-1 remains to be answered.
In this study we failed to detect any expression of E-selectin on sperm cells by flow cytometry, immunoblotting, or
immunogold EM. The apparent absence of the expression
of E-selectin on porcine sperm cells argues against a biological role for this lectin in gamete interactions. However,
an immunohistologic study has demonstrated the expression of both E- and P-selectin on vascular endothelial cells
in the decidua basalis, but not on decidua parietalis (Burrows et al., 1994). Therefore, it is conceivable that E-selectin,
along with P-selectin, on vascular endothelial cells in the
decidua basalis may interact with the zona pellucida ligand
during trophoblast implantation.
The expression of P-selectin on the acrosomal membrane of porcine sperm cells is supported by several experimental findings. First, blotting of sperm cell extracts separated on SDS-PAGE with two well-characterized P-selectin
antibodies (Ma et al., 1994; Asa et al., 1995
; Toombs et al.,
1995
) resulted, for both reagents, in the detection of a protein band with an apparent molecular mass identical to
those of human platelet P-selectin (Ma et al., 1994
) and
porcine platelet P-selectin (Fig. 7). Since purified sperm
cells (see Materials and Methods) were used for this experiment, it is unlikely that platelet contamination is responsible for the signal on the blot; as argued above, a considerable amount of platelets are required for a positive
signal in this type of experiment. Second, immunoelectron
microscopies of the porcine sperm cells quite unequivocally demonstrate the abundant presence of a molecule
recognized by a P-selectin antibody, on what appears to be
the acrosomal membrane of the sperm cells (Fig. 8). This distribution to a specific membranous compartment on the
sperm cells is in itself an argument against nonspecific labeling, caused by unspecific binding of the antibody as
well as by unspecific adsorption of (soluble) antigen to the
sperm cell surface. The fact that only acrosome-reacted
sperm cells are capable of binding the antibodies also argues against nonspecific results. Finally, the capacity of
acrosome-reacted porcine sperm cells to attach to oocytes
and HL-60 cells in a manner that is inhibitable by P-selectin antibodies strongly suggests a P-selectin function on
the acrosome-reacted sperm cells (Table I; Fig. 9).
The ultrastructural distribution of the zona pellucida
P-selectin ligand indicates that it is less likely a component
of the zona pellucida matrix. Instead, the molecule appears to be located exclusively in membranes embedded
within the matrix of the zona pellucida. The membrane
structures in the zona pellucida may originate from the
long oocyte microvilli and/or from the follicle cell projections that transverse the zona pellucida and make contact (gap junctions) with the oocyte plasma membrane (Austin,
1968). The morphological localization of P-selectin ligand-
containing membranes in the zona pellucida implicates a
potentially functional role for a P-selectin ligand during
the penetration by the acrosome-reacted sperm cells through
the thick, gel-like matrix of the zona pellucida. In this regard, the expression of functional P-selectin on the acrosomal membrane of sperm cells supports this hypothesis.
The findings in this report implicate a potentially biological role for the zona pellucida P-selectin ligand and the
sperm P-selectin in porcine gamete interactions. However,
since homozygous mice deficient in P-, E-, or L-selectin,
by homologous recombination, have no apparent deficiencies in breeding (Myadas et al., 1993; Arbonès et al., 1994
;
Labow et al., 1994
), this interaction may be only one of
several molecular mechanisms involved in fertilization in
vivo (Wassarman, 1995
; Snell and White, 1996
). A similar,
apparently redundant pathway has been described for leukocyte-endothelial cell interactions. The phenotypes of P-,
E-, and L-selectin knockout mice appear normal until the
animals are challenged by inflammatory mediators (Myadas et al., 1993
; Arbonès et al., 1994
; Labow et al., 1994
).
Taken together, our in vitro studies suggest that a mechanism, similar to that involved in leukocyte recruitment, may be involved in sperm-egg binding. However, the specific role(s) of these molecules in the different steps of sperm-egg interaction, especially in vivo, clearly require(s) further investigation.
Received for publication 10 January 1997 and in revised form 24 March 1997.
1. Abbreviations used in this paper: BCECF-AM, 2We thank Drs. C.W. Smith for peptide synthesis and M.R. Deibel for detailed protocol of affinity isolation of PSGL-1 peptide Ab on immobilized peptide beads. We are grateful to Drs. S. Kornfeld and A.E. Buhl for critical comments on the manuscript. We also thank P.A. Aeed, R.A. Evans, and D.D. Gleason for their technical expertise during this study.