BIOLOGY OF REPRODUCTION 61, 749–756 (1999)
Role of Egg Sulfolipidimmobilizing Protein 1 on Mouse Sperm-Egg Plasma
Membrane Binding 1
Vichuda Ahnonkitpanit,3,6 Dawn White,3 Somchai Suwajanakorn,3,6 Frederick W.K. Kan,5 Malivalaya
Namking,3,8 George Wells,4 Nuanthip Kamolvarin,3,7 and Nongnuj Tanphaichitr2,3
Human IVF Program, Reproductive Biology Unit, Hormones/Growth/Development Research Group, Loeb Research
Institute, Ottawa Civic Hospital, Departments of Obstetrics and Gynecology, and Biochemistry3 and Department of
Medicine, 4 University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
Department of Anatomy and Cell Biology,5 Queen’s University, Kingston, Ontario, Canada K7L 3N6
Department of Obstetrics and Gynecology6 and Department of Biochemistry,7 Faculty of Medicine, Chulalongkorn
University, Bangkok, Thailand
Department of Anatomy,8 Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
protein family [5]. The disintegrin domain of fertilin a
(ADAM1), fertilin b (ADAM2), and cyristestin (ADAM3)
has been shown to be involved in sperm binding to the egg
plasma membrane [6–11], whereas the fusion peptide domain of fertilin a is believed to play a role in gamete membrane fusion (for review see [3]). Egg P95 is also important
for sperm-egg interaction [12], and it is speculated, on the
basis of its molecular size, that egg P95 may be an aintegrin subunit [13]. Since other sperm surface proteins
shown to be involved in gamete plasma membrane interaction are not related to fertilin (e.g., sperm DE protein
[14]) (for reviews see [3, 4]), it is likely that there are other
egg surface components besides integrins that are significant for this gamete interaction.
Sulfolipidimmobilizing protein 1 (SLIP1) is a 68-kDa
plasma membrane protein that has in vitro binding specificity to sulfogalactosylglycerolipid (SGG) [15–17], the major sulfoglycolipid of mammalian male germ cells and
sperm [18]. A rabbit polyclonal antibody (antiSLIP1) was
generated against the 68-kDa SDS-PAGE band of SLIP1
isolated from rat testis homogenate by SGG affinity column
[15] and was used for characterizing its property and function. Immunoblotting indicates similarity in selective tissue
distribution of SLIP1 to that of SGG, i.e., in testicular germ
cells and to a lesser extent in white matter of brain [16,
18]. In contrast, only SLIP1, and not SGG, is present in
eggs [16]. Testis SLIP1 is also more evolutionarily conserved than SGG (present only in mammals), as the protein
is commonly found in various vertebrates including mammals, amphibians, birds, and fish [16].
Subsequent studies revealed that SLIP1 has an ATPbinding site [19], and a crude extract of SLIP1 can be prepared by treating testicular cells or sperm with a sucrose
solution containing low concentrations of ATP and EDTA
(AES). Its extractability from the cells using this simple
salt solution strongly suggests that SLIP1 is a peripheral
plasma membrane protein [16, 20]. DEAE-cellulose column chromatography was then used to purify antiSLIP1cross-reactive protein from the crude extract of rat testis
SLIP1 [19]. Results from Lingwood’s and our group indicate that rat testis SLIP1 prepared by this method consists
of at least three components: 1) a zona pellucida (ZP)-binding component [20], 2) a testis heat shock protein 70 (P70)
component [17], and 3) an albumin component (possibly a
contaminant during SLIP1 isolation from the testis). Therefore, SLIP1 was defined as a group of 68-kDa proteins that
bind specifically to SGG [17]. Recently, we have purified
antiSLIP1-cross-reactive protein from the AES extract of
ABSTRACT
We have shown that sperm sulfolipidimmobilizing protein 1
(SLIP1, molecular mass of 68 kDa), a sulfogalactosylglycerolipid
(SGG)-binding protein, is significant in sperm-zona pellucida
(ZP) interaction. The objective of this study was to localize SLIP1
on the egg and determine its role in gamete interaction. Immunofluorescence and immunoprotein A gold electron microscopy localized SLIP1 to the egg plasma membrane. In vitro gamete binding, using zona-free eggs preincubated with antiSLIP1
Fab before coincubation with sperm, showed a significant, dosedependent decrease in sperm-egg plasma membrane binding.
Similar results were obtained when affinity-purified antiSLIP1
IgG was used for egg pretreatment. The significance of egg SLIP1
in sperm-egg plasma membrane binding was further demonstrated by a decrease (36–52%) in in vitro fertilization when zonaintact eggs were pretreated with antiSLIP1 IgG. Since SLIP1 has
been shown to bind SGG in vitro, we investigated the possibility
that sperm SGG may participate in sperm-egg plasma membrane
binding through egg SLIP1. Pretreatment of sperm with antiSGG
Fab prior to coincubation with zona-free eggs resulted in a dosedependent decrease in sperm-egg plasma membrane binding.
Collectively, these findings strongly suggest a role for egg SLIP1
in sperm-egg plasma membrane interaction, which may be
through its binding to sperm SGG.
INTRODUCTION
Sperm-egg plasma membrane interaction is one of the
important steps of mammalian fertilization. This interaction
occurs at the sperm equatorial segment or the postacrosomal region of acrosome-reacted sperm, where initial binding is followed by fusion of the gamete plasma membranes
(for reviews see [1, 2]). Among several sperm surface proteins reported to be involved in gamete plasma membrane
interaction, fertilin (PH30) is the most extensively studied
(for reviews see [3, 4]). Fertilin is a surface heterodimeric
complex (consisting of a and b transmembrane subunits)
in the ADAMs (a disintegrin and metalloprotease domain)
Accepted April 26, 1999.
Received November 3, 1997.
1
This work was supported by Medical Research Council, Canada (MT10366 to N.T.) and the Rockefeller Foundation (to N.T., S.S., and N.K.);
V.A. and S.S. were awardees of Research Development Fellowships from
the Faculty of Medicine, Chulalongkorn University; M.N. was awarded a
visiting scientist fellowship from Khon Kaen University; and D.W. is the
recipient of a Chorafas Foundation Award.
2
Correspondence: Nongnuj Tanphaichitr, Loeb Research Institute, 725
Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365;
e-mail: [email protected]
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AHNONKITPANIT ET AL.
boar sperm that possesses both SGG and zona-binding ability but that does not contain the P70 and albumin components. This protein, appearing as a doublet with molecular
masses of 68 and 62 kDa, is termed P68/62 [21].
Work in our laboratory has demonstrated that SLIP1 is
localized to the plasma membrane overlying the sperm head
and is involved in sperm-zona binding as assessed in vitro
[20, 22] and in vivo [23]. Purified SLIP1 and P68/62 bind
to isolated ZP and the ZP of intact eggs [20, 21]. Recently,
we have also demonstrated the significance of sperm SGG
in sperm-ZP binding [24], and direct immunofluorescence
of SLIP1 and SGG reveals a similar localization pattern of
the two molecules in the sperm head [22, 24]. Nonetheless,
it is not clear whether SLIP1 and SGG act synergistically
in ZP binding.
SLIP1 can also be extracted from mammalian eggs with
the AES solution. Like male germ cell SLIP1, this AES
SLIP1 extract from eggs is able to bind SGG in vitro [16].
However, SLIP1 localization on eggs and its physiological
role have not been described. In the present study, using
immunofluorescence and electron microscopic immunocytochemistry, we have localized SLIP1 to the egg plasma
membrane. We have also shown for the first time that egg
SLIP1 is significant in mouse sperm-egg plasma membrane
binding, the initial step in sperm-egg plasma membrane interaction.
MATERIALS AND METHODS
Egg and Sperm Collection
Zona pellucida-intact mature eggs, collected from the
oviduct of superovulated CF-1 female mice, were dissociated from cumulus masses; and, where indicated, the ZP
was removed with acidic Tyrode’s solution as previously
described [25]. Eggs were then washed and incubated
(378C, 5% CO2) in Krebs’ Ringer bicarbonate (KRB) medium supplemented with 0.4% BSA (KRB-BSA) for functional studies, or in KRB supplemented with 0.4% ovalbumin (KRB-ovalbumin) for selected functional experiments, as well as for SDS-PAGE/immunoblotting and immunolocalization work.
Motile capacitated Percoll gradient-centrifuged (PGC)
mouse sperm were prepared from caudal and vas deferens
sperm collected from CD-1 male mice as described previously [26] and further capacitated by incubating in KRB0.4% BSA (378C, 5% CO2, 30 min) at a concentration of
107 sperm/ml.
Antibodies
AntiSLIP1 antiserum, a rabbit polyclonal antibody produced by Dr. C. Lingwood (Hospital for Sick Children,
University of Toronto, Toronto, ON, Canada) against SLIP1
prepared by SGG affinity chromatography, was formulated
to reduce nonspecific binding as described by Lingwood
[15]. Purification of antiSLIP1 IgG and normal rabbit serum
(NRS) IgG was performed as described previously [20].
Conjugation of Cy3 to antiSLIP1 IgG and NRS IgG was
performed using a kit from Biological Detection Systems
(Pittsburgh, PA) according to the manufacturer’s instructions.
Affinity-purified antiSLIP1 IgG was also prepared. Approximately 250 ng (3.7 pmol) of purified boar sperm P68
(the ZP- and SGG-binding component of SLIP1) [21] was
subjected to SDS-PAGE followed by Western blotting to
nitrocellulose. The excised P68 band was incubated over-
night at 48C with 1 ml of antiSLIP1 IgG (10 mg/ml in Trisbuffered saline solution: 137 mM NaCl in 20 mM Tris-HCl,
pH 7.4). Affinity-purified antiSLIP1 IgG was removed from
the blot by treatment with 1 ml of 100 mM glycine-HCl,
pH 2.5 (258C, 30 min). The pH of the eluate was adjusted
to 8.0 with 1 M Tris-HCl, pH 8.8, and the eluate was concentrated to 50 ml in KRB using a Microcon 30 microconcentrator (Amicon, Beverly, MA) according to the manufacturer’s instructions. A negative control was prepared by
incubating antiSLIP1 IgG of the same volume and concentration with a blank blot (no protein) and processing as
described above. Since the antiSLIP1 IgG solution should
not adsorb to the blot, its expected concentration was 200
mg/ml. This antiSLIP1 IgG solution was used either directly
or diluted with KRB to 100 mg/ml for egg treatment. A
‘‘blank eluate’’ used for egg treatment was also prepared
by treating a blank blot with only acidic glycine followed
by pH adjustment and solution concentration as described
above.
Rabbit polyclonal IgG antibody directed against SGG
and its lipid analogue, sulfogalactosylceramide (SGC), was
generated in our laboratory [24] according to the immunization method and regimen described by Lingwood et al.
[27]. This IgG antibody (termed antiSGG/SGC) recognizes
SGG, lysoSGG, and SGC chromatographed on a silica
plate, but it does not cross-react with galactosylglycerolipid, galactosylceramide, cholesterol sulfate, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
phosphatidic acid, or sphingomyelin (unpublished results).
IgG was purified from the pooled antiserum by ammonium
sulfate precipitation [28] followed by protein A column
chromatography using an Immuno Pure Immobilized Protein A Pak column (Pierce, Rockford, IL). AntiSGG/SGC
Fab was generated from antiSGG/SGC IgG using an Immuno Pure Fab preparation kit (Pierce). Affinity-purified
antiSGG Fab was prepared by chromatographing the polyclonal antiSGG/SGC Fab through an SGG-celite matrix according to the method previously described [29].
Rabbit polyclonal antiHSP 74.5 antibody, directed
against the recombinant Mycoplasma hypopneumoniae heat
shock protein (HSP) 74.5 [17], was generously provided by
Dr. C. Lingwood. A Cappel polyclonal antibody against rat
serum albumin, antiRSA, was purchased from Organon
Teknika (Mississauga, ON, Canada).
Immunological Studies of Egg SLIP1
Since antiSLIP1 has been shown previously to crossreact with albumin on nitrocellulose [21], immunological
studies of mouse eggs were performed using KRB-ovalbumin as the medium for egg collection and washing, unless otherwise stated.
Immunoblotting. Proteins from zona-free eggs, extracted
with SDS sample buffer, were electrophoresed on duplicate
0.75-mm-thick, 10% polyacrylamide gels [30] using a
minigel apparatus (Bio-Rad Laboratories, Mississauga, ON,
Canada). One gel was silver stained [31] for protein detection, and the other was electroblotted to nitrocellulose [32].
The blot was probed for SLIP1 and albumin using antiSLIP1 and antiRSA, as described previously [21].
Immunolocalization of SLIP1 on eggs. Direct immunofluorescence of SLIP1 was performed on zona-free mouse
eggs that were washed in PBS and prefixed with 3% paraformaldehyde in PBS (15 min, 378C). Eggs were blocked
for nonspecific binding in PBS supplemented with 10% fetal calf serum (PBS-FCS) (378C, 1 h). After seven to eight
EGG SLIP1 FUNCTIONS
washes in PBS-PVP (PBS supplemented with 3 mg/ml
polyvinylpyrrolidone), the zona-free eggs were exposed to
Cy3-antiSLIP1 IgG (200 mg/ml in 50 ml of PBS-PVP;
378C, 30 min), washed in PBS-PVP, and placed onto a glass
slide in 1–2 ml of PBS-PVP. They were then gently flattened with a coverslip and viewed under a Zeiss epifluorescence IM35 (Carl Zeiss, Thornwood, NY) microscope
using a rhodamine filter. A negative control was performed
by exposing ZP-free eggs to Cy3-NRS IgG. To aid the localization of the microvilli-free region of the egg (i.e.,
above the egg chromosomes), eggs were loaded with a
chromosome fluorescent dye, Hoechst 33342 (Sigma
Chemical Co., St. Louis, MO), at 5 mg/ml [33], before being subjected to treatment with Cy3-antiSLIP1 IgG.
SLIP1 on zona-free eggs was also localized by immunoprotein A-gold labeling. Zona-free eggs preblocked with
KRB-FCS were incubated with antiSLIP1 IgG (378C, 1 h)
and washed successively with KRB-ovalbumin as described
above. They were then exposed to protein A-gold (11 nm)
solution (378C, 1 h), prepared in PBS as described previously [34]. After extensive washing with PBS, the eggs
were fixed with 2.5% glutaraldehyde in PBS (258C, 1 h),
washed again with PBS, and embedded in 2% low-meltingpoint agarose (Sigma) in PBS. After overnight solidification
at 48C, the egg-agarose block was stored in 0.2 M sucrose
1 1% paraformaldehyde in PBS until further processing
for electron microscopy. The agarose blocks of immunogold-labeled eggs and controls were embedded in Epon
(Electron Microscopy Sciences, Ft. Washington, PA) according to routine procedure. Ultrathin sections were then
prepared for examination on a Hitachi (Tokyo, Japan) 7000
electron microscope operated at 75 kV.
751
FIG. 1. Immunoblotting of egg SLIP1. Lane 1: purified boar sperm P68
(5 ng); lane 2: 115 zona-free mouse eggs. The immunoblot was probed
with antiSLIP1. The arrow with an open head indicates the position of
boar sperm P68 band. Arrows with triangle heads indicate positions of
molecular weight standards (Mr 103, 83, and 50 [3 1023]).
0.3% BSA at 106 sperm/ml before coincubation with zonafree eggs under the same conditions as described above.
Fusion assay. Gametes were prepared, treated with antibody, coincubated, and processed as described for the
binding assay except that 1) the zona-free eggs were preloaded with 5 mg/ml Hoechst 33342 (378C, 5% CO2, 30
min) prior to the antibody treatment and 2) gamete coincubation was performed for 60 min. The number of sperm
penetrated per egg was scored under a Zeiss epifluorescence IM35 microscope using the Hoechst dye filter, and
the percentage of eggs with penetrated sperm was determined.
Mouse Sperm-Egg Plasma Membrane Binding and Fusion
Mouse In Vitro Fertilization
Binding and fusion assays were performed using either
KRB-ovalbumin or KRB-BSA for egg collection and washing, and antiSLIP1 dilution and treatment (see Results for
explanation). In all experiments, both sperm capacitation
and gamete coincubation were performed in KRB-BSA.
Binding assay. Capacitated PGC mouse sperm were pelleted by centrifugation and resuspended in KRB-BSA at a
concentration of 106 sperm/ml. These sperm were plated as
a 60-ml droplet for coincubation with 20–30 zona-free eggs,
which were either pretreated (378C, 5% CO2, 30 min) with
an antibody (antiSLIP1 Fab/IgG, antiRSA Fab/IgG, or NRS
Fab/IgG control) or untreated (blank control). All eggs
were prewashed extensively to remove unbound antibody
with KRB-ovalbumin prior to addition of the sperm droplet.
After gamete coincubation (378C, 5% CO2, 30 min), the
sperm-egg complexes were washed six times in KRB-BSA
to remove loosely bound sperm using a drawn Pasteur pipette (220-mm bore). Sperm bound to the egg plasma membrane at the focal plane of the egg diameter were then
counted under an inverted Nikon (Garden City, NY) TMS
phase-contrast microscope. The number of sperm bound per
egg on antiSLIP1-Fab/IgG-treated or antiRSA-Fab/IgGtreated eggs was compared to that for the NRS-Fab/IgGtreated control eggs.
In another set of experiments, capacitated PGC sperm
were resuspended in KRB-0.3% BSA at 5 3 106 sperm/ml
and treated with various concentrations of antiSGG/SGC
Fab or affinity-purified antiSGG Fab (378C, 5% CO2, 30
min). Sperm were washed free of excess antibody by centrifugation (500 3 g, 10 min) and resuspended in KRB-
Cumulus-free zona-intact mouse eggs were treated with
200 mg/ml antiSLIP1 IgG or NRS IgG (378C, 5% CO2, 30
min). Approximately 60 antibody-treated or untreated eggs
were incubated (378C, 5% CO2, 5% O2, 90% N2) in 60 ml
KRB-BSA containing PGC sperm at 50 000–125 000
sperm/ml under paraffin oil. Five to six hours afterward,
eggs were scored for evidence of two-pronuclei formation
or single-sperm penetration by exposure to 0.25% acetoorcein, which stained the nuclei [35]. Fertilization rate was
defined as percentage eggs fertilized in each sample.
Statistical Analysis
The Student’s t-test was used to determine whether data
obtained from two groups of samples were significantly different from each other; ANOVA was applied for comparison among three or more sample groups.
RESULTS
Presence of an AntiSLIP1-Cross-Reactive Protein in
Mouse Eggs
Immunoblotting indicated the presence of a mouse egg
protein having a molecular mass of 68 kDa (P68e) that was
cross-reactive with antiSLIP1 (Fig. 1). This result agrees
with that previously described by us [20] and by Lingwood’s group [16]. Since antiSLIP1 has been shown previously to cross-react with albumin, when present at . 10
ng on nitrocellulose [21], immunoblotting was also performed with antiRSA. Results revealed the absence of
cross-reactivity of antiRSA with P68e or boar sperm P68
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AHNONKITPANIT ET AL.
FIG. 2. Direct immunofluorescence of aldehyde-fixed zona-free egg
SLIP1 using Cy3-antiSLIP1 IgG. Top panels) Phase-contrast micrographs
of corresponding fluorescent images (bottom panels). Eggs shown in a/b
and c/d were treated with Cy3-antiSLIP1 IgG (200 mg/ml), whereas eggs
shown in e/f were incubated with Cy3-NRS IgG (200 mg/ml). Experiments
were performed three times, and $ 50 eggs were viewed in each experiment. Consistently, all Cy3-antiSLIP1-IgG-treated eggs showed positive
fluorescent staining, although , 5% of the eggs exhibited fluorescent
staining of low intensity (e.g., the egg marked with asterisk in b). In contrast, Cy3-NRS-IgG-treated eggs revealed no fluorescent staining (f).
(data not shown). The amount of P68e was ;40 pg/egg, as
assessed by comparing the intensity of P68e on the chemiluminograph with that of boar sperm P68 (5 ng) (Fig. 1),
as well as the SDS-PAGE silver-staining intensity of the
extracted P68e band with that of standard rat serum albumin bands (data not shown).
Direct immunofluorescence using Cy3-antiSLIP1 IgG of
aldehyde-fixed zona-free unfertilized mouse eggs revealed
the presence of antiSLIP1-reactive protein on the surface
of all eggs (Fig. 2, a–d). The majority of the eggs showed
high fluorescent intensity with Cy3-antiSLIP1 IgG, whereas
, 5% were stained lightly (see the asterisked egg in Fig.
2b as an example). The fluorescent staining appeared over
the entire egg surface, including both microvilli-containing
and microvilli-free regions (as assessed by their locations
in relation to egg chromosome staining by Hoechst 33342,
data not shown), but not on the polar body. Eggs that were
pressed slightly for microscopic viewing showed uniform
fluorescent staining (Fig. 2, c and d), while eggs that were
flattened more showed a punctate fluorescent staining pattern at the periphery (Fig. 2, a and b). This punctate pattern
could be attributable to folding of the egg surface under
pressure during the flattening process. Identically prepared
mouse eggs exposed to Cy3-NRS IgG showed no fluorescent staining (Fig. 2, e and f), indicating that the fluorescence seen with eggs treated with Cy3-antiSLIP1 IgG was
specific to egg surface SLIP1. The presence of antiSLIP1reactive protein on the egg plasma membrane was further
confirmed by immunoprotein A-gold electron microscopy
(Fig. 3, a and b). SLIP1 was localized to both the microvilli
and the basal part of the egg plasma membrane (Fig. 3, a
and b). Denuded eggs labeled with protein A-gold complex
alone showed the egg plasma membrane essentially devoid
of gold particles, indicating the specificity of the immunolabeling (Fig. 3c).
Inhibition of Sperm-Egg Plasma Membrane Binding by
Pretreatment of Eggs with AntiSLIP1
Since SLIP1 is present on the egg plasma membrane, it
may be engaged in sperm-egg plasma membrane interac-
FIG. 3. Immunoelectron microscopic localization of SLIP1 on zona-free
mouse eggs. Electron photomicrographs of ultrathin sections of Epon-embedded eggs, previously incubated with antiSLIP1 IgG followed by labeling with protein A-gold complexes (11 nm in diameter), showed the
presence of many gold particles (arrowheads) over the egg plasma membrane (a, b) at both the tips of the microvilli and the basal part between
microvilli (a). The number of gold particles associated with the egg plasma
membrane was drastically reduced in the control (c), in which eggs were
incubated with the protein A-gold complexes alone. a) 333 040, b)
320 650, c) 315 340.
tion. In order to test this hypothesis, unfertilized zona-free
mouse eggs were pretreated with antiSLIP1, and the ability
of sperm to penetrate the egg was assessed. Since antiSLIP1 appears to cross-react with albumin on nitrocellulose
[21], we performed both sperm-egg plasma membrane
binding and fusion experiments with eggs cultured in either
albumin or ovalbumin. The results revealed that the number
of sperm bound per egg and the number of sperm penetrated per egg were the same for three sets of control and
antibody-treated samples (i.e., control eggs with no antibody treatment, antiSLIP1-Fab-treated eggs, and NRS-Fabtreated eggs), regardless of whether albumin or ovalbumin
was used in the medium. Therefore, data from all egg samples were pooled. In all experiments, eggs pretreated with
100 mg/ml antiSLIP1 Fab incorporated a similar number of
sperm (p . 0.5) as compared to eggs pretreated with NRS
Fab (negative control) (data not shown). These results suggest that egg SLIP1 may not have an obvious role in spermegg plasma membrane fusion.
However, when the number of sperm bound to the egg
plasma membrane was determined, the results revealed that
preexposure of the eggs to various concentrations of
antiSLIP1 Fab significantly inhibited sperm-egg plasma
membrane binding (p , 0.001, Fig. 4A). Inhibition was
;35% of the control value at 100 mg/ml antiSLIP1 Fab.
This inhibitory effect was dose-dependent, reaching the
highest level of 72% of the control value at 400 mg/ml of
antiSLIP1 Fab. This level of inhibition is comparable to
that seen with eggs pretreated with anti-pan-integrin antibody [6] or with anti-a6 integrin [6, 9].
EGG SLIP1 FUNCTIONS
Initially, antiSLIP1 Fab fragments were used to avoid
nonspecific inhibition due to the presence of Fc receptors
on the egg plasma membrane [36]. Our later experiments,
however, demonstrated that NRS IgG did not inhibit sperm
binding to the egg plasma membrane. Therefore, we pretreated zona-free eggs with 100 mg/ml and 200 mg/ml of
antiSLIP1 IgG that was preexposed to a blank nitrocellulose blot (see Materials and Methods). This resulted in significant inhibition of sperm-egg plasma membrane binding
(p , 0.001), i.e., 45% and 57%, respectively, as compared
to that in control eggs preexposed to 200 mg/ml of NRS
IgG (Fig. 4B). AntiSLIP1 IgG (molecular mass ;150 kDa,
bivalent for antigen binding) at 100 and 200 mg/ml would
correspond to approximately 66 and 133 mg/ml of antiSLIP1 Fab (molecular mass ;50 kDa, monovalent for antigen binding), respectively. The inhibitory effects of
sperm-egg plasma membrane binding appeared to be somewhat greater for eggs pretreated with antiSLIP1 IgG as
compared to eggs preexposed to antiSLIP1 Fab (Fig. 4, A
and B). This may be due to the possibility that antiSLIP1
IgG possesses higher affinity to its antigen than the Fab
fragments. Affinity-purified antiSLIP1 IgG inhibited sperm
binding to the egg plasma membrane to the same extent
(i.e., ;60% of the control value, p , 0.001, Fig. 4B) as
the non-affinity-purified antibody (exposed to a blank blot).
When zona-free eggs were preexposed to the ‘‘blank eluate’’ (prepared as described in Materials and Methods), no
inhibition of sperm-egg plasma membrane binding was observed as compared to that in the untreated or NRS-IgGpretreated eggs. The results strongly suggested that a 68kDa egg protein that has shared antigenicity with sperm
SLIP1 was involved in sperm-egg plasma membrane binding.
The effect of antiRSA IgG (200 mg/ml) pretreatment was
also evaluated in eggs cultured in ovalbumin-supplemented
medium. The results revealed that the number of sperm
bound to the egg plasma membrane, as well as the number
of penetrated sperm, was the same in antiRSA-IgG-treated
eggs as in NRS-IgG-treated eggs or in eggs not exposed to
antibody (data not shown).
Zona-intact eggs, which possess a barrier to polyspermy,
were also pretreated with antiSLIP1 IgG (200 mg/ml, n 5
103 combined from two experiments), and, as expected, a
decrease in the in vitro fertilization rate of these eggs was
observed as compared to that in control eggs (treated with
of 200 mg/ml NRS IgG, n 5 153). From two experiments
performed, the average fertilization rate for control eggs
was 75% (79% in experiment 1 and 72% in experiment 2)
and that for antiSLIP1-IgG-pretreated eggs was 34% (38%
in experiment 1 and 31% in experiment 2). Therefore, the
average inhibition of the in vitro fertilization rate resulting
from egg pretreatment with antiSLIP1 IgG was 55% of the
control value. Our previous findings indicated the absence
of SLIP1 in the ZP, and capacitated mouse sperm could still
bind to zona-intact eggs pretreated with antiSLIP1 at the
same level as the control untreated eggs [20]. Together with
the results shown in Figure 4, a decrease in the mouse in
vitro fertilization rate of eggs pretreated with antiSLIP1 was
most likely due to the interference of egg SLIP1’s function
in gamete plasma membrane binding.
Inhibition of Sperm-Egg Plasma Membrane Binding by
Pretreatment of Sperm with Antibody Recognizing Sperm
SGG
SGG has been shown to bind to isolated SLIP1 and boar
sperm P68 in vitro [16, 17, 21]. To investigate whether
753
FIG. 4. Inhibitory effects of antiSLIP1 pretreatment of zona-free eggs on
sperm-egg plasma membrane binding. A) Zona-free eggs were treated
with various concentrations of antiSLIP1 Fab prior to coincubation with
capacitated PGC sperm. Zona-free eggs treated with NRS Fab of the highest concentration served as controls. Data were expressed as mean 6 SD
percentage control of three replicate experiments. The total number of
eggs in each sample is shown on top of each bar. The average number of
sperm bound per control egg was 20.3 6 3.6. B) Zona-free eggs were
treated with affinity-purified antiSLIP1 IgG, which was prepared by adsorbing the antiSLIP1 IgG solution to boar sperm SLIP1 (P68), immobilized on nitrocellulose as described in Materials and Methods. For comparison, zona-free eggs were treated with antiSLIP1 IgG, which was preincubated with a blank blot (no protein), with the volume of the antibody
solution adjusted so that the final antibody concentration was 200 mg/ml.
This antiSLIP1 IgG solution was diluted to 100 mg/ml for treatment of
another set of zona-free eggs. Control zona-free eggs were treated with
NRS IgG at 200 mg/ml. Data were expressed as mean 6 SD of the number
of sperm bound per egg; the total number of eggs counted in each sample
is indicated on top of the bars. Data shown in B were from a single
experiment and were similar to results obtained from another replicate
experiment. *Significant difference (in both A and B) relative to the control
by ANOVA ( p , 0.001).
sperm SGG is a binding element of egg SLIP1 during
sperm-egg plasma membrane interaction, sperm were treated with antiSGG/SGC Fab prior to coincubation with zonafree eggs. Since mouse sperm contain only SGG and not
SGC [26], antiSGG/SGC presumably interacted specifically
with sperm SGG. In addition, immunoblotting indicated
that antiSGG/SGC did not cross-react with sperm proteins
(data not shown). Figure 5A displays a significant dose-
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AHNONKITPANIT ET AL.
FIG. 5. Inhibitory effects of antiSGG pretreatment of sperm on spermegg plasma membrane binding. Capacitated PGC sperm were treated with
A) 50 and 150 mg/ml of antiSGG/SGC Fab, as well as with B) 0.5, 1, and
10 mg/ml of affinity-purified antiSGG Fab. Control sperm were treated
with NRS Fab of the highest concentration. Data in A were expressed as
mean 6 SD of percentage control of three replicate experiments. Data in
B, shown as mean 6 SD of the number of sperm bound per egg, were
from a single experiment and were similar to results obtained from another replicate experiment. The figure on top of each bar is the total number of eggs used in each sample. *Significant difference as compared to
the control by ANOVA ( p , 0.001).
dependent inhibitory effect on sperm-egg plasma membrane binding when sperm were preexposed to 50 mg/ml
and 150 mg/ml of antiSGG/SGC Fab (p , 0.001); the number of sperm bound per egg was 41% and 17%, respectively, of that observed in eggs exposed to 150 mg/ml NRS
Fab. When various concentrations (0.5, 1, and 10 mg/ml)
of affinity-purified antiSGG Fab were used, significant inhibition of sperm-egg plasma membrane binding was observed to be 49%, 45%, and 24% of that in controls (NRS
Fab at the highest concentration), respectively (p , 0.001,
Fig. 5B). The results suggested the significance of sperm
SGG in gamete plasma membrane binding.
DISCUSSION
Sperm SLIP1 is a 68-kDa peripheral plasma membrane
protein involved in sperm-ZP interaction [20, 23]. In this
study, we demonstrated by immunological methods that
egg SLIP1 had the same molecular mass of 68 kDa as
sperm SLIP1 and was localized to the egg plasma membrane (Figs. 1–3). The presence of SLIP1 at the egg mi-
crovilli tips (Fig. 3a) suggested its possible role in gamete
plasma membrane interaction. This postulation was confirmed by results from the gamete binding assay, showing
reduced sperm binding to the plasma membrane of zonafree eggs pretreated with antiSLIP1 in a dose-dependent
manner (Fig. 4). This decrease was reflected in the in vitro
fertilization rate using antiSLIP1-treated zona-intact eggs.
However, egg SLIP1 was less significant in gamete membrane fusion, as revealed by a lack of inhibition in sperm
penetration into eggs pretreated with antiSLIP1. We also
demonstrated that sperm SGG was involved in sperm-egg
plasma membrane binding, since pretreatment of sperm
with antiSGG resulted in a dose-dependent decrease in
sperm binding to the egg plasma membrane (Fig. 5). Since
SLIP1 has been shown to bind to SGG in vitro [16, 17,
20], our results suggested that egg SLIP1 may interact with
sperm SGG during gamete plasma membrane binding.
Since the amount of SLIP1 was estimated to be only
;40 pg/egg, it was impractical to isolate sufficient egg
SLIP1 either for antibody production or for use directly in
the gamete binding/fusion assay. AntiSLIP1, produced
against rat testis SLIP1 [15], was therefore used instead.
Results from both Lingwood’s group and our group have
demonstrated that antiSLIP1 is cross-reactive with an albumin-like component and HSP70 in addition to boar
sperm P68, which is the SGG/SGC- and ZP-binding component of SLIP1 [17, 21]. Our unpublished indirect immunofluorescent results using antiRSA and anti-HSP74.5
[17] indicate the absence of albumin and HSP70 on the
plasma membrane of zona-free mouse eggs. Therefore, the
inhibition of sperm-egg plasma membrane binding of eggs
pretreated with antiSLIP1 Fab should be due to specific
masking of an egg surface antigen that is immunologically
related to boar sperm P68. Nonetheless, we further confirmed this inhibitory result using antiSLIP1 affinity purified against boar sperm P68 (Fig. 4). Therefore, we concluded that the egg SLIP1 component participating in
sperm-egg plasma membrane binding probably has an immunological property similar to that of sperm P68, which
is involved in sperm-ZP binding [21].
Sperm-egg plasma membrane interaction commences
with binding followed by fusion of the plasma membranes.
Results shown in Figure 4, A and B, strongly suggest that
egg SLIP1 is important for the initial step of gamete plasma
membrane interaction. Although sperm penetrated zonafree eggs pretreated with 100 mg/ml antiSLIP1 Fab at the
same level as control zona-free eggs, the possibility that
egg SLIP1 may also be directly involved in gamete plasma
membrane fusion cannot be totally excluded at the present
time. The involvement of egg SLIP1 in gamete plasma
membrane binding is analogous to the postulated participation of sperm SLIP1 in the initial binding of sperm to
the egg ZP [22]. It has also been documented that other
egg proteins such as integrins [6, 22] and egg P95 [12] are
involved in gamete plasma membrane binding. As with
sperm-ZP binding [1, 2], more than one sperm surface protein can be involved in gamete plasma membrane binding,
and, in some cases, the sperm surface proteins can be alternates for one another [2].
Purified SLIP1 and sperm P68 exhibit specific affinity
to SGG in vitro [17, 21]. Since sperm SGG is localized to
the concave and convex ridges of the sperm head and to
the postacrosomal region (the site of sperm-egg plasma
membrane interaction) (unpublished results), and since a
crude SLIP1 AES extract of eggs was shown to bind to
SGG in vitro [16], we investigated the possible role of
EGG SLIP1 FUNCTIONS
sperm SGG in sperm-egg plasma membrane binding. Results shown in Figure 5 supported our postulation. Preexposure of sperm to antiSGG decreased sperm binding to
the egg plasma membrane in a dose-dependent manner. Interestingly, the highest inhibition (76% of the control value)
was achieved at 1:1 molar ratio of affinity-purified antiSGG
Fab (10 mg/ml) to sperm SGG (;3.1 3 10216 mol/sperm
[22]). A great efficiency of antiSGG antibody in inhibiting
gamete plasma membrane binding may plausibly be due to
its dual action. First, it masked sperm SGG from interacting
with SLIP1 on the egg plasma membrane. Second, the antibody may have also modified sperm plasma membrane
fluidity to be less fitting to egg plasma membrane binding.
This postulation was based on our results from a model
membrane study indicating that interaction of SGG with
other phospholipids results in enhancement of fluidity of
both lipids [37]. Our results (Fig. 5) also support the hypothesized role of SGG in cell-cell/extracellular matrix adhesion [38]. SGG has been shown to bind to several adhesive glycoproteins including L/P selectin [39], an adhesive protein involved in the lymphocyte homing event [40].
Significantly, we have recently demonstrated that sperm
SGG in the sperm head convex ridge participates in spermZP binding [24], possibly as a complex with sperm SLIP1,
although it is unclear to which ZP glycoprotein and to
which part (sugar moiety or polypeptide) sperm SGG binds.
Since SGG is absent on the egg plasma membrane (unpublished results), it is likely that egg SLIP1 may bind directly
to sperm SGG. This interaction may be one of the mechanisms of sperm-egg plasma membrane binding, although
it is unlikely that egg SLIP1 has a role in regulating sperm
binding to only the microvilli-containing region of the egg
surface, considering that it was present in both microvillicontaining and microvilli-free regions (Fig. 2, a–d). Nonetheless, the postulated mechanism of egg SLIP1-sperm
SGG interaction is different from that documented between
the disintegrin domain of sperm fertilin and egg integrins
[6, 10, 11, 22]. Further investigation will be required to determine whether egg SLIP1 and egg integrins are involved
in gamete plasma membrane binding at the same stage.
ACKNOWLEDGMENTS
The authors wish to thank Drs. Marie-Claude Léveillé and Manee Rattanachaiyanont for valuable comments on the manuscript.
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