BIOLOGY OF REPRODUCTION 43, 1019-1025 (1990)
Evidence That an Arg-Gly-Asp Adhesion Sequence Plays a Role
in Mammalian Fertilization
R. A. BRONSON' and F. FUSI
Divkion of Reproductive Endocrinology Department of Obstetrics and Gynecology
Health Science Center, State University of New York at Stony Brook
Stony Brook, New York 2 2794-8091
ABSTRACT
The Arg-Gly-Asp (RGD) sequence is known to play a role in many recognition systems involved in cell-to-cell and cell-tomatrix adhesion. In our experiments we demonstrated that an RGD-dependent recognition is involved in sperm-oolemmaladhesion and egg penetration. Following coincubation of RGD-containingoligopeptides in a heterologous system (human sperm and
zona-free hamster eggs), a s i m c a n t decrease in the number of oolemma-adherent sperm was noted at 15 pM RGDV (Arg-Gly.
AspVal) and at 5 pM GRGDTP (Gly-Arg-Gly-Asp-Thr-Pro),
and fertilization was completely inhibited at 250 pM RGDV and 30
pM GRGDTP. In a homologous system (hamster sperm and zona-free hamster eggs), a concentration-dependent decrease in
oolemmal adhesion and egg penetration was also noted, with complete inhibition of fertilization at 200 pM GRGDTP. The speciticity of the receptor was confirmed by the fact that small changes in aminoacid composition impaired the peptide's effectiveness
and that peptide-dependent inhibition of fertilization was partially reversible in competition studies. The presence of a molecule
on the oolemma capable of binding the RGD sequence was demonstrated by using immunobeads coupled with an RGD-containing
hexapeptide (GRGDTP), which rosetted over the egg surface in a manner reversible by the addition of free GRGDTP in the
medium.
INTRODUCTION
The aminoacid sequence Arg-Gly-Asp (RGD) has been
shown to play a widespread role in a variety of cell-to-cell
adhesion-recognition systems and in cell-to-extracellularmatrix interactions [ 1,2].The receptors that recognize RGD
in their ligands are glycoprotein cell membrane heterodimers [3,4] and are classificated as members of the same
supergene family, the Integrins [2,5].
Fibronectin, as well as other extracellular matrix proteins, such as type I collagen and laminin, have been shown
to contain RGD sequences in their cell binding domains,
and the addition of various RGD-containing oligopeptides
inhibits the attachment of a number of cell types to extracellular matrix proteins [6,7].The mammalian integrins defined by the beta-2 subunit [8] play a role in lymphocytemonocyte functions and are structurally related to receptors for fibronectin [3].The complement component C3bi
contains an RGD sequence that interacts with the receptor
MAC-1 (CR?), a leukocyte-associated integrin [9],playing a
role in concert with other cell surface moieties (IgG, Fc
receptors, clq) in phagocytosis of opsonized particles by
macrophages [lo, 111.
Epididymal spermatozoa from rabbits and hamsters are
known to be able to bind C3 and to activate complement
via the alternative pathway [12]. Recently, C3b dimers had
been shown to bind to acrosome-reacted human spermaAccepted July 3, 1990.
Received May 14, 1990.
'Correspondence: Richard A. Bronson, M.D., Department of Obstetrics and Gynecology, Health Science Center, T-9, 060, Stony Brook, NY 11794-8091.
tozoa [13]. In addition, fibronectin has been detected on
the surface of epididymal human spermatozoa and to
undergo alterations in its surface distribution during epididymal transit [14-161. Given these observations, as well
as the widespread role in nature of RGD sequences in cell
interactions, we asked whether this tripeptide might play a
role in sperm-egg interaction leading to fertilization.
In a preliminary experiment, we demonstrated a concentration-dependent decrease in the adhesion of human
spermatozoa to the oolemma of zona-free hamster eggs and
their subsequent penetration in the presence of Arg-GlyAsp-Val (RGDV), an RGD-containing oligopeptide, but not
Gly-Arg-Gly-Glu-Ser (GRGES), a fibronectin-derived peptide that does not contain the adhesion sequence [ 171. This
effect was reversed by washing out the RGD oligopeptide
prior to insemination.
In the present experiments, we compare the ability of
several diEerent oligopeptides to inhibit fertilization in both
a homologous system (zona-free hamster eggs with hamster sperm) and a heterologous one (zona-free hamster eggs
with human sperm), and determine whether this effect is
competitively reversable. Because prior studies have shown
that longer peptides containing the RGD sequence are more
effective in inhibiting the binding of cells to extracellular
matrix proteins [6], and our data confirmed this evidence,
we chose to use an RGD-containing hexapeptide (Gly-ArgGly-Asp-Thr-Pro: GRGDTP) to study its effects on fertilization. In addition, we used immunobeads (Bio-Rad, Richmond, CA) to which GRGDTP was coupled, which acted as
markers to determine whether RGD-binding receptors were
present on the plasma membranes of sperm and/or eggs.
1019
1020
BRONSON AND FUSI
MATERIALS AND METHODS
Fibronectin-derived Peptides
The peptides GRGDTP, Gly-Arg-Gly-Asp-Ser(GRGDS), and
RGDV, which contain the RGD sequence, were purchased
from Peninsula Labs (Belmont, CA),as well as GRGES and
Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), other fibronectin-derived peptides not containing RGD sequence and known to
be inactive in cell-to-cell and cell-to-matrix adhesion [6].
Lyophilized peptides, demonstrated by the manufacturer
to be 99% pure by thin-layer chromatography, were resuspended at 1 mM concentration in Biggers-Whitten-Whittingham medium (BWW) containing 30 mg/mL human
serum albumin (HSA, Sigma Chemical Co., St. Louis, MO,
lot #127F-9320), aliquotted, and stored at -20" until used.
On the day of test, aliquots were thawed at room temperature and preequilibrated at 37°C in 5% COz in air for 2 h.
Preparation of Zona-free Hamster Eggs
Female golden hamsters 4-6 weeks old received injections of 30 IU eCG (G-4877, Sigma Chemical Co.) on the
morning of the estrous discharge, and 30 IU hCG (Sigma
Chemical Co.) 56-60 h later. Fourteen to sixteen hours after hCG injection, hamsters were killed and oviducts containing cumula oophora were placed in BWW containing 5
mg/ml HSA (HSA/BWW, 5 mg/ml). Each cumulus was enzymatically dissolved with 1 mg/ml hyaluronidase (Sigma
Chemical Co.) in HSA/BWW (5 mg/ml), and eggs were
harvested. Zonae pellucidae were removed from the eggs
with 1 mg/ml trypsin (T8003, Sigma Chemical Co.) in HSA/
BWW. The eggs were observed in trypsin for zona dissolution for approximately 1 min and were then washed
through five 200-p.1 changes of HSA/BWW ( 5 mg/ml) as
previously described [ 181. Eggs from different females were
pooled and divided equally among treatment groups.
Preparation of Human Spermatozoa
Spermatozoa from four healthy donors whose specimens were normal according to World Health Organization
criteria [ 191, were recovered following a 60-90-min swimup. Semen was allowed to liquify at room temperature for
at least 30 min, and 300-pl aliquots were injected under 2
ml HSA/BWW, 5 mg/ml, in 15-mi loosely capped conical
tubes (Falcon, Becton-Dickinson, Lincoln Park, NJ, #2099).
After a 60-90-min incubation at 37" in 5% CO, in air, the
upper 1-1.5 ml of medium was collected and spermatozoa
were washed by centrifugation for 8 min at 600 X g. Thereafter spermatozoa were resuspended at 20 million/ml in
HSA/BWW (30 mg/ml), divided into 250-pl aliquots, and
incubated overnight at 37°C in 5% CO,.
Coincubation of Hamster Eggs with Human Spermatozoa
Zona-free hamster eggs preincubated for 30 min in 100
kl HSA/BWW (30 mg/ml) containing fibronectin-derived
peptides at different concentrations or in HSA/BWW (30
mg/ml) alone (control) were inseminated with 50 pl capacitated human sperm suspension containing the same
peptide concentration in 60-mm dishes (Falcon, BectonDickinson, #3002). Gametes were coincubated for 3 h at
37°C in 5% COz before evaluation of oolemmal-adherent
and penetrated sperm.
To measure sperm binding to the egg plasma membrane, a modification of the procedure of Wolf and Hamada
[20] was used: Eggs were washed free from unbound and
loosely bound sperm by serial passage through at least 3
microdrops (75 pl) HSA/BWW (30 mg/ml). The same person pipened all the eggs in each treatment group for all
experiments performed in this study, and the same micropipette, of approximately 100-120-pm diameter, was used
for all eggs washed in an individual experiment. Eggs were
then stained by short-term (5-15 s) exposure to 1 mM acridine orange-3% dimethyl sulfoxide (DMSO) in BSA/BWW
(30 mg/ml), and washed and prepared as whole mounts
under 22 x 22-mm coverslips. Under W illumination, the
unexpanded heads of oolemma-adherent spermatozoa appeared bright green or orange against the dark field and
could be counted at 400X. Spermatozoa that had penetrated into the ooplasm exhibited expanded green heads,
associated with early nuclear decondensation, against the
orange background of the ooplasm.
Preparation of Gametesfor Zona-free Hamster Egg
Penetration by Hamster Spemzatozoa
The procedure to obtain hamster eggs was the same as
for the test with human sperm, except that a modified Tyrode solution was used instead of BWW. Modified Tyrode
(AA-Tyrode) was prepared containing the following components (for 100 ml): sodium chloride, 666 mg; potassium
chloride, 23 mg, sodium phosphate monobasic, 5 mg; glucose, 90 mg; magnesium chloride, 10 mg; sodium pyruvate,
5.5 mg; calcium chloride, 29 mg; sodium bicarbonate, 210
mg; sodium lactate 60% syrup, 140 pl; DL-isoleucine,3 mg;
L-glutamine, 15 mg; L-methionine, 1 mg; L-phenylalanine, 2
mg; penicillin, 8 mg (all the reagents were from Sigma
Chemical Co.). BSA (ICN Immunobiologicals, Lisle, IL) was
added to a final concentration of 3 mg/ml and medium was
filtered and equilibrated overnight the day before use at
37°C in 5% COz.
Medium for hamster sperm preparation was m-Talp [21],
100 ml of which contains: sodium chloride, 586 mg; potassium chloride, 20 mg; magnesium chloride, 10 mg; sodium
phosphate, 4 mg; glucose, 77 mg; sodium pyruvate, 1 mg;
penicillin G, 8 mg; calcium chloride, 26 mg; sodium lactate
syrup, 130 p1; and BSA, 300 mg. The day of the test, the
m-Talp, previously filtered and equilibrated, was prepared
by adding 15 mg/ml BSA, 5 X 10 -* M taurine (Sigma
Chemical Co.) and 5 x
M epinephrine (Sigma Chemical Co.) (Hi-Talp). Male golden hamsters were killed and
caudae epididymides were dissected and placed in 3 3.5-
1021
RGD AND FERTILIZATION
cm dish (Falcon, Becton-Diclunson, #3001) whose bottom
part was filled with Hi-Talp. Spermatozoa were expressed
from the epididymis with needles and the medium was collected. After a 30-min incubation, spermatozoa were counted,
diluted to 1 X 106/ml in Hi-Talp and incubated for 5 h at
37°C in 5% C 0 2 .
After hamster sperm capacitation, 50 p1 sperm suspension was added to a 100-p1 drop of AA-Tyrode containing
zona-free hamster eggs, and gametes were incubated in the
presence or absence of various amounts of peptides at 37°C
in 5% COz for 3 h. Thereafter, eggs were processed as previously described for the heterologous human-hamster system.
GRGDTP Coupling to Imrnunobeads
Lyophilized GRGDTP was coupled to activated immunobeads using a modification of the Bio-Rad protocol. One
hundred micrograms GRGDTP was resuspended in 250 p1
coupling buffer (0.003 M phosphate) at a pH equal to the
isoelectric point of the protein (pH 7). Two and one-half
micrograms activated immunobead matrix (Bio-Rad) was
reconstituted in the same buffer, centrifuged, and resuspended in the GRGDTP solution by mixing gently. After a
1-h incubation at 4"C, 0.5 mg EDAC (l-ethyl-3-[3 dimetylamino-propyl] carbodiimide HCl; Bio-Rad) was added and
the suspension was incubated overnight at 4°C. Thereafter,
the beads were washed (5 min at 1000 X g) with PBS,
followed by two washings with 1.4 M NaCl/PBS and two
additional washings with PBS. The beads were then resuspended in PBS containing 5 mg/ml BSA.
GRGDTP-coupled Beads Binding to Zona-pee
Hamter Eggs
Zona-free hamster eggs, prepared as previously described, were placed in a 50-pl drop of bead suspension
in a 60-mm dish (Falcon, Becton-Dickinson). As a control,
immunobead matrix resuspended at the same concentration was used. After a 20-min incubation at 37°C in 5% COa,
eggs were washed through five 75-p1 drops of HSA/BWW
(30 mg/ml) and immunobead binding was measured by
observation with an inverted microscope (Diaphot, Nikon,
Garden City, M I ) under phase-contrast illumination at 400X.
A competition experiment between bead-coupled and free
GRGDTP was performed on hamster eggs by preincubating
eggs in 30-pl drops of HSA/BWW (30 mg/ml) containing
several GRGDTP concentrations (1-400 pM) or in BWW
control for 30 min and thereafter adding 50 pl beads. After
20 min further incubation, eggs were processed as described above and immunobead binding was evaluated.
GRGDTP-coupledBeads Binding to Human Spermatozoa
Human spermatozoa capacitated overnight as previously
described were challenged with GRGDTP-coated beads before or after exposure to progesterone to induce an acro-
some reaction [22]. Lyophilized progesterone (Sigma
Chemical Co.), resuspended in HSA/BWW (30 mg/ml) to
obtain a 100 pg/ml concentration, was added to spermatozoa to reach a concentration of 5 pg/ml. After a 20-min
incubation, sperm were washed and checked for bead
binding and acrosome reaction. Acrosome-reacted sperm
were detected by means of Pisum sativum agglutinin (PSA)fluorescein isothiocyanate (FITC) staining [231, as described
previously in detail [24]. Briefly, dead spermatozoa were
excluded by staining 90 pl of 20 million sperm/ml in HSA/
BWW (30 mg/ml) with 25 pl Hoechst H258 (Bisbenzimide,
Sigma Chemical Co.), a supravital stain that binds DNA of
dead sperm, in PBS (10 pg/ml). After 5 min incubation to
allow staining, sperm were washed free of excess of Hoechst
H258 by means of centrifugation (10 min at 900 X g ) through
250 pl of 2% polyvinylpyrrolidone (PW, Sigma Chemical
Co.) in PBS. The pellet was resuspended in 100 p1 of 95%
ethanol in PBS and placed at 4°C for 30 min. Thereafter, a
drop of sperm suspension was placed over each slide,
ethanol was allowed to evaporate, and slides were covered
with 20 p1 of 100 pg/ml PSA-FITC (Vector Labs, Burlingame, CA, no FJA051) and incubated in a humid chamber
in the dark for 5 min. The slides were then rinsed, mounted,
and scored by classlfying live sperm (excluding H258 stain)
as acrosome-intact (acrosome well fluorescent) or acrosome-reacted (no fluorescent staining or equatorial band).
To check the ability of GRGDTP-coated beads to bind to
capacitated or acrosome-reacted spermatozoa, 5 p1 sperm
suspension were mixed with 50 pl bead suspension and
incubated for 5-10 min at room temperature (37°C) or for
1 h. Thereafter slides were prepared and immunobead
binding to the sperm surface was scored.
Statiwical Analysis
Data obtained in the coincubation experiments were
analyzed by Student's t-test for unpaired samples.
RESULTS
A total of eleven experiments in which human sperm
from four dfierent semen donors were incubated with zonafree hamster eggs in the presence of fibronectin-derived
peptides were conducted. Five oligopeptides were initially
screened at 50-pM concentration for their ability to inhibit
fertilization. As seen in Table 1, GRGES was without effect,
whereas RGDV, GRGDS, and GRGDTP were inhibitory. The
fibronectin-derived peptide GRGESP, not containing RGD,
did not significantly inhibit penetration ( p > 0.05, Student's
t-test), but decreased sperm oolemmal adherence. The RGDcontaining peptides were all inhibitory compared both to
peptide-free control and GRGES, with the greatest effect exhibited by GRGDTP, which was chosen for further study.
When spermatozoa and eggs were mixed in the presence of GRGDTP or RGDV, a concentration-dependent decrease in the number of human spermatozoa adherent to
1022
BRONSON AND FUSI
TABLE 1. Effects of fibronectin-derived peptides on zona-free hamster egg penetration
and oolemmal adherence by human spermatozoa.
Number
of
eggs
Percent egg
penetration
Penetration
index
(?SD)
Control
GRGES'
13
13
77
85
1.07
1.07
GRGESP
13
54
RGDV
14
43
G W S
14
28
G K T P
14
7
Treatment
Mean adherent sperm
(+SD)
*
29.4
3.8
29.6 f 4.8
(NS)
18.0 f 7.3
( p < 0.001)
12.5 & 4.1
( p < 0.001)
12.0 + 6.4
( p < 0.001)
6.9
2.9
( p < 0.001)
(NSb)
0.57
(NS)
0.49
( p < 0.05)
0.28
( p < 0.003)
0.07
( p < 0.001)
*
'Peptide concentration was 50 p M in all dishes.
bStudent's t-test, compared to control.
the oolemma of zona-free hamster eggs was noted. These
findings were associated with diminished penetration of
sperm into the eggs, as judged both by percentage of eggs
penetrated as well as the number of penetrating sperm per
egg, when compared with peptide-free control (Fig. 1).The
effects were more evident for GRGDTP than for RGDV:
complete inhibition of penetration was observed at a 30p M concentration of GRGDTP and at 250-pM RGDV, with
a statistically significant effect compared to the peptide control at 1-pM GRGDTP and at 30-pM RGDV. The number of
oolemma-adherent spermatozoa was significantly reduced
at 5-pM GRGDTP (24.7 k 6.1 compared with 36.5 ? 5.3
control), and was 9.2 k 4.8 at 30-pM GRGDTP. For RGDV,
significant diminution was first observed at 15-pM concentration. In all groups, no difference was observed in the
percentage of motile spermatozoa or in the quality of sperm
motion.
The peptide GRGES had a limited effect, impairing both
oolemmal adhesion and penetration at 60-pM concentration compared to the peptide-free control, but plateauing
without exhibiting a complete inhibition of penetration. The
effect of GRGES was significantly less than that of the RGDE f f e c t s o f f ibronectin-derived peptides
on s p e r m - 0 0 1 e m m a 1 adhes 1on
E f f e c t s of fibronectin-derived peptides
on sperm-egg p e n e t r a t i o n
1 2 ,
GRGES
.
12
GRGDTP
10
RGDV
08
06
04
02
00
O75uM
I UM
5uM
I5uM
30uM
60ut-l
-
0 7 5 uM
125uM 250uM
5uM
15 uM
30uM
60uM
Pept Ide c o n c e n t r a t i o n s
Pept Ide c o n c e n t r a t Ions
a, Control c o n s i s t e d of gamete Incubatlon
b, pc 0.01. Student t t e s t
C, pc 0.03
I UM
In
t h e absence o f peptides
d. pc 0.005
e, pc 0.001
FIG. 1. Concentration-dependent effects of fibronectin-derived peptides on zona-free hamster egg penetration by human spermatozoa.
125 UM 250uM
RGD AND FERTILIZATION
containing peptides, at each concentration at which inhibition of adhesion or penetration was noted.
The addition of GRGES to medium containing 50-pM
GRGDTP partially reversed the inhibitory effects of the latter peptide on sperm/egg adhesion and penetration in a
concentration-dependent manner (Table 2), with a significant recovery first noted at 30-pM GRGES ( p < 0.05 for
penetration and p < 0.001 for adherence, Student's t-test)
and a plateau over 65-pM concentration. N o complete reversibility was observed.
The effect of GRGES and GRGDTP, observed in experiments repeated twice, on the homologous, hamster/hamster system is shown in Table 3. At each peptide concentration, from 2 p M to 200 pM, there was a statistically
significant difference between GRGES and GRGDTP in their
effects on adhesion and penetration. At 2-pM concentration, the significance w a s p < 0.05 for penetration, with a
penetration index of 0.38 for GRGDTP and 0.77 for GRGES.
At the same concentration, a significant difference was noted
in the number of oolemma-adherent sperm (19.1
7.3
compared to 37.2 f 5 . 1 , <
~ O.OOOl), which increased at
higher concentrations.
1023
Immunobeads to which GRGDTP were coupled bound
to the oolemma of zona-free hamster eggs, while immunobeads free of oligopeptides failed to bind (Fig. 2, Table
4). In addition, the oolemmal binding of GRGDTP-coated
*
FIG. 2. Binding of GRGDTP-coupled immunobeads to zona-free hamster
eggs ( ~ 2 7 5 0 ) .
TABLE 2. GRGES-GRGDTP competition: effects of zona-free hamster eggs, oolemmal adhesion, and
oocyte penetration by human spermatozoa.
Penetration rate
Peptide concentration
Peptide-free control
G E T P (50 pM)
G E T P (50 pM) + GRGES (5 pM)
G E T P (50 pM)
+ GRGES (30 pM)
G E T P (50 +M)
+ GRGES (65 pM)
G E T P (50 pM) + GRGES (125 FM)
Mean adherent sperm
Penetration
index
Percent
penetration
69.6 f 20.7
11.7 f 8.5'
25.1 f 12.2
(NSb)
34.5 f 19.5
( p < 0.001)
35.8 f 8.9
( p < 0.001 )
37.1 f 10.5
( I ) < 0.001)
1.07
0.17'
0.17
(NSb)
0.23
(NS)
0.54
( p < 0.05)
0.43
( 1 ) < 0.05)
77
16
16
23
39
30
' p < 0,001 (Student's f-test) between values obtained in presence of GRGDTP and in peptide-free control.
bNS = Not significant. Student's f-test versus values obtained in presence of GRGDTP.
TABLE 3. Effects of RGD-containing peptides on hamster sperm/hamster eggs
coincubation.
Number
of eggs
Percent egg
penetration
Control
GRGES ( 2 pM)
14
13
86
69
GRGES (75 pM)
13
54
GRGES (200 pM)
13
46
G E T P (2 pM)
13
38
G-TP
13
7
13
0
Treatment
(75 pM)
G E T P (200 pM)
"Student's t-test, compared to control.
Penetration
index
1.07
0.77
(NS')
0.61
(NS)
0.46
( p < 0.01)
0.38
( p < 0.004)
0.07
( p < 0.0001)
0
p < 0.0000
Mean adherent
sperm
37.7 f 6.2
37.2 f 5.1
(NS)
31.6 f 5.3
(NS)
26.4 5 5.2
( p < 0.005)
19.1
7.3
( p < 0.001 )
3.8
4.6
( p < 0.0001)
1.8 k 1.9
( p < 0.0000)
*
*
BRONSON AND FUSI
1024
GRGDTP-coupled immunobead binding to zona-free hamster egg oolemma.
TABLE 4.
Bead category
Number
of eggs
Oolemmal binding
of immunobeads'
BWW control
uncoupled bead matrix
10
-
BWW control
G E T P - c o u p l e d beads
32
Medium for egg incubation
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G
G
G
G
g
S
F
g
T
T
T
T
P
P
P
P
(1 p M in BWW)
(100 pM in BWW)
(200KM in BWW)
(400 pM in BWW)
+/++
-
10
10
+
= no binding,
= binding of 1 to 15 beads to the oolemma,
beads to the oolemma.
8-
++
++
10
20
++
=
binding of more than 15
TABLE 5. GRGDTP-coupled immunobead binding to human spermatozoa.
Percent live
sperm
Percent acrosome-reacted
(PSA staining)
Sperm capacitated
overnight (18 h)
90
8
Sperm capacitated
overnight (18 h)
and exposed to
5 pg/mL progesterone
84
41
Treatment
immunobeads was reversed by the addition of GKGDTP to
the medium, confirming the specificity of their binding.
GRGDTP-coated beads bound only occasionally to the
heads of spermatozoa (Table 5 ) , irrespective of whether
sperm were capacitated overnight and exposed to progesterone to induce an acrosome reaction, an occurrence confirmed by PSA lectin staining.
DISCUSSION
Our results suggest that the RGD sequence is involved
in a specific recognition mechanism in sperm oolemmal
adhesion and egg penetration. The ability of RGD-containing peptides to inhibit adhesion and penetration in both
the human/hamster and the hamster/hamster system, and
the fact that small changes in amino acid composition impaired the peptide's effectiveness (GRGES contains two of
the three amino acids in sequence in RGD) suggest the
presence of at least one specific receptor that allows gamete interaction and acts through a well-defined signaling
sequence. The fact that RGD-containing peptides affected
fertilization when present at low concentration in incubation medium and the partial reversibility obtained by competition with other fibroneain-derived peptides suggest that
the recognition via RGD is part of a physiologic event. When
the receptors were occupied by proteins containing their
ligand sequence, sperm-egg interaction was dramatically
decreased.
The finding that other fibronectin-derived peptides, not
containing the RGD sequence, had an effect on fertilization
at high concentration and were able to partially reverse the
effects of RGD-containing peptides, might signify that other
portions of receptor, functionally inactive but situated near
GRGDTP-coupled bead binding
Head
(YO)
Midpiece
Tail
Unbound
9
0
0
91
12
0
0
88
the RGD-recognizing sequence, interact with such oligopeptides. Very high peptide concentration could sterically
impede the RGD recognition even if the recognizing sequence were not occupied, and GRGES might partially
compete with GRGDTP for the binding to close epitopes.
The specific recognition of RGD-containing peptides by
receptors present on the oolemmal surface was confirmed
by the ability of GRGDTP-coated beads to bind to the oolemma of hamster eggs and by the competition for receptor
between GRGDTP coupled to the immunobeads and
GRGDTP free in the medium.
Although oolemmal binding in the mouse can be impaired by trypsinization of zona-free mouse eggs prior to
their insemination, suggesting the presence of specific oolemma1 glycoprotein receptors that play a role in fertilization [ 2 5 ] ,fertilization of zona-free hamster eggs is relatively
insensitive to protease exposure [ 261. While these results
call into doubt the presence of specific oolemmal sperm
receptors, our current findings now suggest that RGD recognition molecules exist on the egg plasma membrane.
The low binding of GRGDTP-coupled beads to spermatozua might be explained in different ways. There may
be no RGD receptors on sperm, but only on eggs. It is also
possible that the receptors are present on spermatozoa at
a lower density than on eggs, and the sensitivity of beads
might not be high enough to detect them. Alternatively,only
a limited proportion of spermatozoa may present the receptors and be able to interact with eggs.
These experiments and others suggest that homologies
may occur between sperm/egg interaction and leukocyte
phagocyosis. Active sperm motion is not necessary for sperm
incorporation by the oolemma, the egg playing an active
RGD AND FERTILIZATION
role [ 27,281. Polymorphonuclear leukocytes, lymphocytes
and macrophages recognize their targets in part by means
of integrins, which show a complex interaction modulated
by MHC, Fc receptors, fibronectin, C3b, and C l q [lo, 111.
Since hamster eggs [29] and human eggs [Bronson and Fusi,
unpublished observations] possess both Fc receptors and
C3b receptors [13], and since fibronectin [14-161 and C3
dimeric fragments are present on human sperm, it is possible that sperm incorporation uses receptor mechanisms
similar to those involved in phagocytic events.
12
13
14
15
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16
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