* Molecular and Cellular Biology and Department of Cell Biology and Human Anatomy, School of Medicine, University of
California, Davis, California 95616; and § Cell Biology Program, University of Connecticut Health Center, Farmington,
Connecticut 06030
Sperm-egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane.
Cell-cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm
surface protein with a role in sperm-egg plasma
membrane adhesion is fertilin, a transmembrane heterodimer ( and
subunits). Fertilin
and
are the
first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin
functions in sperm-egg adhesion leading to fusion. Full length cDNA clones have been isolated for
five ADAMs expressed in mouse testis: fertilin
, fertilin
, cyritestin, ADAM 4, and ADAM 5. The presence
of the disintegrin domain, a known integrin ligand, suggests that like fertilin
, other testis ADAMs could be
involved in sperm adhesion to the egg membrane. We
tested peptide mimetics from the predicted binding
sites in the disintegrin domains of the five testis-expressed
ADAMs in a sperm-egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin
strongly inhibited (80-90%) sperm adhesion and fusion
and was a more potent inhibitor than the fertilin
active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin
also
strongly inhibited (80-90%) both sperm-egg adhesion
and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted
sperm using antibodies against either cyritestin or fertilin
showed staining of the equatorial region, a region of the sperm membrane that participates in the early
steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin,
functions with fertilin
in sperm-egg plasma membrane adhesion leading to fusion.
The interaction of sperm with the egg culminating in
sperm-egg membrane fusion, is a multi-step process in mammals. After penetrating the cumulus cell
layer and the zona pellucida, a sperm adheres to the egg
plasma membrane and fuses. Only acrosome-reacted sperm
can fuse with the egg. During the acrosome reaction, the outer acrosomal membrane fuses at multiple points with
the anterior head sperm plasma membrane, and the fused
membranes are released along with the soluble acrosomal
contents. The inner acrosomal membrane that is incorporated into the sperm plasma membrane during the acrosome reaction remains a distinct membrane domain. The region of the sperm membrane that makes the initial contact with the egg plasma membrane is the inner acrosomal
membrane (IAM),1 followed by the equatorial/posterior
head region of the membrane (Shalgi and Phillips, 1980 The molecular mechanisms of these interactions between the sperm and egg plasma membranes are not well
understood. Our initial studies of sperm-egg fusion in
guinea pigs indicated that fertilin, a heterodimeric ( The evidence indicating participation of fertilin Fertilin Five members of the ADAM protein family, all of which
are expressed in testis, have been sequenced in mouse (Heinlein et al., 1994 Synthesis of Predicted Active Site Peptides from the
Disintegrin Domain of ADAM Proteins
Peptides with sequences from the predicted active sites of the disintegrin
domains of ADAM proteins were synthesized by the W.M. Keck Biotechnology Resource Center (Yale University, New Haven, CT). The sequences
of the peptides were chosen by sequence alignment with the known active
sites of snake disintegrins (RGD) and guinea pig fertilin
Preparations of Antibodies against Fertilin Antibodies to the cytoplasmic tail domains of fertilin Table I.
Antibodies to Fertilin ;
Talbot and Chacon, 1980; Koehler et al., 1982
; Yanagimachi, 1994
). The fusion begins with the sperm head plasma
membrane, but only the equatorial/posterior head region actually fuses with the egg plasma membrane. The IAM
region is incorporated into the egg cytoplasm by a phagocytosis-like process. The sperm tail also eventually fuses
with the egg plasma membrane and contributes to the zygote membrane in most mammals (Yanagimachi, 1994
).
and
subunits) sperm membrane protein, is involved in the fusion process. This protein is located on the posterior head
of acrosome-reacted guinea pig sperm. Both a monoclonal antibody to fertilin
(Primakoff et al., 1987
) and peptide
mimetics of a predicted binding site in the disintegrin domain of fertilin
(Myles et al., 1994
) inhibited sperm-egg
fusion. Similar studies using fertilin
active site peptides
to inhibit sperm-egg fusion in mouse have been reported
(Almeida et al., 1995
; Evans et al., 1995
). Thus a role for
fertilin
in sperm adhesion to the egg plasma membrane
is well supported.
in the
process of sperm-egg fusion does not provide information
as to whether other sperm surface molecules may also
function in this process. In the adhesion process between
other cell types, multiple receptors and counter receptors
are active. For example, in neutrophil adhesion to endothelial cells, there are five pairs of adhesion partners that
participate. From the selectin family, L-selectin (on the
neutrophil) and E- and P-selectin (on the endothelial cell)
bind to carbohydrate ligands on the adhering cell. After these interactions, the integrins
L
2 and
M
2 on the
neutrophil bind to adhesion partners, ICAM-1 and/or
ICAM-2 on the endothelial cell (for review see Springer,
1994
).
and
are the first identified members of a
new family of membrane proteins, the ADAM family (a
disintegrin and metalloprotease). These proteins share the
same multidomain structure, including pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane
and cytoplasmic domains (Wolfsberg et al., 1995
). At least
17 full length ADAM cDNAs have been cloned and sequenced from mammals (Yagami-Hiromasa et al., 1995
;
Wolfsberg et al., 1995
; Gupta et al., 1996
; Weskamp et al.,
1996
), and ADAM sequences have also been found in
other species including Xenopus laevis (Blobel, C., personal communication), Drosophila melanogaster (Rooke et
al., 1996
), and Caenorhabditis elegans (Podbilewicz, 1996
).
This family of proteins has sequence homology with soluble snake peptides and proteins that contain one or more
of the above first four domains (Blobel et al., 1992
). Disintegrin peptides from snake venom are known to bind to
platelet integrin
IIb
3 (GP IIb/IIIa) and prevent binding
of fibrinogen and subsequent clotting (Adler et al., 1991
;
Beer et al., 1992
). By analogy, it is likely that fertilin
binds
through its disintegrin domain to an egg integrin. Several
different integrins have been identified on mammalian
eggs, and at least one of them (
6
1) appears to participate
in sperm adhesion to the egg membrane (Almeida et al.,
1995
).
; Wolfsberg et al., 1995
) and mapped to distinct chromosomes (Lemaire et al., 1994
; Cho et al., 1996
):
fertilin
, fertilin
, cyritestin, ADAM 4, and ADAM 5. Fertilin
, ADAM 4, and ADAM 5 are expressed in testis
and other tissues, whereas fertilin
and cyritestin (cyritestin sequence data are available from Genbank/EMBL/
DDBJ under accession number X64227) are testis specific (Heinlein et al., 1994
; Wolfsberg et al., 1995
). Because each of these proteins has a potential integrin ligand site in the disintegrin domain, we characterized and studied their
functions in sperm-egg adhesion and fusion. Our data suggest that in addition to fertilin
, a second ADAM family
member, cyritestin, is also involved in sperm-egg plasma
membrane adhesion and fusion.
Materials and Methods
(TDE; Fig. 1).
The peptides synthesized were each linear, eight residue peptides from
mouse fertilin
, fertilin
, cyritestin, ADAM 4, and ADAM 5. Their sequences are underlined in Fig. 1. All of the peptides were purified by
HPLC, and their sequences were confirmed by mass spectroscopy.
Fig. 1.
Sequence alignment of disintegrin domain
active sites of snake disintegrins and guinea pig fertilin with mouse ADAM family members. The sequences
shown are the 13 amino acids
that form the RGD-containing loop of two snake venom disintegrins, kistrin and bitistatin,
and the corresponding 14 amino acids of guinea pig fertilin
and
mouse ADAM family proteins. Italicized residues are those
which align with RGD, and the underlined sequences are the
peptide sequences tested in the adhesion and fusion assay.
[View Larger Version of this Image (26K GIF file)]
and Cyritestin
and cyritestin were
generated by synthesizing peptides containing the COOH-terminal 15 amino acids of fertilin
and cyritestin. The peptides, conjugated to Diphtheria toxin, were used as antigens for the immunization of rabbits by Chiron Mimotopes Peptide Systems (Victoria, Australia). Sequences of the
peptides are: fertilin
, FSSEEQFESESESKD, and cyritestin, PYYRSIPEDGNDSQQ. The corresponding antisera are termed m
-CT1 (mouse
fertilin
COOH terminus 1) and mCyri-CT1 (mouse cyritestin COOH
terminus 1; Table I). m
-CT1 and mCyri-CT1 antibodies were affinity purified using the corresponding peptide and a ProtOnTM Kit 1 (Chiron Mimotopes Peptide Systems, Raleigh, NC) following the producer's protocols
and application guide. A second antibody to the cytoplasmic tail domain
of cyritestin was generated using a peptide containing cyritestin residues
787-800: GNTDQNFMTVPGSF. A multiple antigenic peptide (MAP) with
this sequence was prepared by Research Genetics (Huntsville, AL) and
used to immunize rabbits. The resultant antiserum is termed mCyri-CT2.
mCyri-CT2 antibodies were affinity purified as described.
and Cyritestin
To produce antibodies to the active site region of fertilin and cyritestin, chimeric peptides containing a T cell epitope and a B cell epitope were
used. The chimeric peptide sequences were: fertilin
, PSDKHIEQYLKKAKGEVCRLAQDEADVTEYCNGTSE, and cyritestin, PSDKHIEQYLKKARGRLCRKSKDQADFPEFCNGETE. In these peptides, the
NH2-terminal sequence PSDKHIEQYLKK is a T cell epitope from the
malaria circumsporozoite protein which elicits a strong T cell response
and thus obviates the need for conjugating the peptide to a carrier protein
(Good et al., 1987
). The T cell epitope is followed by a single A as a spacer
and then a COOH-terminal sequence (23 residues) from the disintegrin
domain active site region, for fertilin
, KGEVCRLAQDEADVTEYCNGTSE, and for cyritestin, RGRLCRKSKDQADFPEFCNGETE (Heinlein et al., 1994
; Wolfsberg et al., 1995
). The chimeric peptides were synthesized and cyclized using the two cysteines in the disintegrin domain by
the W.M. Keck Biotechnology Resource Center. The residue after the
QDE in fertilin
and the KDQ in cyritestin is also a cysteine in the wildtype sequence but was substituted with alanine (A) in these peptides to
prevent the formation of a disulfide bond with one of the other cysteines.
The cyclized peptides were purified by HPLC, and their sequences were
confirmed by mass spectroscopy. B10.A(4R) mice (The Jackson ImmunoResearch Laboratory, Inc., Bar Harbor, ME) were immunized on day 0 intraperitoneally with 100 µg of chimeric peptide emulsified in Complete
Freund's Adjuvant (Sigma Chemical Co., St. Louis, MO) followed by two
additional intraperitoneal injections (56 d and 89 d) with 100 µg chimeric peptide emulsified in Incomplete Freund's Adjuvant (Sigma Chemical Co). Serum was collected by tail bleeds from the mice 2 wk after the third
injection. These antisera to the active site regions of fertilin
and cyritestin are termed m
-AS1 and mCyri-AS1.
Sperm and Egg Isolation for the In Vitro Adhesion and Fusion Assay
Sperm for the in vitro adhesion and fusion assay were isolated from the
cauda epididymis and vas deferens of 10-12-wk-old male ICR mice (Harlan Sprague Dawley, Inc., Indianapolis, IN). Dissected cauda and vas were
diced with scissors, and the sperm were released into Whittingham's medium (Whittingham, 1971) containing 3% BSA. Released sperm were incubated at 37°C, 5% CO2, for 3-4 h in the same medium for capacitation
and acrosome reaction. This procedure resulted in a population of 60-
70% acrosome-reacted sperm (acrosomal status was determined by Coomassie blue staining; Moller et al., 1990
).
Eggs were collected from the oviducts of 6-8-week-old superovulated
female ICR mice (Harlan Sprague Dawley, Inc.). Mice were superovulated by the injection of 10 IU of pregnant mare's serum gonadotropin
(PMSG; Sigma Chemical Co.) followed 48 h later by an injection of 5 IU
of human chorionic gonadotropin (hCG; Sigma Chemical Co.). About 12 h
after hCG injection, mice were killed and their oviducts removed and put
in prewarmed Whittingham's medium with 0.3% BSA. Cumulus-egg
complexes were collected and transferred to 500-µl drops of Whittingham's
medium containing 300 µg/ml Type I-S hyaluronidase (Sigma Chemical
Co.) under mineral oil. After a 3-5 min incubation at 37°C, 5% CO2, cumulus-free metaphase II eggs (eggs with one polar body) were collected,
transferred to a 100-µl drop of medium, and then washed through two
more 100-µl drops. Zonae pellucidae of metaphase II eggs were removed
by gently passing through a narrow bore pipette after treating with 10 µg/ ml chymotrypsin (Sigma Chemical Co.) for 2-4 min. Zona-free eggs were
washed three times with medium, and then preloaded with 4,6-diamidino-2-phenylindole (DAPI) dihydrochloride (Polysciences, Inc., Warrington, PA) at 10 µg/ml for 15 min at 37°C, 5% CO2.
In Vitro Adhesion and Fusion Assay
To test the peptides' effects on sperm-egg adhesion and fusion, peptides
were added to eggs at varying concentrations 30 min before addition of
sperm and were present throughout the sperm-egg incubation. Capacitated and acrosome-reacted sperm were added to eggs in 500 µl Whittingham's medium with 3% BSA under mineral oil to produce a final concentration of 2-4 × 104 sperm/ml. After 30 min incubations at 37°C, 5% CO2,
eggs were gently washed through three 100-µl drops of Whittingham's
medium with 3% BSA, and then the eggs were mounted onto microscope
slides (Corning Glass Works, Corning, NY). Sperm binding was scored
under the light microscope with 20× magnification. Fusion was scored by
the fluorescent labeling of sperm nuclei by DAPI present in the preloaded
eggs (Kline and Kline, 1992). Two measures of fusion were used: fertilization index (FI, mean number of fused sperm per egg) and fertilization rate
(FR, percentage of eggs fused with at least one sperm).
To test the effects of anti-peptide antibodies on sperm-egg adhesion and fusion, antisera (1:50 final dilution) were added to capacitated and acrosome-reacted sperm and incubated for 30 min at 37°C, 5%CO2. Zonafree eggs were added to the preincubated sperm. Subsequent steps were the same as above.
Immunoblot Analysis
To study the molecular forms of fertilin and cyritestin on spermatogenic
cells or sperm at different developmental stages, three populations of cells
were analyzed. The first was a pool of all testicular spermatogenetic cells,
referred to as "testicular cells" (Blobel et al., 1990
). The second population consisted of isolated testicular sperm, the most fully developed cells
in the testis (Blobel et al., 1990
). These two populations were isolated through 52% Percoll (Sigma Chemical Co.) gradients (Phelps et al., 1990
).
The third population was "epididymal sperm," which are sperm gently expressed from the cauda epididymis and vas (Phelps et al., 1990
). These
three populations of cells were collected from 10-12-wk-old male ICR
mice, washed twice with PBS, resuspended in 1× SDS sample buffer,
heated at 100°C for 4 min, and pelleted in a microcentrifuge at 14,000 rpm
for 10 min. The supernatants were used for electrophoresis.
SDS-PAGE was conducted on 10% resolving gels with 5% stacking gels. About 106 cells were loaded in each lane. After electrophoresis, proteins were transferred to nitrocellulose membranes (0.2 µm; Bio-Rad Laboratories, Richmond, CA), which were then blocked with 5% nonfat dry milk in TBS. Primary anti-peptide antibodies, followed by alkaline phosphatase-conjugated secondary antibodies (Promega Biotech, Madison, WI) were added in TBS containing 3% BSA and 10% FBS. Alkaline phosphatase activity was detected by color developed with 5-bromo, 5-chloro indolyl phosphate, and nitroblue tetrazolium (BCIP/NBT; Sigma Chemical Co.).
Sperm Immunofluorescence
Epididymal sperm were collected as described above in Whittingham's
medium containing 3% BSA. After 15-min incubations at 37°C, 5% CO2,
sperm were washed with PBS three times. Both acrosome-intact and
acrosome-reacted sperm were used for immunofluorescence. About 5-10%
of sperm in the acrosome-intact populations had acrosome reacted. The
acrosome reaction was induced as described or by treatment with 10 µM
A23187 (Sigma Chemical Co.) for 15 min. Preimmune serum, m-AS1 or
mCyri-AS1, the antiserum to the active site region of fertilin
or cyritestin, at 1:50 final dilution, was mixed with a 50-µl sperm sample (107/ml) in
PBS. After 30 min incubation at room temperature, sperm samples were
layered onto 0.5 ml of PBS with 3% BSA and pelleted gently. A Fab fragment of goat anti-mouse IgG conjugated with rhodamine (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was added to the sperm
at 1:75 dilution in PBS and incubated for 30 min at room temperature in
the dark. Sperm were washed through PBS with 3% BSA as described.
Before observation, the sperm were fixed with 1.5% paraformaldehyde for
10 min and then washed once with PBS. Fluorescence with prefixed sperm
was done as described by Linder et al. (1995)
. Images were acquired with a scanning confocal microscope (LSM410; Carl Zeiss, Inc.,Thornwood, NY) with 100× magnification. Paired phase-contrast images were acquired
simultaneously with a transmitted light detector. Images were printed
without processing. Sperm fluorescence was also checked with an Axiophot microscope (Carl Zeiss, Inc.) with 60× magnification. 50 sperm were
checked to determine the percentage of stained sperm for each experiment.
Inhibition of Sperm-Egg Plasma Membrane Adhesion and Fusion by Active Site Peptides
We asked if the disintegrin domain of any testis-expressed
ADAM family member other than fertilin has a role in
sperm-egg membrane adhesion. We tested the ability of
peptides from the putative active site in the disintegrin domain of each protein to inhibit an in vitro adhesion and fusion assay. Three parameters were measured: (a) the number of sperm bound per egg; (b) the FI, and (c) the FR.
Structural studies of snake disintegrins have shown that
the RGD tripeptide is the integrin-binding site and is located at the tip of a flexible loop created by disulfide bonds (Adler et al., 1991
; Chen et al., 1991
; Calvete et al., 1992
; Saudek et al., 1992
). The active-site regions of the
five mouse testis proteins were predicted by sequence
alignment with the snake disintegrin binding loop and
guinea pig fertilin
(Fig. 1). Linear, eight residue peptides
covering this region (Fig. 1, underlined) were tested.
The mouse fertilin peptide inhibited sperm-egg fusion
(Fig. 2 A), as expected from previous work with guinea pig
fertilin
peptides (Myles et al., 1994
) and mouse fertilin
peptides (Almeida et al., 1995
; Evans et al., 1995
). The fertilin
peptide gave a 59% inhibition in FI and a 55% inhibition of FR, but only a slight inhibition of sperm-egg
binding (13%; Fig. 2 A). The cyritestin peptide inhibited
sperm-egg fusion more strongly than the fertilin
peptide
and additionally inhibited sperm binding to the egg plasma
membrane (Fig. 2 A). The peptide from cyritestin resulted
in 84% inhibition of sperm-egg binding, 93% inhibition of
the FI, and 92% inhibition of the FR. The fertilin
peptide showed limited inhibition (~30%) of sperm-egg binding and fusion. Whether or not this is biologically significant is not yet clear. In comparison, the other two family
members, ADAM 4 and 5 peptides, showed essentially no
inhibition (Fig. 2 A). The control peptides for all of the
five proteins, which contained the same eight amino acids
but in a rearranged (scrambled) order, showed only slight or no inhibition (Fig. 2 B).
Inhibition by the fertilin and cyritestin peptides was
dose dependent. Relative to the fertilin
peptide, the
cyritestin peptide inhibited to a greater extent at high concentration (500 µM) and was also a more potent inhibitor
at lower concentration (Fig. 3, A-C). For example, using
the peptides, 50% inhibition of FI was obtained with ~400
µM fertilin
and ~70 µM cyritestin, and 50% inhibition
of FR was obtained with ~470 µM fertilin
and ~80 µM
cyritestin (Fig. 3, B and C).
Additional control experiments were done to check if inhibiting peptides had an adverse effect on the eggs. Eggs were incubated with the cyritestin peptide as in the previous experiments and were then washed into peptide-free medium before addition of sperm. Under these conditions, sperm-egg binding and fusion were not inhibited.
Characterization and Processing of Fertilin and
Cyritestin Proteins
Because these peptide studies indicated a role for both
mouse fertilin and cyritestin in sperm-egg fusion, we
used antibodies made against unique peptide sequences
from these two proteins (Table I) to determine their relative molecular weights and if the proteins were proteolytically processed after synthesis. We have previously found
that guinea pig fertilin
and
are first expressed as large
precursors in the testis and are subsequently processed
(Phelps et al., 1990
; Blobel et al., 1990
; Wolfsberg et al.,
1993
). Such processing appears to be required for protein function in some ADAMs (Yagami-Hiromasa et al., 1995
).
To determine whether mouse fertilin
and cyritestin also
undergo processing during sperm development, we immunoblotted testicular cells and sperm cells from different
developmental stages with antibodies against fertilin
or
cyritestin.
Detergent extracts were prepared from three cell populations: testicular cells (TC), testicular sperm (TS), and epididymal sperm (ES). All extracts were electrophoresed by
SDS-PAGE, and then subjected to immunoblot analysis with
the anti-peptide antibodies. We used antibodies directed
against the COOH-terminal cytoplasmic tail (Table I), because the processing of guinea pig fertilin and meltrin
occurs in the NH2-terminal extracellular domain at the
junction of the metalloprotease and disintegrin domains (Blobel et al., 1992
; Yagami-Hiromasa et al., 1995
). If a
similar pattern of processing occurs for mouse fertilin
and cyritestin, then the COOH-terminal antibodies should
be able to recognize both the precursors and processed
forms. Using the m
-CT1 antibody, we found two bands in
the testicular cell extract with molecular mass of 101 and
88 kD (Fig. 4 A). At the testicular sperm stage, there were
two bands observed: one at 101 kD and the other at 55 kD.
At the epididymal sperm stage, there was only one band that ran at 55 kD (Fig. 4 A). To test the specificity of these bands, 50 µg/ml of the same peptide used for generating
the antibody was added during the first antibody incubation
in the immunoblot. The peptide inhibited binding of m
-CT1
to all the bands in all three cell populations (Fig. 4 A
).
Like fertilin , cyritestin was expressed as a larger precursor that was processed on epididymal sperm, but the processing was not as complete as with fertilin
. Using the
mCyri-CT1 antibody (Table I) as primary antibody in the
immunoblot gave a major band at 110 kD in all three cell
populations (Fig. 4 B). There was an additional band at
55 kD observed in epididymal sperm (Fig. 4 B). Previously
it had been reported that cyritestin was completely processed to the 55-kD form on epididymal sperm (Linder et al., 1995
). To understand the difference between our results
and this previous report, we also used a second anti-cytoplasmic tail antibody (mCyri-CT2; Table I) made against
the identical peptide sequence used previously (Linder et al.,
1995
). Consistent with their result, this antibody (mCyriCT2) predominantly recognized the band at 55 kD (Fig. 4 B,
ES
). These results indicate that the mCyri-CT1 antibody
can recognize both processed and unprocessed cyritestin on epididymal sperm, whereas the mCyri-CT2 antibody
recognizes mainly the processed form. There are two additional strong bands with relative molecular weight of 30-
35 kD in testicular cells and testicular sperm (Fig. 4 B). All
the bands recognized by the cyritestin antibodies disappeared after the addition of 50 µg/ml of the immunizing
peptide during first antibody incubation (Fig. 4 B
).
By analogy with fertilin and meltrin
, the cyritestin
band of 55 kD is the expected size of a cyritestin processed
form, beginning at the NH2 terminus of the disintegrin domain and ending with the cytoplasmic tail COOH-terminal residues recognized by mCyri-CT1. Like meltrin
on
myoblasts, cyritestin on epididymal sperm is only partially
processed to this 55-kD form, and some precursor remains. The two bands of 30-35 kD appear to be proteolytic products whose size indicates loss of the disintegrin
domain, and these fragments are of unknown significance.
Effects of Active Site Antibodies on In Vitro Sperm-Egg Adhesion and Fusion
An alternative approach to using peptide mimetics as inhibitors of the fusion assay was tested. We generated antibodies to the active site region of fertilin or cyritestin by
immunizing mice with chimeric peptides comprised of a
malaria T cell epitope and a B cell epitope(s) containing
23 amino acids from the disintegrin active site. These antisera, m
-AS1 and mCyri-AS1, immunoblotted fertilin
and
cyritestin, respectively, and bound specifically to sperm
extracts in an ELISA, i.e., binding was inhibited in the
presence of the immunizing chimeric peptide (data not
shown). The ability of these anti-active site antisera to inhibit sperm-egg adhesion and fusion was tested. Sperm
were induced to acrosome react by incubating for 3 h in
capacitating medium. Acrosome-reacted sperm were incubated for 30 min with m
-AS1 or mCyri-AS1, and then
eggs were added to the sperm. Both m
-AS1 and mCyriAS1 inhibited sperm-egg binding and fusion dramatically
(80-90%). The preimmune sera did not have any observable effect on binding or fusion (Fig. 5).
Localization of Fertilin and Cyritestin on the
Sperm Surface
Because interaction of the sperm and egg membranes is a
regional process, it is important to know the localization of
fertilin and cyritestin. From staining of permeabilized
sperm, it was previously reported that cyritestin was located exclusively on the IAM (Linder et al., 1995
). This
localization was observed using an anti-cytoplasmic tail
antibody. When we stained permeabilized sperm with antibody mCyri-CT2 raised against the same peptide, no difference was found between preimmune and immune sera.
The reason for this discrepancy is unclear. Using antibodies to the active site regions of fertilin
and cyritestin,
we obtained localized staining patterns on the plasma
membrane. Live, swimming, acrosome-intact sperm were
stained with the active site antibodies m
-AS1 or mCyriAS1. With either m
-AS1 or mCyri-AS1, we observed bright, fluorescent staining restricted to the equatorial region on the majority of sperm (Fig. 6, A and B). The preimmune serum controls for both fertilin
and cyritestin
did not show any fluorescence.
Because only acrosome-reacted sperm are able to fuse
with the egg plasma membrane, we also stained live sperm
after they had been induced to acrosome react by incubation in capacitating medium for 3 h or treatment with 10 µM
A23187. After induction of the acrosome reaction, the
main staining pattern (80% of the sperm) showed unchanged localization of cyritestin, i.e., restricted to the equatorial region (Fig. 6 D). A small population of the sperm
(10-20%) had both equatorial segment and IAM staining
after acrosome reaction. The antibody against fertilin ,
on the other hand, was localized in both the equatorial and
IAM regions of ~70% of the sperm (Fig. 6 C). The preimmune serum controls for both fertilin
and cyritestin also
did not show any fluorescence on acrosome-reacted sperm.
The staining of sperm with mCyri-AS1 was abolished in
the presence of the immunizing cyritestin chimeric peptide, but was unchanged in the presence of the fertilin
chimeric peptide, indicating that mCyri-AS1 binds specifically to the active site of cyritestin on the sperm surface.
Because antibodies could potentially induce changes in
the localization patterns by causing antigen clustering, we
also stained sperm after they had been fixed. The same
patterns were observed for both fertilin and cyritestin.
Common domain structures of the ADAM family of membrane proteins indicate that family members may have
similar functions. Two of the proteins in this family that
have been previously studied, functionally appear to participate in cell-cell fusion: fertilin in sperm-egg fusion
(Primakoff et al., 1987; Blobel et al., 1992
; Myles et al., 1994
;
Almeida et al., 1995
; Evans et al., 1995
), and meltrin
in
myoblast fusion (Yagami-Hiromasa et al., 1995
). Because
other ADAM proteins were found to be expressed in mouse testis, we asked if any of these additional family members
were also involved in sperm-egg adhesion and fusion. We
focused on the hypothesis that the predicted active site in
the disintegrin domain of these proteins may participate in
sperm-egg adhesion and fusion. To test this, we carried
out peptide and antibody inhibition studies using an in
vitro adhesion and fusion assay.
Our studies indicated that cyritestin, one of the ADAM
family proteins whose expression is limited to testis, participates in sperm-egg binding and fusion along with fertilin . Both the predicted active site peptide and an antibody to a peptide from the active site region could inhibit
sperm-egg binding and fusion strongly (80-90%). The
cyritestin active site peptide was a more potent inhibitor of
both binding and fusion than the corresponding fertilin
peptide. 50% inhibition of sperm-egg fusion was obtained with a fivefold lower concentration of cyritestin peptide than fertilin
peptide. Most of the eggs displayed no sperm
bound when incubated with the cyritestin active site peptide at saturating concentrations or when sperm were preincubated with anti-active site antibody. If the cyritestin
peptide-treated eggs were washed into peptide-free medium, these eggs could be subsequently fertilized.
Sperm-egg fusion was also inhibited by the active site
peptide from fertilin , a result consistent with our previous results in guinea pig (Myles et al., 1994
) and with results using shorter (Evans et al., 1995
) and longer fertilin
peptides in mouse (Almeida et al., 1995
). Additional evidence supporting the role of fertilin
in mouse gamete fusion comes from the experiment with the anti-active site
antibody for fertilin
. This antibody strongly inhibits (80-
90%) both sperm-egg binding and fusion.
The inhibitory potency of a peptide mimetic of the disintegrin active site of an ADAM protein will depend upon
the sequence of the peptide and its affinity for the corresponding receptor on the egg. The cyritestin peptide,
SKDQCDFP, and fertilin peptide, AQDECDVT, are
identical at underlined residues 3 = D, 5 = C, and 6 = D. Although these residues may be necessary for binding activity, they are not sufficient for the inhibitory effect.
These same residues are also present in the ADAM 5 peptide, SVDECDLL, which did not inhibit either sperm-egg
binding or fusion.
Because specific sperm surface domains are associated
with specific functions, the localizations of sperm surface
proteins must be consonant with their proposed activities.
Localization of cyritestin in the equatorial region is consistent with its participation in sperm-egg fusion. Although
both the equatorial regions and posterior head of the
sperm membrane fuse with the egg membrane, the initiation of fusion has been reported in some species to occur
in the equatorial region (for reviews see Yanagimachi, 1981, 1988, 1994). Fertilin is also located in the equatorial region of mouse sperm, although it is restricted to the posterior head of guinea pig sperm (Primakoff et al., 1987
). This
may reflect the fact that, unlike mouse sperm, in guinea
pig sperm the equatorial region is very narrow and fusion
occurs in the central region of the sperm head, which could
include the anterior part of the posterior head as well as
equatorial region (Noda and Yanagimachi, 1976
).
We found that both mouse fertilin and cyritestin were
made as large precursors (101 and 110 kD, respectively)
and processed during sperm maturation to 55-kD forms.
The processing of fertilin
was complete, whereas only a
portion of the cyritestin precursor was processed. This processing could be a prerequisite for function of these proteins. For the ADAM family member meltrin
, only the
processed form was functional in myoblast fusion (Yagami-Hiromasa et al., 1995
).
Our studies indicate at least two testis-specific ADAM
family members, cyritestin and fertilin , have roles in
sperm-egg adhesion leading to sperm-egg fusion. Participation of multiple members of a protein family in a single
cell-cell adhesion event has been observed in other cases.
One system that has been intensely studied is the adhesion
of leukocytes to endothelial cells that leads to leukocyte
extravasation (for review see Springer, 1994
; Frenette et
al., 1996
). As with sperm adhesion to the egg plasma membrane, this system involves a moving leukocyte attaching
to a stationary endothelial cell. When, for example, a neutrophil binds to the endothelial cell, L-selectin on the neutrophil and P- and E-selectin on the endothelial cell initially
bind to carbohydrate ligands on the adhering cell. Subsequently, members of the integrin family on the neutrophil,
L
2 and
M
2, bind to one or two endothelial ligands,
ICAM-1 and ICAM-2, members of the Ig super family.
It is likely that cyritestin and fertilin bind via the disintegrin domain to egg integrins (Almeida et al., 1995
). We
do not yet know if either cyritestin or fertilin
bind to one
or more (integrin) receptors on the egg. In addition, the
two proteins could bind to the same or different sets of receptors. In neutrophil-endothelial cell adhesion, the
ligand ICAM-1 binds to both
L
2 and
M
2 integrins, whereas the ligand ICAM-2 binds only to
L
2 (for review see Springer, 1994
).
Cyritestin and fertilin may be acting in sperm adhesion sequentially or concurrently. Sequential action could
involve the first binding step activating the egg receptor or
sperm ADAM for the second binding step. Concurrent action could involve cooperativity in promoting cell adhesion. In either case, disruption of either adhesion results in
a strong block to sperm-egg fusion, indicating both proteins are required. This finding of unexpected complexity in the sperm adhesion process may provide a key to understanding how adhesion occurs and how it leads to fusion of
the two gametes.
Received for publication 17 December 1996 and in revised form 20 January 1997.
Please address all correspondence to Diana Myles, Molecular and Cellular Biology, University of California, Davis, CA 95616. Tel.: (916) 752-1553; Fax: (916) 752-3085; E-mail: dgmyles{at}ucdavis.eduThe authors are grateful to Dr. Harini Bagavant and Dr. Kenneth S.K. Tung in the Department of Pathology, University of Virginia (Charlottesville, VA) for help in designing and using the chimeric peptides. We also thank Dr. Judith M. White (University of Virginia, Charlottesville, VA) and members of her laboratory, especially Dr. Tyra G. Wolfsberg for helpful discussions.
This work was supported by National Institutes of Health grants HD16580 (D.G. Myles) and U54-29125 (P. Primakoff).
FI, fertilization index; FR, fertilization rate; IAM, inner acrosomal membrane; MAP, multiple antigenic peptide.