AMER. ZOOL., 839-849 (1979).
The Interactions of Sea Urchin Gametes During Fertilization
VICTOR D. VACQUIER
Marine Biology Research Division, A-002, Scripps Institution of Oceanography, University
of California, San Diego, Lajolla, California 92093
SYNOPSIS. The interactions between sea urchin spermatozoa and ova during fertilization
usually exhibit a high degree of species specificity. Under natural conditions and reasonable
gamete concentrations, most interspecific inseminations fail to yield zygotes. Macromolecules on the external surfaces of the apposing gametes must surely be responsible for
successful gamete recognition, adhesion and fusion. Species specific recognition between
surface components of sperm and egg could occur during at least three events comprising
the fertilization process. The first event is the interaction of the sperm plasma membrane
with the egg jelly coat. This induces the sperm acrosome reaction resulting in the exocytosis
of the "bindin"-containing acrosome granule and also the extrusion of the acrosome process
from the anterior tip of the sperm. The second event is the adhesion of the bindin-coated
acrosome process to glycoprotein "bindin receptors" on the external surface of the egg
vitelline layer. The third event is the penetration of the vitelline layer and the fusion of
sperm and egg plasma membranes. With the isolations of the component of egg jelly which
induces the acrosome reaction, sperm bindin from the acrosome vesicle and the egg surface
bindin receptor from the vitelline layer, there is hope of discovering the molecular basis of
this most interesting intercellular interaction which results in the activation of embryonic
development.
INTRODUCTION
The successful interaction of sea urchin
sperm and egg usually exhibits a high degree of species specificity. In most interspecific inseminations sperm fail to
adhere to or fuse with foreign eggs. Loeb
(1916) probably was the first to suggest that
species specificity of fertilization must reside in the proteins carried on gamete surfaces. During the past two years several
gamete surface components have been isolated which appear to function in spermegg recognition and adhesion.
The purpose of this mini-review is to discuss some of these recent findings in relation to the species specificity exhibited by
invertebrate gametes. Special attention will
be given to three interactions: The induction of the sperm acrosome reaction by egg
jelly, the attachment of spermatozoa to eggs
and the fusion of sperm and egg plasma
membranes. Comprehensive reviews of invertebrate and mammalian fertilization
have recently appeared and their contents
Work supported by NIH Grant HD 12986.
will not be restated here (Epel, 1978; Epel
and Vacquier, 1978; Gwatkin, 1978; Metz,
1978; Yanagimachi, 1977, 1978). Although
most of the work to be mentioned concerns
sea urchins, some exciting new data on gamete interactions in other animals will also
be presented. An attempt will be made to
identify interesting questions for future research.
INTERACTION OF EGG JELLY WITH SPERM
The usually transparent egg jelly coat
surrounding the sea urchin egg can be solubilized by treatment of the eggs with seawater at pH 5. After removal of the eggs,
addition of sperm to the jelly solution may
cause two visually observable and independent effects: the swarming of sperm into
clusters (Loeb, 1916) and the acrosome
reaction (both reviewed by Epel, 1978;
Metz, 1978).
The acrosome reaction
In most marine invertebrates the acrosome reaction consists of two events: the
839
840
VICTOR D. VACQUIER
exocytosis of the acrosome granule located
in the anterior apex of the cell and the extension of the acrosome process (Epel,
1978; Metz, 1978). Under natural conditions the acrosome reaction can be induced
by soluble egg jelly, by contact of sperm
with the in situ jelly coat surrounding the
egg or by contact of the sperm with the egg
surface.
Epel (1978) has reviewed the work on the
dependence of the echinoid acrosome reaction on extracellular Ca 2+ . Since then,
Schackmann et al. (1978) have found the
acrosome reaction of S. purpuratus sperm in
response to eggjelly results in release of H+
from the sperm coincident with the uptake
or exchange of Ca2+. Their studies with
drugs and ionophores show the acrosome
reaction to be absolutely dependent on entrance of Ca2+ and Na+ with the possible
involvement of K+ release.
Enzymatic activities associated with
marine invertebrate sperm have been reviewed in detail (Metz, 1978), but until recently, clear evidence of the association of
proteolytic activity with the sea urchin acrosome reaction did not exist. Levine et al.
(1978) report a very sensitive assay utilizing
tritiated benzoyl arginine ethylester as substrate, which allows the detection of a
trypsin-like activity present in S. purpuratus
sperm only after the induction of the acrosome reaction. They find 80% of the activity
is exposed by treatment of sperm with seawater containing three times the normal
Ca2+ concentration (30 mM). The activity is
membrane-bound and treatment with the
serine protease inhibitor diisopropyl phosphofluoridate inhibits the enzyme and also
the ability of sperm to fertilize eggs.
In a series of now classical papers, Tilney
and his associates demonstrated that the
extrusion of the acrosome process of the
echinoderm sperm involves the polymerization of actin filaments (Tilney et al., 1978).
The polymerization of acrosomal actin depends on the expulsion of H+ from the
sperm which results in an increase in intracellular pH. In the unreacted state, the
actin is complexed with a regulatory protein which keeps it from polymerizing. The
rise in pH causes the regulatory protein to
dissociate from the actin allowing the G
actin to polymerize into F actin filaments.
The nucleation site of actin polymerization
is an organelle called the "actomere" which
appears to be a small collection of preformed actin filaments (Tilney, 1978).
The swarming reaction: Are chemoattractants
involved?
When sperm of most echinoderm species
are suspended in solutions of eggjelly the
cells immediately form visible spherical
clusters up to 2 mm in diameter. This
phenomenon is independent of the acrosome reaction and extracellular Ca2+. In
echinoids the clusters are composed of
freely moving cells which are not agglutinated or bonded together. After 2—10 min
the clusters spontaneously disappear. In asteroids, cluster formation does not reverse
and may involve the immobilization of the
cells (Epel, 1978; Metz, 1978). Early work
on cluster formation showed it to be a
species specific phenomenon which is the
reason it was originally termed "isoagglutination" (Metz, 1978). However, Collins
(1976) found (and we have confirmed) that
the swarming reaction of echinoid sperm is
not necessarily species specific and does not
occur in all species. For example, sperm of
Lytechinus pictus do not swarm when suspended in L. pictus or Strongylocentrotus purpuratus egg jelly. Strongylocentrotus purpuratus sperm however swarm as well in L.
pictus jelly as they do in their own.
One hypothesis to explain the swarming
reaction of echinoderm sperm is to propose
the existence of a chemotactic substance
which is released by sperm in response to
jelly. Support for such a factor in the sea
urchin Arbaciapunctulata has been provided
by an ingenious experiment utilizing goldcoated nucleopore filters (Lopoefa/., 1977).
Three wells containing either jelly-clustered sperm, jelly or seawater containing
ovalbumin are covered by a gold-coated
nucleopore filter over which is placed a
fresh suspension of actively moving sperm.
When sperm come in contact with the gold
surface they become permanently bound to
it. After several minutes the sperm suspension is washed out and the number of sperm
bound over each well determined. The re-
SPECIES SPECIFICITY OF SPERM-EGG INTERACTIONS
841
suits of several experiments show that four Elevation of cyclic AMP in sperm
times as many sperm are bound to the filter treated with eggjelly
over the well containing jelly-clustered
Garbers and Hardman (1976) have resperm. Although this phenomenon can be
repeatedly demonstrated in A. punctulata, it ported the most interesting elevation of cycis not found in sperm of S. purpuratus lic AMP in both S. purpuratus and L. pictus
sperm upon treatment with eggjelly. Addi(Lopo, personal communication).
A saponin has recently been isolated tion of theophylline alone to sperm profrom eggjelly of the starfish Asterias amuren- duces a twofold increase in cyclic AMP and
sis which causes the agglutination of sperm treatment with egg jelly alone a sevenfold
(Uno and Hoshi, 1978). This compound increase within 1 min. The combination of
induces sperm agglutination in a concen- theophylline and eggjelly produces a 100tration range of 10~3 to 10~6 M. The fold increase. The stimulatory factor was
molecular weight is about 1200 and below not inactivated by boiling but was destroyed
10~5 M it is undialyzable indicating micelle by ashing. Sephadex G-50 separated the
formation. In a highly purified form it will cyclic AMP elevating activity into two peaks,
not induce aggregation of sperm of the one excluded from the column and the
closely related starfish Distolasterias nipon. other included with an approximate moHowever, impure preparations of A. am- lecular weight of 1,000 to 10,000. The eggurensis eggjelly saponin will induce aggluti- released cyclic AMP stimulator caused cluster formation (swarming) of the sperm.
nation of D. nipon sperm.
The
physiological functions, if any, of this
The chemotaxis of sperm to ovarian fluid
elevation
of cAMP in sea urchin sperm and
of sessile marine hydroids and alcoholic exthe
swarming
of sperm into clusters in retracts of eggs of tunicates and chitons has
sponse
to
eggjelly
remain unknown.
been elegantly demonstrated using cinematography (Miller, 1977; see review by
Metz, 1978). Ovarian or egg extracts in the Biochemical analysis of eggjelly
bore of a pipette cause the immediate species specific swarming of sperm of many
SeGall and Lennarz (1979) have analyzed
hydromedusae to the pipette tip (Miller, the jelly coat macromolecules of four sea
1979a, 19796). Miller (1979c) has most re- urchin species and have found them to be
cently described the very interesting inter- very similar in composition. Jelly of all four
actions of sperm and egg of the leptomedu- species contains two major macromolsan Orthophxis caliculata. If sperm are added ecules, a sialic acid rich protein and a fucose
to eggs before second polar body forma- sulfate polysaccharide. They find all of the
tion they show no attraction for the egg, acrosome reaction inducing activity of egg
but swim randomly into the eggjelly where jelly resides in the fucose sulfate polysacthey become trapped and immobilized. charide. Regarding species specificity of
But, if added after polar body II emission, induction of the acrosome reaction, these
the sperm move rapidly through the jelly workers found a range from complete speand over the entire egg surface. The im- cificity to complete nonspecificity dependmobile sperm, trapped in the eggjelly, re- ing on the particular cross species mixture
activate and also move to the egg surface of sperm and jelly. For example, sperm of
membrane. The egg appears to release a Arbacia punctulata and Strongylocentrotus
species specific chemoattractant of about drobachiensis respond only to their own egg
1000 mol wt 1 or 2 min after polar body jelly, whereas S. purpuratus sperm react
II formation. Once at the egg surface the equally well with their own and also L. varsperm undergo a head-to-head agglutina- iegatus jelly but not with A. punctulata jelly.
tion only at the animal pole. This agglu- We have also found (Brandriff and Vactination lasts 10 min after which the egg is quier, unpublished) that on an equal weight
no longer attractive to sperm. However, if basis, egg jelly from the California sea urthe eggs are left unfertilized they continue chins S. purpuratus, S. franciscanus and L.
to be attractive to sperm for at least 3 hours. pictus shows absolutely no specificity in in-
842
VICTOR D. VACQUIER
duction of the acrosome reaction in sperm on the egg surface which can be reduced to
suboptimal amounts by acid treatment and
of these three species.
Summers and Hylander (1975) also found washing. If the same experiment is perin 9 of 11 cross species inseminations among formed on eggs of S. purpuratus, exacdy opechinoderms of Bermuda, the sperm un- posite results are obtained; acid treatment
derwent a normal acrosome reaction but and washing greatly increase the sperm
did not attach to the foreign eggs. SeGall binding rate and egg fertilizability. Our
and Lennarz (1979) conclude that since the idea is that with the latter species, the optijelly coats from different species such as ma! surface concentration of egg jelly
A. punctulata and 5. drobachiensis are very necessary for acrosome reaction induction
similar in chemical composition, the species is not removed by low pH and washing. Egg
specificity of acrosome induction must be jelly definitely exists on the surfaces of
related to structural differences in the fu- acid-dejellied S. purpuratus eggs and some,
or all of it, is removed during the cortical
cose sulfate polysaccharide.
In most sea urchin species egg jelly will granule exocytosis following sperm-egg fuinduce the acrosome reaction. However, in sion (Glabeand Vacquier, 1978; Vacquier et
Pseudocentrotus depressus sperm swim di- al., 1979).
rectly through the jelly coat without reacting (Aketa and Ohta, 1977). These workers Questions for future research on the interaction
isolated intact jelly layers, added sperm and of eggjelly with sperm
then fixed the preparation for electron microscopic observations which revealed not a
The interaction of the fucose sulfate polysingle sperm entering the jelly hulls had saccharide with the sperm plasma memundergone the acrosome reaction. In this brane to induce the acrosome reaction,
species the sperm appear to undergo the the swarming of sperm in response to jelly,
acrosome reaction only when directly in and the chemotaxis of hydroid and tunicontact with the egg vitelline layer.
cate sperm may all involve specific sperm
membrane receptors. If so, do the receptors aggregate in the plane of the memIs egg jelly essentialfor fertilization ?
brane to form or unmask ion channels? Or,
The answer to this question is obviously do these egg jelly components act as deterno, since it has been known for several years gents to remove proteins which block ion
that blastomeres of 1, 2, 4 and 8 cell em- channels?
bryos can be refertilized after removal of
Can the fucose sulfate polymer be cleaved
the investing fertilization envelope and into a basic unit retaining biological funchyaline layer (Metz, 1978). One must con- tion? In a species such as S. purpuratus, how
sider sperm to be cells specialized for fusion is the jelly associated with the egg vitelline
with other cells, and it is known that induc- layer? Is it only on the outer surface, or
tion of the acrosome reaction directly entirely throughout the vitelline, or possiagainst the naked egg or blastomere plasma bly between the vitelline and the egg plasma
membrane may lead to gamete fusion and membrane? Many of these questions are
sperm incorporation. However, although now potentially answerable with the recent
an indispensible role for egg jelly does not development of methods to radioiodinate
appear to exist, one can still ask whether and fluorescently label the surface of sea
egg jelly enhances the fertilizability of eggs. urchin and mouse sperm while keeping
We have found (Vacquier et al., 1979) that them viable and capable of fertilization
L. pictus eggs treated 2 min with sea water at (Gabel et al., 1978; Lopo and Vacquier,
pH 4.7 and washed extensively with fresh 1978).
sea water, show a great decrease in their
What is the function of the trypsin-like
sperm binding capacity and fertilizability protease exposed during the acrosome
that can be restored by addition of soluble reaction? Does it digest a hole in the viteljelly. We interpret this result by postulating line layer or does it prepare gamete surface
the existence of an optimal amount of jelly components for mediation of membrane
SPECIES SPECIFICITY OF SPERM-EGG INTERACTIONS
843
fusion in a manner similar to the serine method which yielded the membrane-free
protease found in association with the F insoluble granular contents of the acroprotein of Sendai virus (Gething et al., some granule. This material was composed
1978)?
of a single major protein (30,500 mol wt)
which we named bindin because of its suspected function in sperm-egg binding
ADHESION OF SPERM TO THE EGG SURFACE
(Vacquier and Moy, 1977). Paniculate bindin is isolated by suspending sperm in
Many invertebrate eggs possess a glyco- calcium-free sea water, pH 5.8, containing
protein coverlet closely adhering to the 1.25% Triton X-100. The membranes of
plasma membrane which is usually called the cell solubilize and the insoluble acrothe vitelline layer (VL). In sea urchins, the some granule content is released as a spherVL is the site of species specific sperm adhe- ical mass about 0.2 /Am in diameter. The
sion (reviewed by Metz, 1978). The bond paniculate bindin is collected by filtration
between sperm and egg involves attach- through a glass fiber column followed by
ment of the acrosome process of the sperm centrifugation at 13,000 x g. The pellet is
to the outer VL surface. The VL can be scraped from the tube and washed in calisolated as an intact structure which retains cium-free sea water by resuspension, using
its sperm binding quality (Glabe and Vac- mild sonication, and centrifugation to pelquier, 1977a). The present idea is that let. EM analysis shows this material to be
species specific surface components of the composed entirely of spheres of granular
sperm acrosome process bind to com- acrosome content. Chemical analysis yields
plementary "sperm receptors" on the egg tyrosine as the only N-terminal amino acid
VL and tightly cement the sperm to the VL. and shows the complete absence of carbohydrate and phospholipid. Sea urchin
sperm bindin has now been isolated from at
Isolation of sperm surface components mediating least four species.
gamete adhesion in sea urchins
Two separate results support the hyIn a long series of papers over the past 12 pothesis that bindin mediates sperm atyears Aketa and his co-workers have iso- tachment to eggs. First, with monospecific
lated glycoproteins from sea urchin sperm rabbit anti-bindin, we have localized bindin
and egg which appear to function in species on the surface of the acrosome process
specific gamete adhesion (reviewed by using peroxidase-conjugated swine antiMetz, 1978). More recently, this group has rabbit serum and the electron microscope.
reported the isolation of a sperm factor Bindin is exposed only after the exocytosis
which, when added to eggs, causes the eggs of the acrosome granule. On sperm bound
to lose their sperm binding capacity and to eggs, the anti-bindin localized bindin
fertilizability. This effect requires the pres- bridging the space between acrosome proence of Ca+2 and Mg*+ and is species specific cess and egg microvillus. The immunoper(Aketa et al., 1978). When this factor is in- oxidase electron microscopic localization
cubated with its putative egg surface recep- shows bindin is in the proper place at the
tor "sperm binding factor") it loses its fer- proper time to be the mediator of sperm
tilization inhibiting effect, another result adhesion (Moy and Vacquier, 1979). Rewhich appears to be species specific. The garding the species specificity of the antifactor is 5% protein and 95% carbohydrate. body to S. purpuratus bindin, we find cross
Electron micrographs of echinoid reactivity with all other echinoid species
sperm-egg adhesion by Summers and his (sand dollars and sea urchins) but no reaccolleagues (Summers et al., 1975) impli- tivity with asteroid or holothuroid sperm.
cated the contents of the acrosome granule
The second, and more compelling result,
as the sperm adhesive used for egg attach- is that bindin is a species specific agglutinin
ment. These data prompted us to attempt of unfertilized sea urchin eggs (Glabe and
to isolate the acrosome granule of S. pur- Vacquier, 19776; Glabe and Lennarz, 1978;
puratus sperm. We finally developed a Vacquier and Moy, 1978). The agglutina-
844
VICTOR D. VACQUIER
tion of eggs can be blocked by glycopeptides Isolation of egg surface components mediating
released by trypsin or pronase digestion of gamete adhesion in sea urchins
the surfaces of unfertilized eggs. Proteasetreated eggs fail to agglutinate, suggesting
The work on this topic to 1977 has been
the removal of a "bindin receptor." We extensively reviewed by Epel (1978), Epel
have found that aldehyde-fixed eggs also and Vacquier (1978) and Metz (1978). Since
agglutinate, but when such eggs are oxi- then Tsuzuki etal. (1977) have reported the
dized with metaperiodate, agglutination purification of the sperm receptor factor
by bindin does not occur. The above data from eggs of the sea urchin Hemicentrotus
support the idea that bindin interacts with a pulcherrimus. The factor is removed by solspecific oligosaccharide receptor probably ubilization of the VL in 1 M urea with final
located on the outer VL surface. To our purification by gel filtration and ion-exknowledge bindin is the first protein iso- change chromatography. The final preparalated in fairly pure form and milligram tion runs as a single component on Sephaquantities that mediates a species specific dex CL-4B in 6 M guanidine.hydrochloride
intercellular adhesion of a metazoan. It is and appears to be a very large glycoprotein.
also the first sperm surface component The purified factor completely blocks the
whose biological function is fairly well es- fertilization capacity of only H. pulcherrimus
tablished. We hope that future work on this sperm. The factor does not decrease sperm
protein will greatly increase our knowledge motility. Antisera to the factor make only
of the fertilization process in sea urchins H. pulcherrimus eggs incapable of binding
and other animal groups.
sperm. It is not clear from the report if
the
factor binds to sperm before or after the
We have proposed (Vacquier and Moy,
1978) that sperm bindins are a new class of acrosome reaction.
Schmell et al. (1977) reported the fertilisperm-borne proteins mediating spermegg adhesion in many phylogenetically dis- zation of A. punctulata eggs can be inhibited
tantly related animal groups. The acrosome by addition of a membrane isolate from A.
granule of the oyster Crassostrea gigas has punctulata eggs. The same phenomenon
been isolated and shown to be composed of can be demonstrated by homologous mixan insoluble material which is 85% protein tures of S. purpuratus eggs and membranes.
by weight, stains for carbohydrate, and But, when A. punctulata eggs are fertilized
separates into two major components of in the presence of S. purpuratus membranes
65,000 and 53,000 apparent molecular (or 5. purpuratus eggs in A. punctulata memweight (Brandriff«« al., 1978). If this is oys- branes) no inhibitory effect on fertilization
ter bindin it is quite dissimilar from sea occurs. This is excellent evidence for the
urchin bindin. The oyster acrosomal existence of a species specific sperm recepglycoprotein agglutinates oyster eggs and tor on the egg surface. Further evidence for
the agglutination is blocked by glycopep- the surface location of the receptor is that
tides digested from egg surfaces. We have trypsinization of eggs before preparation of
preliminary evidence that a similar protein the membraneous fraction destroys the inmay be present in abalone sperm (genus hibitory quality of the membranes.
Haliotis) and in sperm of the echiuroid
The species specific quality of bindin as
worm Urechis caupo (Stephano-Hornedo an egg agglutinin is a direct demonstration
and Gould-Somero, unpublished). Sperm- that species specific bindin adhesive (recepegg adhesion in mammals also exhibits a tor) molecules must exist on the VL surface
high degree of species specificity (Yanagi- of the egg. A large glycoprotein, a putative
machi, 1977) and it seems reasonable to as- "bindin receptor," has been identified by a
sume the involvement of bindin-like pro- direct assay requiring the affinity of bindin
teins. If mammalian bindins do exist they for radio-iodinated bindin receptor (Glabe
might be ideal antigens to use in attempts and Vacquier, 1978). To remove the recepto induce female immunosterility without tor from the VL, 125I-labeled eggs of S. purrisking the development of autoimmune puratus are parthenogenetically activated by
complications.
the ionophore A23187 in the presence of
SPECIES SPECIFICITY OF SPERM-EGG INTERACTIONS
845
tus (Denis-Donini and Campanella, 1977).
The site, called "the animal dimple," is the
only place on the entire egg surface where
fertilization can occur. The dimple is actually a pit or cleft in the egg surface. Lectin
receptors not found on the cell surface are
found in the dimple. Receptors for soybean
and wheat germ agglutinins are found only
on the dimple walls, while fucose binding
protein binds only to the dimple bottom.
This lectin binding pattern is present only
in mature oocytes and disappears immediately after fertilization.
Many species of tunicates are self-sterile
hermaphrodites and studies by Morgan
(1939) showed the block to selfing involved
failure of the sperm to attach to and penetrate the egg chorion (outer investment).
With renewed interest in fertilization
studies during the past five years, and
armed with the new methodology of cell
surface research, the basis for self-sterility
in tunicates is being reinvestigated. The ultrastructure of the gametes at fertilization,
showing sperm-egg attachment and fusion
has recently been published (Villa, 1977).
In one tunicate species, Ciona intestinalis,
Sperm-egg adhesion in other animal groups
about 15% of the individuals are self-fertile
Returning for a moment to Miller's work (Rosati and De Santis, 1978). Eggs from the
with the leptomedusan Orthopyxis (Miller, self-sterile individuals remain unfertiliza1979c), it will be remembered that the ble at very high sperm densities showing the
sperm only become attracted to the egg sur- exacting nature of this cell surface recogniface after extrusion of the second polar tion. This recent work shows conclusively
body. The sperm undergo a head-to-head that it is the chorion, and not some subagglutination during attachment to the egg stance emanating from the surrounding
surface at the site where polar body II was follicle cells, that is the site of self-nonself
emitted (the animal pole). The motile recognition. If the chorion is removed, ferspermatozoa in the aggregate mass do not tilization occurs. In the nonself inseminachange position in relation to each other, tion, successful recognition results in the
suggesting they are tightly agglutinated by formation of a strong adhesion between the
cell surface factors. After about 12 min the outer surfaces of the chorion and the
clot of sperm breaks up, the egg no longer is plasma membrane of the unreacted sperattractive to fresh sperm and cleavage oc- matozoon.
curs in 45 min. The attachment of the
Hylander and Summers (1977) have presperm to only a restricted area of the egg sented a detailed ultrastructural study of
surface suggests that sperm receptors ap- sperm-egg adhesion in two species of bipear on the egg surface at the site of polar valve molluscs, Chama macerophylla and Spisbody, release and. disappear after sperm- ula solidissima. In these eggs the cytoplasegg fusion has occurred.
mic microvilli of the egg surface project
Another example of a very restricted completely through the VL and the exarea on the egg surface, highly specialized posed villi tips are covered with a dense
for sperm attachment and fusion, is found fibrillar bundle of cell surface material
in oocytes of the amphibian Discoglossus pic- closely resembling the tips of brush border
soybean trypsin inhibitor. About 10% of the
surface labeled material is released into the
8,000 x g supernate. To assay bindin receptor activity, unlabeled, paniculate bindin is
mixed with the iodinated surface released
material and the mixture filtered through a
GFC filter which retains 100% of the bindin. Radioactivity per filter is thus a measure of bindin receptor activity. Controls
show the 125I on the filter does not result
from nonspecific trapping. The affinity of
bindin for 125I-receptor exhibits saturation
kinetics with 50% maximum complex formation in 25 s. Competition for bindin125
I-receptor complexes with unlabeled receptor from a different species, whole egg
protein and BSA shows specificity for the
homologous unlabeled receptor. The receptor activity bands in a pH 3 - 5 isoelectric
focus column at pH 4.00 and its exclusion
from a BioGel A5m column shows it must
be an aggregate of several million molecular weight which is 34% neutral sugar and
thus presumed to be a glycoprotein (Glabe
andVacquier, 1978).
846
VICTOR D. VACQUIER
microvilli of the vertebrate intestine. The
sperm acrosome granule ofChama contains
materials of two different electron densities. Exocytosis of the granule releases the
more distal, less electron-dense component
which dissipates with the extension of the
acrosome process. The more electrondense and proximally located granule
component binds to the fibrillar material on
the microvillar tips and holds the sperm
tightly bound to the egg surface. Close
examination of these micrographs leaves
little doubt that the acrosome granule component must beChama bindin and the fibrillar material on the villi tips a bindin receptor glycoprotein (Hylander and Summers,
1977).
Future research on sperm-egg adhesion
action with bindin studied in detail under
in vitro conditions?
One important question concerns the
possibility of the existence of mammalian
sperm bindins. Quantitative techniques are
now available to study the attachment of
sperm to the egg zona pellucida (Saling et
al., 1978). One possible method to attempt
to isolate a mammalian sperm bindin would
be to separate all the membrane-associated
sperm head proteins by 2-dimensional slab
gel electrophoresis and then prepare antibody to each protein. Immunoperoxidase
localization of each protein at the EM level
on sperm bound to eggs might pinpoint the
location of a putative bindin. More of the
protein could then be isolated by immunoprecipitation, antibody affinity columns, or standard biochemical methods.
Further knowledge about sea urchin
FUSION OF GAMETE PLASMA MEMBRANES
sperm bindin and its glycoprotein receptor
The reasons for the induction of the acon the egg VL seems crucial for our advancement towards a true molecular un- rosome reaction and the binding of sperm
derstanding of species specific gamete rec- to the egg VL must surely be to enhance the
ognition. How does bindin interact with the probability of membrane fusion between
sperm membrane? What is the nature of the gametes. At this time, almost nothing is
the self-interaction of bindin monomers to known about the relation between penetraform the insoluble mass comprising the ac- tion of the VL and membrane fusion. In sea
rosome granule? What is the 3-dimensional urchin eggs the VL is very thin, being
structure of bindin and how does it interact only 100-300 A in diameter (Glabe and
with the recognition sequence of its recep- Vacquier, 1977a) and the acrosome process
tor? What are the molecular differences is short, being only about 1 mfi in length.
conferrring species specificity on two dif- The events of sperm attachment to VL, penferent bindins? Does bindin not only medi- etration of the VL and fusion of membranes
ate gamete adhesion, but also act as a fusion probably occur so rapidly in time that they
promoting protein during sperm-egg cell would be impossible to separate from one
membrane coalescence? Is the bindin re- another. One electron micrograph, howceptor a transmembrane protein extend- ever, has been published showing a sea uring from the outside of the VL, through chin sperm attached by its bindin to an egg
the VL and plasma membrane for linkage microvillus before extension of the acrowith the cortical cytoskeleton of the egg? some process (Epel and Vacquier, 1978). In
Newly obtained evidence using microelec- more favorable species, such as the horsetrodes to monitor the membrane potential shoe crab Limulus polyphemus, the vitelline
of the sea urchin egg indicates 1 — 2 mVstep layer is roughly 40 fim in diameter and the
change in potential preceding gamete fu- sperm acrosome process is at least that dision which is hypothesized to be caused by mension in length. In this species there are
occurrence of the acrosome reaction at the easily discernible phases of sperm attachVL surface or contact of the acrosome pro- ment to the VL by the acrosome content,
cess with the VL (Dale et ai, 1978). Could extension of the acrosome process through
this be caused by transmembrane signal- the thick VL and fusion of the tip of the
ing? Can the bindin receptor be removed process with the egg cell membrane (Brown,
intact from the VL, purified, and its inter- 1976).
SPECIES SPECIFICITY OF SPERM-EGG INTERACTIONS
Returning to our dearth of knowledge of
the mechanism of gamete membrane fusion, we can ask the following questions:
Are fusigenic proteins or special configurations of intramembraneous particles necessary? What roles do the membrane potentials of the gametes, the actin cytoskeleton
of the egg cortex and the possible opening
of ion channels play in membrane fusion?
Jaffe (1976; Jaffe and Robinson, 1978)
showed the resting potential of the unfertilized S.purpuratus egg of - 7 0 mV greatly
favors fusion with sperm. If eggs are voltage clamped to potentials approaching
0 mV, sperm will attach to, but not fuse
with, the egg. A rapid positive change in
membrane potential occurs within 0.1 sec
of sperm fusion and constitutes a very fast
block against polyspermic fusions (Jaffe,
1976).
A cytoskeleton of actin filaments underlies the sea urchin egg plasma membrane
(Begg and Rebhun, 1978). Could these
filaments be involved in the fusion process?
Longo (1978) and Byrd et al. (1977) report
that cytochalasin B inhibits the incorporation of sperm without blocking the cortical
granule exocytosis of the egg. The implication from this work is that the sperm acrosome process membrane must fuse with the
egg plasma membrane, but no fertilization
cone forms and so the sperm is not taken
up. Initiation of the cortical reaction removes the sperm from the egg surface.
Treatment of Urechis caupo eggs with
cytochalasin B also inhibits sperm penetration but does not block the activation of the
egg(Gould-Someroe£a/., 1978). In this case
it is uncertain whether membrane fusion
between the acrosome process and the egg
membrane has occurred, but it is difficult to
see how the egg could activate in response
to sperm without having had a fusion event
occur. From the above evidence it may be
tentatively concluded that the actin filaments in the egg cortex may play a yet undefined role in sperm-egg fusion and
sperm entry to the egg cytoplasm.
Fluofescein dyes have been found to reversibly block the fertilization of sea urchin
eggs (Carroll and Levitan, 1978). At low
concentrations, sperm bind to the eggs indicating occurrence of the acrosome reac-
847
tion, but the eggs do not activate, suggesting blockage of either sperm-egg or egg
cortical granule fusion. At higher concentrations both sperm binding to the VL and
egg activation are inhibited. The abilities of
different dye analogs to block gamete fusion is directly related to the lipid solubility of the dye, suggesting the hydrophobic
domains of membranes are the sites of action. Additional evidence for the involvement of gamete membrane lipids in spermegg fusion come from experiments in which
hamster eggs were first treated with a variety of either carbohydrases, proteases or
lipases and their fertilizability determined
(Hirao and Yanagimachi, 1978). Only
phospholipase C was found to be inhibitory
to fusion.
In the majority of cases, in both invertebrates and vertebrates, the interspecies barriers to fertilization reside in either failure
of the sperm to undergo the acrosome reaction or failure of the reacted sperm to bind
to the egg VL. Removal of the VL or other
egg investment by mild trypsinization is a
standard technique to bridge the interspecies barrier and achieve interspecies,
intergenus and even interphyletic fertilization (Longo, 19776). Another method is to
inseminate eggs in sea water containing
NaOH or NH 4 OH. This latter method was
discovered by Jacques Loeb (1916) who
used it to achieve fertilization between sea
urchin eggs and either starfish or sea
cucumber sperm. His method was to inseminate eggs in 50 ml sea water to which
0.6 ml of 0.1 N NaOH had been added.
Although he did not know the exact pH of
this mixture, repetition of his experiment
with a modern pH meter shows the pH to
be elevated from 8.0 to 9.2 which is exactly
the pH we use to artificially induce the
acrosome reaction (NH4OH, being a penetrating base, works better; Longo, 19776).
Not knowing about the acrosome reaction
or its necessity for achievement of fertilization, Loeb interpreted the success of
interspecific fertilization at elevated pH in
terms of changes in the ionic states and
conformations of cell surface proteins —
concepts which would certainly be acceptable to us today.
The past two years have yielded much
848
VICTOR D. VACQUIER
new information about the process of gamete interaction during fertilization in a
wide variety of animals. The prospects for
new and even more significant discoveries
in the coming years appear to be excellent,
given the present momentum and excitement in the fields of fertilization and cell
surface research.
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