AMER. ZOOL., 35:381-390 (1995)
Gamete Recognition and Egg Activation in Sea Urchins'
KATHLEEN R. FOLTZ
Department of Biological Sciences,
Division of Molecular, Cellular and Developmental Biology, and the
Marine Biotechnology Center, University of California,
Santa Barbara, California 93106-9610
SYNOPSIS. Free-spawning marine invertebrates face the challenge of
ensuring that gametes of the same species come into contact, recognize,
bind to and fuse with one another once they have been released by the
adults. Coordinated spawning, chemoattraction and specific cell-cell recognition events help to overcome this challenge. One marine invertebrate,
the sea urchin, has served as a model system for the study of gamete
recognition and fertilization for over 100 years. Recent biochemical and
molecular advances in this area have begun to address the questions that
have been raised by the results of elegant physiological observations. The
picture of fertilization that is emerging is characterized by highly specific
cell-cell interactions between proteins on the surfaces of the gametes.
These proteins then mediate the binding and subsequent events that lead
to activation of the egg and delivery of the male genetic material. Because
of these recent insights, the sea urchin egg is in a position to provide
answers to one of the central debates in developmental biology—the mechanism of egg activation. Does the sperm deliver an activating factor? Does
sperm binding trigger a receptor-mediated signal? Or is the mechanism
a complex combination? With the tools and knowledge gained from the
study of sea urchin fertilization, testing of these hypotheses should be
feasible in the near future.
INTRODUCTION
tive experimental model system because
The sea urchin has served as a classic sys- t h e y w e r e r e a d i l y available in large quantem for the study of fertilization and early t l t i e s , were easy to keep in the lab and prodevelopment for well over 100 years. The V l d e d m a n Y gametes. In the days when the
discoveries that the fusion of the pronuclei microscope was the major piece of laborawas critical (Hertwig, 1876) and that par- t o r v equipment, the sea urchin provided relthenogenesis was possible (Loeb, 1899) both a t l v e l y lar8e> transparent eggs that could be
were made by the study of the sea urchin e a s i l y fertilized and monitored. In fact, the
egg. As pointed out by the late Alberto Mon- s e a " r chm egg is one of the few cells in which
roy in a 1986 lecture, the importance of the t h e experimenter can monitor single cell
sea urchin in cell and developmental biol- e v e n t s (including physiological changes) as
ogy is due not only to the discoveries that w d l a s Perform large-scale biochemical
have been made but also to "the formula- analyses with relative ease (see Vacquier,
tion of problems" that have arisen by study- * 9 7 9 ) - T h u s > m a n y o f t h e k e y e v e n t s i n f e r "
ing these marine invertebrates (Monroy, tihzation and egg activation first were
1986)
described in the sea urchin and have since
Historically, sea urchins were an attrac- b e e n extended to other species (see Lillie,
1919). The sea urchin continues to contribute to the general knowledge of fertilization
today (for recent reviews, see Chandler,
• From the Symposium The Role of Cell-Cell Inter-
\ 991.
Foitz and
Le nna rz, 1993; Whitaker
actions and Environmental Stimuli in the Development _ _ J o
IQQ'U
ofMarine Invertebrates presented at the Annual Meet- Tana awann, w*).
.
,-,,•,
ing of the American Society of Zoologists, 27-30
« e purpose of this review IS to highlight
December 1993, at Los Angeles, California.
and discuss some of the recent discoveries
381
382
KATHLEEN R. FOLTZ
attraction
egg jelly
acrosome reaction
vitelline layer
penetration of
egg jelly
egg membrane
attachment to &
penetration of VL
binding to the
egg membrane
SEA URCHIN SPERM-EGG INTERACTIONS
(after Palumbi, S. 1992. Trends Ecol. Evol. 7, 114-118)
FIG. 1. Sea urchin gamete interactions (modified from Palumbi, 1992). Proteins on the surfaces of the sperm
and egg mediate each step. Bindin is exocytosed during the acrosomal reaction (see text) and interacts with the
sperm receptor, located in the egg plasma membrane. Binding and fusion are followed by the calcium wave,
leading to cortical granule exocytosis and subsequent fertilization envelope elevation.
that have been made regarding species-specific sea urchin gamete interactions and egg
activation. The major advances have concerned the identification and characterization of proteins on the gamete surfaces which
are involved in recognition and binding. In
addition, these recent data will be presented
in the context of the biology of the regular
echinoids; that is, their relevance to the animal's life history will be considered. Finally,
some of the outstanding questions that
remain will be presented.
BASIC INTERACTIONS BETWEEN THE
SPERM AND EGG
Many marine invertebrates in general and
sea urchins in particular face the challenge
of achieving successful species-specific
sperm-egg contact after spawning the
gametes into the sea water (Strathmann,
1987). This interaction can be viewed at
several levels in which kinetics and specificity must be considered. Reproductive
success requires not only that adults undergo
coordinated spawning but that the gametes
themselves be able to distinguish among different species of sperm and egg. Figure 1
briefly presents the various steps, discussed
below, at which this recognition occurs. The
sperm must be attracted to the egg of the
correct species, make contact with the egg
jelly coat, undergo the acrosome reaction
and then penetrate to the egg surface proper
where membrane binding and fusion occurs.
GAMETE INTERACTIONS AND EGG ACTIVATION
383
tides (e.g., speract, from Strongylocentrotus
Spawning and chemoattraction
Most echinoids are free-spawning; there- purpuratus) have been isolated and charfore, time and location of gamete release are acterized and the receptors on the sperm
critical and represent the only behavioral surface which recognize these peptides also
constraint that the adult plays in fertiliza- have been identified (Garbers, 1989). A surtion success (Tyler and Tyler, 1966; O'Rand, vey across echinoid species suggests that the
1988). Environmental cues, particularly the sperm attractant properties of the peptides
photoperiod, probably are proximal signals are not absolutely species specific but are
that coordinate gamete production and species-preferential (Suzuki, 1990). Ward
release (Pearse et al, 1986). The fact that and Kopf (1993) have provided a thorough
spawning often is observed to occur during review of the egg peptide factors which affect
phytoplankton blooms supports the sperm motility and attraction to the egg.
hypothesis that the adults are induced to
spawn once abundant food is available for Sperm-egg contact and the
the pelagic feeding larvae (Thorson, 1946; acrosome reaction
Himmelman, 1975,1981). Recent evidence
The jelly coat which surrounds the sea
indicates that phytoplankton may induce urchin egg induces sperm to undergo the
spawning via a heat-stable metabolic factor acrosome reaction, presumably by engaging
(Starr et al, 1990, 1992). This partially- a specific receptor on the sperm surface
purified factor induced spawning in sea (Trimmer and Vacquier, 1986; Ward and
urchins (Strongylocentrotus drobachiensis) Kopf, 1993). The egg jelly component that
in the laboratory. In addition, females appears to be essential for this acrosome
spawned more rapidly when treated with induction is a fucose sulfate polysaccharide
the factor and exposed to sperm, suggesting (SeGall and Lennarz, 1979, 1981) although
a synergistic effect (Starr et al, 1992). In there is some evidence to suggest that a glyfact, pheromones released with gametes coprotein may be involved (Keller and Vacprobably enhance synchronized spawning quier, 1992). Interestingly, the species-spec(Starr et al, 1992; Hyman, 1955; Kennedy ificity of the jelly component(s) ranged from
and Pearse, 1975). However, the identities complete to non-existent, depending upon
of the actual "spawning factors" are not yet which cross-species was tested. For examknown. Likewise, their corresponding ple, sperm from Arbacia punctulata and
"receptors," perhaps located in the adult Strongylocentrotus drobachiensis responded
gonad, that then trigger the gamete release only to their own egg jelly. However, S. purremain undefined. An interesting question puratus sperm responded to their own egg
is whether these spawning factors are spe- jelly and jelly from eggs of Lytechinus varcies-specific.
iegatus but not to that of A. punctulata.
Is the acrosome reaction a critical step in
Once spawned, the gametes must interact
within a relatively narrow window of time. fertilization? The answer in marine inverSea urchin sperm is viable for only a short tebrates whose sperm have acrosomes
while in seawater (< 1 hr) and successful undoubtably is yes; sperm that do not
fertilization requires relatively high sperm undergo the acrosome reaction do not fuse
densities (Pennington, 1985). Indeed, sperm with the egg (Ward and Kopf, 1993). The
concentration appears to be the most acrosome reaction appears to be highly regimportant parameter for fertilization suc- ulated (Summers and Hylander, 1975; Epel,
cess (Levitan etal, 1991). This necessitates 1978; Trimmer and Vacquier, 1986) and
that the sperm contact the egg as rapidly as involves the polymerization of actin filapossible. Sperm motility rates and patterns ments and extension of the sperm tip (Tilrespond to egg-associated molecules (Ward ney et al, 1978; Tilney, 1978). The acroet al, 1985). In fact, specific peptides resid- some reaction occurs as the sperm penetrates
ing in the jelly coat of sea urchin eggs serve the jelly coat. As part of the reaction, it is
as species-specific enhancers of motility and likely that proteases also are released and
probably as chemoattractants. These pep- activated; the role of these proteases is
384
KATHLEEN R. FOLTZ
unknown and there is some controversy over
whether they are required (Farach et al,
1987; Green and Summers, 1982; Levine et
al, 1978; Levine and Walsh, 1979, 1980;
Yamada et al, 1982). Finally, the acrosome
reaction also exposes a protein (bindin) on
the tip of the process which is responsible
for adhesion to the egg surface proper (see
below).
Contact with the egg surface
Like most marine invertebrate eggs, the
sea urchin egg has the jelly layer and a vitelline layer covering the oolemma proper
(Larabell and Chandler, 1991). Fertilization
of the egg requires that the plasma membranes of the two gametes fuse (see Myles,
1993). How is it that the two membranes
are brought into position for this event to
occur? Is this also a species-specific event?
Loeb (1916) suggested that specific proteins
on the sperm and egg surface were responsible; this idea was carried further by Lillie
(1919) and Tyler (1948). In 1977, Vacquier
and Moy succeeded in purifying the first
protein from any species responsible for
sperm-egg adhesion. This sea urchin sperm
protein, which they called bindin, was localized to the tip of the acrosomal process and
occupied the space between the egg surface
microvillus and the acrosome (Moy and
Vacquier, 1979). In several elegant papers,
it was demonstrated that bindin had the
ability to agglutinate egg species-specifically
(Glabe and Vacquier, 1977; Glabe and Lennarz, 1979; Vacquier and Moy, 1978).
The isolation of bindin from sea urchin
sperm sparked new interest in identifying
proteins and determining the mechanism of
gamete interactions and egg activation (see
Vacquier, 1979). Using techniques and ideas
developed from the study of sea urchin fertilization, investigators soon began identifying glycoproteins on the surfaces of mammalian gametes (see Wassarman, 1990;
Myles, 1993; Ward and Kopf, 1993).
BINDIN AND THE EGG RECEPTOR
FOR SPERM
Bindin
Several excellent reviews are available for
bindin (Trimmer and Vacquier, 1986; Minor
et al, 1989) and the reader is directed to
these for a thorough description of this
interesting cell adhesion protein. Since its
discovery (Vacquier and Moy, 1977), bindin has been cloned and sequenced from at
least four different sea urchin species (Minor
et al., 1991; Glabe and Clark, 1991). Comparisons of bindin from different species
have revealed specific as well as conserved
primary structure. There is a conserved central core flanked by two species-specific
domains. Recent investigations have
focused on the binding site and the basis of
specificity. Using recombinant bindin protein, Lopez et al. (1993) demonstrated that
the conserved central core must be flanked
by a portion of either one of the specific
regions to achieve binding to the egg. Unfortunately, the question of whether the central
core itself is sufficient could not be determined (Lopez et al., 1993). In an alternative
approach to identifying binding domains,
peptides representing portions of both the
core and the species-specific domains have
been tested for the ability to bind to eggs
and block sperm binding (Minor et al.,
1993). Peptides that represented S.franciscanus bindin sequences just to the N-terminal side of the central core blocked sperm
binding in both S. franciscanus and S. purpuratus. Together, the results of these two
studies suggest that at least a portion of the
species-specific domain of bindin is capable
of binding to the egg while the role of the
conserved core sequence remains unknown.
Relative affinities and some idea of true
functional binding (see below) must be
assessed. In addition, the issue of speciesspecificty will need to be addressed further
by testing the binding activity of the peptide
against other species' eggs.
The egg receptor for sperm
Efforts to isolate an egg surface component that mediated gamete adhesion in sea
urchins have a long history (excellent older
reviews include Epel, 1978; Metz, 1978). At
about the same time that bindin was isolated, several groups reported the characterization of a high Mr sperm-binding activity from sea urchin eggs (Tsuzuki et al., 1976;
Schmell et al, 1977; Glabe and Vacquier,
1978). Presumed to be a large glycoprotein,
GAMETE INTERACTIONS AND EGG ACTIVATION
this activity was referred to as the "sperm
receptor" or the "bindin receptor" located
on the vitelline surface of the egg (Glabe
and Vacquier, 1978; see also Gache et al.,
1983). Further, Schmell et al. (1977) had
demonstrated that binding activity was species-specific and trypsin-sensitive, further
supporting the idea that the binding protein
on the egg surface was a receptor for bindin.
Over the next ten years or so, the sperm
receptor was characterized further (reviewed
by Ruiz-Bravo and Lennarz, 1989).
Eventually, it was possible to isolate a
proteolytic fragment of the sperm receptor
which bound to sperm species-specifically
as well as to purified bindin (Foltz and Lennarz, 1990). This led to the production of a
specific antiserum which allowed the identification and characterization of the intact
receptor {ca. Mr 350 kD; Foltz and Lennarz,
1992; Ohlendieck et al, 1993). The subsequent cloning and sequencing of the sperm
receptor (Foltz et al., 1993) indicated the
presence of a species-specific, sperm binding domain as well as conserved domains.
Biochemical characterization indicated that
the sperm receptor was a membrane protein
(Foltz and Lennarz, 1992; Ohlendieck et al.,
1993); the deduced sequence also indicated
a signal sequence and transmembrane
domain (Foltz et al., 1993). Localizing the
sperm receptor to the oolemma meant that
the sperm either penetrated the vitelline
layer to reach the receptor or that the receptor protruded through the vitelline layer.
The finding that the sperm receptor was a
transmembrane protein was perhaps the
most surprising result and immediately
begged the question of whether or not it
transduced a binding signal (see below). With
both the sperm component (bindin) and the
egg component (the sperm receptor) in hand,
the stage has been set for addressing a variety of gamete recognition questions.
Bindin-receptor interactions
As mentioned, both bindin and the sperm
receptor have species-specific sequences.
With both bindin and the sperm receptor
identified, an obvious question to pursue is
the molecular nature of the binding and the
species-specificity. Evidence had accumulated which suggested that the carbohydrate
385
component of the sperm receptor was
responsible for adhesion (reviewed in RuizBravo and Lennarz, 1989). For example,
Pronase glycopeptides and small, heavilyglycosylated tryptic fragments derived from
the receptor bound to sperm but without
species-specificity (Ruiz-Bravo and Lennarz, 1986), which led to the conclusion that
the protein must be responsible for the specificity, perhaps by presenting the carbohydrate moieties to the sperm in a speciesspecific conformation. However, the complementary experiment {i.e., using a deglycosylated receptor) was not possible until
recombinant receptor became available.
Surprisingly, recombinant receptor protein
bound to sperm and to purified bindin species-specifically (Foltz et al., 1993). This
finding does not rule out the role of carbohydrates (which are predominantly
O-linked; Foltz and Lennarz, 1992; Ohlendieck et al., 1993) in sperm binding, but it
does indicate that protein-protein interactions do occur. It will be critical to assess
relative binding affinities for bindin of the
glycosylated and non-glycosylated sperm
receptor in order to determine the carbohydrate requirement. It is possible that a
first-step binding of low affinity occurs
between bindin and the carbohydrate moieties of the receptor followed by a higher
affinity, protein-protein interaction.
Moreover, the sperm binding domain that
has been mapped to a 419 amino acid
domain has sequence similarity to the heat
shock 70 protein (hsp70) family (Foltz et
al., 1993). Further structural analyses should
reveal if this similarity is meaningful in
terms of recognition and binding. Meanwhile, the question still remains as to the
refined mapping of the bindin-receptor
interaction sites; deletion analyses of the
receptor protein ought to provide information as to the minimal requirements for
binding and specificity (Stears and Lennarz,
1993). The determination of the precise
binding site on the receptor and likewise the
precise bindin sequences required should be
possible, perhaps by use of specific bindin
peptides (Minor et al, 1993). Likewise,
cloning and sequencing the receptor from
several species will enable the prediction of
those residues which influence specificity.
386
KATHLEEN R. FOLTZ
The resolving power of mutational analysis
in which the specificity is abolished or
changed makes this an attractive avenue of
investigation. With the ability to perform
high resolution binding site analysis by use
of recombinant proteins and the ability to
measure affinities for both sperm and bindin in the glycosylated as well as unglycosylated states, we soon should have a much
greater understanding of the structural basis
of gamete recognition and binding.
Evolutionary implications of
species-specific recognition
As discussed above, free-spawning organisms like the sea urchin have some level of
species-specificty control at each of the steps
of gamete interactions (Fig. 1). This specificty is reflected in the structure of the proteins involved. It has been suggested that
these gamete recognition proteins may be
important determinants of speciation and
evolutionary relationships (Minor et al,
1989; Palumbi, 1992; Palumbi and Metz
1991; Lee and Vacquier, 1992; Vacquier and
Lee, 1993). For example, in Echinometra
species, bindin amino acid sequence differences are greater than those predicted by the
average nuclear DNA differences seen in
these closely related species (Palumbi, 1992).
These differences may correlate with gamete
incompatability (Palumbi and Metz, 1991).
Likewise, sperm lysins (the protein responsible for egg entry) from multiple species of
abalone indicate a significantly higher number of amino acid differences than silent
substitutions (Lee and Vacquier, 1992; Vacquier and Lee, 1993). These data indicate
that egg-sperm interactions, at the molecular level, could be evolving rapidly in these
marine invertebrates (Palumbi, 1992).
Does the sperm receptor also exhibit this
extent of variation? That is, does the species-specific, sperm-binding domain have a
high number of non-synonymous amino
acid substitutions? Sequence analyses are in
progress to assess these questions. Changes
in gamete surface proteins could in fact lead
to reproductive isolation (Lee and Vacquier, 1992; Minor et al, 1989; Palumbi,
1992; Vacquier and Lee, 1993). Whether or
not variation in receptor protein sequence
would be related to population structure and
observed differences in fertilization success
rates among individuals is unknown (Levitan et al, 1991). Continued investigations
into the molecular co-evolution of the sperm
and egg proteins should prove fruitful in
terms of our understanding of recognition
and speciation.
The sperm receptor and egg activation
Despite a rich literature concerning signaling pathways that may lead to egg activation (see Turner and Jaffe, 1989; Epel,
1989; Swann and Whitaker, 1990; Nuccitelli, 1991; Whitaker and Swann, 1993)
very little is known about the precise mechanism that initially triggers activation.
Sperm binding leads to membrane depolarization, increased tyrosine phosphorylation and calcium release, to name but a few
of the responses, but the initiator(s) of this
myriad of egg activation events, which culminate in entry into the cell cycle, remains
a mystery. One hypothesis proposes that the
sperm delivers an activating factor, perhaps
a protein (Dale et al, 1985; Swann, 1990,
1993) or a packet of IP3 or calcium (Iwasa
et al, 1990; Jaffe, 1983). An alternative
hypothesis is that sperm binding triggers a
receptor-mediated signal transduction event.
Typical G-protein and tyrosine kinaselinked signaling pathways are in place in the
egg (see Kline et al, 1988, 1991; Shilling et
al, 1990, 1994; Kinsey, 1984; Peaucellier
et al, 1988, but their activity at fertilization
is not certain. Perhaps more than one mechanism is used (Foltz and Shilling, 1993;
Nuccitelli, 1991). Despite all that has been
learned about bindin and the sperm receptor, little can be said about their relationship
to egg activation events. Application of bindin to eggs results in egg agglutination, not
egg activation, as would be expected if bindin engagement of the receptor alone was
responsible for activation (see Nucitelli,
1991 for discussion). In fact, the only example of a purified sperm protein which activates eggs when externally applied comes
from the echiuran worm Urechis caupo
(Gould and Stephano, 1987, 1991; Gould
et al, 1986). A formal possibility is that
bindin is not being delivered or presented
to the egg in the proper way (Ruiz-Bravo
and Lennarz, 1989; Foltz and Lennarz,
GAMETE INTERACTIONS AND EGG ACTIVATION
387
1993). If so, this should be investigated fur- the timing of tyrosine phosphorylation
ther, perhaps by delivering a small, bindin- places this kinase activation in an upstream
coated bead to the egg surface. Of course, position, investigation of possible downthe absence of "activating activity" also may stream targets will be important. Ultiindicate that bindin is not sufficient for egg mately, it will be necessary to demonstrate
activation and that other egg or sperm com- the absolute requirement for the receptor in
ponents are needed. In fact, bindin may not egg activation if the hypothesis of receptoreven be required for activation but serve mediated signal transduction is to be suponly as the important recognition and adhe- ported. It is possible that other, as yet
sive protein that it is known to be. The same unidentified sperm and/or egg molecules are
can be said for the egg receptor for sperm. required. The most rigorous way to test the
Nonetheless, several intriguing observa- hypothesis will be through heterologous
tions have suggested that the sperm receptor expression studies in which mutational
is more than a "passive" recognition and analyses can be conducted.
binding protein. Although sequence analyIn conclusion, the sea urchin has prosis of the sperm receptor revealed that it vided the field of fertilization with a beauhad no homology to known signaling recep- tiful model system in which to study the
tors such as receptor tyrosine kinases or G related questions of the structural basis of
protein-linked receptors, recent investiga- gamete recognition and egg activation. The
tions have provided clues. Antibody which identification and characterization of the
recognizes the sperm binding domain of the surface proteins that mediate recognition
receptor caused some eggs to undergo inter- and binding not only have provided insight
nal calcium release and the cortical reaction into the mechanism of fertilization in genwhen externally applied (Foltz and Lennarz, eral, they also have opened up new avenues
1992). Our recent data indicate that the of investigation into the mechanism of
sperm receptor is tyrosine phosphorylated marine invertebrate cellular recognition and
in response to sperm binding. This tyrosine speciation.
phosphorylation occurs only when sperm of
the same species are applied and these sperm
ACKNOWLEDGEMENTS
must be acrosome-reacted, demonstrating
I thank Yama Abassi, Robert Belton,
biological specificity (Abassi and Foltz,
1994). Further, application of soluble bin- Patty Debenham, Laurinda Jaffe and Fraser
din to eggs also stimulated receptor tyrosine Shilling for advice and comments on the
phosphorylation. The eggs did not fully acti- manuscript. The author gratefully acknowlvate, although they did exhibit multiple, edges support from the NIH, the Searle
transient fertilization cone structures (Abassi Scholars Fund and the American Cancer
and Foltz, 1994). Perhaps most exciting is Society.
the observation that the tyrosine phosphorylation occurred prior to calcium
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