Gamete Research 4:151-169 (1981)
How Is Polyspermy Prevented?
Brian Dale and Alberto Monroy
Stazione Zoologica, Naples, Italy
INTRODUCTION
In eukaryotes, despite the diversity in the form and function of gametes and in the
behavioral and physiological adaptations of sexual reproduction, the fundamental event of
fertilization is the fusion of one female nucleus with one male nucleus. If more than one
sperm nucleus does interact with the female nucleus, then abnormal cleavage will occur
and death of the embryo ensues.
From laboratory experiments, mainly on sea urchins and mammals, we are familiar
with the image of an egg with one spermatozoon in the cytoplasm and several spermatozoa
outside the plasma membrane or the other egg investments. Such observations led to the
notion that eggs are endowed with membrane-located mechanisms to repel supernumerary
spermatozoa that become operative following the interaction with the fertilizing spermatozoon [see reviews by Rothschild, 1954; Gwatkin, 1977; Monroy and Moscona, 19791.
In this paper we have focused on (a) the changes that take place in the oocyte during
maturation, which result in its ability to interact with spermatozoa, and (b) the events that
occur in the oocyte in response to the fertilizing spermatozoon. These events, which are in
fact a part of the process of egg activation, will exclude the interaction of supernumerary
spermatozoa within a limited range of sperm density.
However, we raise the point that under natural conditions for many groups of animal including mammals, anurans, fish, insects, echinoderms, and nematodes - the successful collision rate is extremely low and this is achieved in part by the organization of the egg and
its investments and part by the absolute sperm-egg ratio at the site of fertilization.
SPERM-EGG INTERACTION: SOME GENERAL REMARKS
Fertilization depends on the specific recognition and binding of spermatozoa with
eggs. Hence, a distinctive characteristic of germ cells, with respect to somatic cells, is that
they are endowed with specific surface structures that allow interaction of gametes belong-
Received June 12,1980;accepted in final form October 7, 1980.
Address reprint requests to Brian Dale, Stazione Zoologica, Napoli 80121, Italy.
0148-7280/S1/0402-0151$05.50 @ 1981 Alan R. Liss, Inc.
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Fig. 1. A glycerol-treated egg of Ciona intestinalis after treatment with ferritin-conjugated Con A
showing a bound spermatozoon. The sperm binding sites, which are positive to Con A, are the tufts of
filaments on the outer surface of the chorion (arrows). Note that the sperm plasma membrane is intact.
(X82,OOO.) [From De Santis et al, 19801.
ing to the opposite sexes, while preventing interaction between gametes of the same sex (a
situation similar to the surface exclusion in bacteria). The phylogenetic history of gamete
interaction has been discussed elsewhere [Monroy and Rosati, 1979a, b].
Except in animals whose spermatozoa lack an acrosome (these will be discussed later),
sperm-egg interaction occurs in two steps. First, the binding of spermatozoa to the spermbinding sites of the vitelline coat (or its equivalent, eg, the so-called chorion of the Ascidians
or the zona pellucida of the mammals). Second, the fusion of the egg plasma membrane
with the inner acrosomal membrane, which becomes exposed during the acrosome reaction.
The vitelline coat of the sea urchin egg is made up of a dense meshwork of fibrils and
tightly adheres to the microvilli that emerge from the egg surface [Chandler and Heuser,
19801. It is connected to the egg plasma membrane by short processes indicated as “vitelline posts” [Kidd, 1978; Chandler and Heuser, 19801. In a scanning electron microscope
How Is Polyspermy Prevented?
153
view, the egg surface is covered by densely packed papillae that appear to be binding sites
of the spermatozoa [Tegner and Epel, 1973,1976; Schatten and Mazia, 19761. In the Ascidian egg the sperm-binding sites consist of tufts of very thin fibrils positive to Ruthenium
Red, Concanavalin A and the fucose binding protein [De Santis et al, 19801 (Fig. 1). In
the mouse egg a glycoprotein component of the zona pellucida has been identified as the
sperm receptor [Bleil and Wassarman, 19801.
One unanswered question is whether the acrosome reaction occurs before or after
the spermatozoon has reached the vitelline coat. It is a widely held notion that in the sea
urchin the acrosome reaction is triggered by the interaction of the spermatozoon with the
egg jelly coat. Hence, that the species-specific attachment of the spermatozoon to the vitelline coat, mediated by the protein “bindin” [see review by Moy and Vacquier, 19791, takes
place after the acrosome reaction has occurred. This notion is based on the well-known
fact that jelly coat solutions are powerful triggers of the acrosome reaction. A criticism of
these experiments is that the component molecules of the jelly coat in solution may have
reactive groups exposed that are not normally exposed in the natural “gel” condition. For
example, small pH changes may trigger the reaction. In this respect it is pertinent to mention the observation of Decker et a1 [1976] that solutions of Arbacia jelly coat at normal
pH of the sea water fail to induce the acrosome reaction. Of particular interest are the experiments of Aketa and Ohta [1977] on the eggs of Pseudocentrotus depressus. The jelly
coat of this egg can be neatly stripped off the egg as an empty hull by centrifugation. Upon
insemination, the hulls are penetrated by spermatozoa that, however, fail to undergo an
acrosome reaction. On the other hand, the spermatozoa attached to the vitelline coat of
dejellied eggs underwent the acrosome reaction [see also Kimura-Furukawa et al, 19781,
It should, however, be kept in mind that it is essentially impossible t o completely remove
the jelly coat from the egg [see Vacquier et al, 19791. Although this is the only sea urchin
egg in which, due to the structure of the jelly coat, such an experiment could be done, the
observation suggests that the jelly coat in its compact “gel” condition (ie, when it does not
quickly dissolve or swell in sea water as is the case of the Arbacia) fails to trigger the acrosome reaction. An answer to this problem may only come from experiments designed to
reproduce as closely as possible the conditions under which the encounter of gametes occurs
in the sea. Nevertheless, the observations mentioned above leave the question open of the
role of the jelly coat in triggering the acrosome reaction.
A detailed analysis of the acrosome reaction in the starfish (to our knowledge the
only well-documented case of the acrosome reaction taking place upon contact of the
spermatozoon with the outer layer of the jelly coat) may provide interesting clues. In this
connection, it is worth mentioning the isolation from the starfish egg of a jelly coat fraction
with acrosome reaction-inducing activity [Uno and Hoshi, 19781. In the Ascidian egg the
spermatozoa bind to the fibrillar tufts with their plasma membrane intact; some of them
then undergo the acrosome reaction [De Santis et al, 19801. In the mouse [Saling and
Storey, 19791 only unreacted spermatozoa are able to bind to the zona pellucida; binding
is then followed by the acrosome reaction and penetration of the spermatozoon through
the zona.
The acrosome and the vitelline coat must have undergone a parallel evolution. Indeed,
in some animals whose eggs have a micropyle as the only site through which the spermatozoon
can reach the egg surface (ie, fish, insects, squid) the spermatozoa lack an acrosome. The
acrosome may be considered a tool that has evolved concomitantly with the sperm receptors on the vitelline coat and enables the spermatozoon to penetrate the vitelline coat.
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Fig. 2. A dark-field illumination photograph of a live egg of Ciona intestinalis showing the large
finger-like follicle cells. [Courtesy of Drs. F. Rosati and R. De Santis.]
FACTORS LIMITING SPERM-EGG FUSION
Before interacting with the vitelline coat, the spermatozoa must traverse and interact with the outer egg investments, which in sea urchins, ascidians, and mammals are the
jelly coat, the follicle cells, and the cumulus cells, respectively. All these layers drastically
reduce the number of spermatozoa that reach the binding sites on the underlying vitelline
coat.
The question of the role of the jelly coat in sea urchm fertilization is still a matter
of controversy [see the review of Metz, 19781. Ideas have been strongly influenced by the
fertilizin theory of F.R. Lillie [1914] that assigned the jelly coat a key role not only in
sperm-egg interaction but also in the activation of the egg. The opposite view is that the
jelly coat, in situ, reduces the number of viable spermatozoa by up to 90% and hence the
jelly coat was suggested t o be the first “block to polyspermy” [Hagstrom, 19591.
The results of comparative studies [Vacquier, 1979; Vacquier et al, 19791 may suggest species differences: in Lytechinus pictus jelly solutions enhance fertilization of dejellied eggs; in Strongylocentrotus purpuratus they decrease fertilizability. However, we
find it difficult to accept that a process of such an importance in the control of fertilization should operate in such different and even opposite ways in closely related species.
Furthermore, the rapid loss in fertilizability of spermatozoa at high dilutions may
dramatically reduce the viability of a large fraction of the spermatozoa [see in particular
How Is Polyspermy Prevented?
155
Fig. 3. A side view of the animal pole of a live Discoglossus (Anura) egg showing spermatozoa
in the animal dimple. (X40.) [From Campanella, 1975.1
Kinsey et al, 19791. In the Ascidians, spermatozoa can react only with the restricted areas
of the vitelline coat that are not covered by the follicle cells [De Santis et al, 19801 (see
Fig. 2).
Having passed the outer investments the spermatozoa, as mentioned in the previous
section, must locate and interact with the binding sites on the vitelline coat. In the dejellied
sea urchin egg there are 1,018-1,745 binding sites per egg for Lytechinus pictus and 1,0983,000 for Strongylocentrotus purpuratus [Vacquier and Payne, 19731, while a maximum
of 2,500-3,000 have been estimated for the Ciona egg denuded of its follicle cells [Rosati
and De Santis, 19801. However, it appears that not all the spermatozoa attached t o the
vitelline coat are triggered into an acrosome reaction - for example, in the case of the
Ascidians [De Santis et al, 19801 and of mammals [Saling and Storey, 19791 - and hence
have the capacity to enter the egg. This may suggest a diversity in the steric organization
of the vitelline coat receptors, whereby a specific molecular fit between the interacting receptors on the spermatozoon plasma membrane and those on the vitelline coat is required
for the induction of the acrosome reaction.
An additional factor limiting sperm-egg fusion is the organization of the egg plasma
membrane. In some eggs, sperm-egg fusion is restricted to a limited area of the egg surface.
In Ascidians, this occurs in a region of about 30" at the vegetal pole [Conklin, 19051, and
can be demonstrated in live eggs deprived of their vitelline coat. In Anurans the site of
sperm entry is restricted to the animal hemisphere. The most strilung case is that of Discoglossus in which the site of interaction is limited to a small dimple [Campanella, 19751
(see Fig. 3). The fine structural organization of these sites is different from that of the rest
of the egg surface [Campanella, 1975; Monroy and Baccetti, 1975; Denis-Donini and Camp
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Dale and Monroy
anella, 19771. In Xenopus, spermatozoa can be “forced” to penetrate the vegetal pole,
although such sperm are unable to develop into pronuclei [Graham, 19661 ;a similar situation exists in Rana pipiens [Elinson, 19751. Alternatively, in the urodele, Pleurodeles,
spermatozoa may enter anywhere over the egg surface [Picheral, 1977a, b]. These observations point to a differentiation of the egg plasma membrane taking place during oogenesis
whereby only a limited part of its area becomes receptive to spermatozoa. It is worth mentioning that such specialization of the membrane may be connected with the origin of
polarity of the oocyte. It is known that in a number of animals, the surface of the oocyte
in contact with the ovarian wall becomes the vegetal pole of the egg - for example, in
Unio [Lillie, 19011 and in Lymnaea [Raven, 19701 - and this is also the site of sperm
penetration.
The restriction in area available for sperm penetration is of course most obvious in
the case of eggs with micropyles (ie, fish, insects, squid). The diameter of the micropyle
is usually the same as the head of the spermatozoon and, as mentioned previously, the
spermatozoa lack an acrosome. In trout and other salmonids, when a spermatozoon reaches
the egg plasma membrane the cortical alveoli begin to open and their contents form a plug
in the micropyle [Ginsburg, 1963a; Brummett and Dumont, 19791. Thus, only one spermatozoon is allowed to reach the egg surface. That the teleost egg surface itself has no
mechanism t o prevent polyspermy has been recognized for some time. Indeed, removal of
the chorion either chemically or manually will lead to polyspermy if, of course, the egg is
challenged with several spermatozoa [Sakai, 1961; see also Nakano, 1969, for a review].
To our knowledge the process of sperm-egg fusion in animals lacking an acrosome has
been studied only in Ascaris [Foor, 19701 and in the teleost Oryzias [Iwamatsu and Ohta,
19781 and Fundulus [Brummett and Dumont, 19791. In both these teleosts the plasma
membrane of the spermatozoon head fuses with the egg plasma membrane. Since in these
studies spermatozoa could enter all over the surface of dechorionated eggs, two possibilities
may be considered. First, that specific sperm-binding sites are present all over the egg plasma
membrane and, of course, on the head of the sperm. Or, second, the surface of both gametes
have no specific binding sites and the fusion of gametes occurs through a process akin to
the fusion of somatic cells, possibly mediated by some substances in the perivitelline space.
SPERM-EGG RATIO AT THE SITE OF FERTILIZATION
In some animals, spermatozoa are produced in great excess; for instance, in man
the sperm-egg ratio can be as high as 109:1 [see Gwatkin, 19771 and in sea urchin 104:1
[see Harvey, 19561. Despite these high ratios behavioral adaptations are also necessary to
ensure fertilization: in the case of echinoderms and some polychaetes, aggregation of mature animals and the simultaneous spawning of the sexes [Hyman, 1955; Austin, 19651; in
mammals, the deposition of sperm in the female tract and the synchrony of mating [see
Austin, 1965 for documentation of other mechanical devices employed in the bringing together of sperm and eggs].
In mammals it is widely accepted that spermatozoa undergo extensive dilution from
the point of insemination to the site of fertilization at the ampulla. For example, a maximum of 700 spermatozoa have been found in sheep ampullae [Braden et al, 19541 and a
minimum of five in man [Doak et al, 19671. However, the technique of flushing tuba1
gametes carries the risk of dislocating spermatozoa from the other segments of the oviduct.
In a study of fertilization in the mouse, fixing ampullar gametes in situ, at the site of conjugation a 1 :1 sperm-egg ratio was discovered - supernumerary spermatozoa were never
observed [Stefanini et al, 19691.
How Is PoIyspermy Prevented?
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Unfortunately, there is no information available on the sperm-egg ratio of echinoderms at the site of fertilization and the scarce information on their behavior at spawning
[Hyman, 19551 is of little help in such an estimation. However, it has recently been shown,
by a direct electrophysiological technique [De Felice and Dale, 19791, that for jelly-free
sea urchin eggs exposed t o spermatozoa at a density of lo6 per ml, the successful collision
rate (termed a) is about 1 every 10 seconds. As mentioned previously, the jelly coat in
situ, particularly shortly after spawning when it is tough and compact, may reduce this
rate to less than 1 to every 100 seconds. A density of lo6sperm/ml implies a dilution factor
of 1,000 to 10,000 of dry sperm from the testis and we feel that such a dilution factor is
reasonable for sea urchins in natural conditions even if the animals aggregate at spawning.
In other animals, notably insects [see the review of Parker, 19791 and nematodes
[Ward and Carrel, 19791, sperm utilization is much more efficient. For instance, in Drosophila, Lefevre and Jonsson [1962] discovered a 1 :1 relationship between the progeny recovered and the number of sperm counted in the seminal receptacles. In hermaphrodite
fertilization of the nematode Caenorhabditis elegans every spermatozoon fertilizes an
oocyte; however, not all oocytes are fertilized because in fact oocytes are produced in excess [Ward and Carrel, 19791. The high efficiency of sperm utilization in insects and nematodes with internal fertilization may be an important adaptation as it enables a minimization of volume and provision of nutrients for the stored sperm [Parker, 19701. The reduction in genetic variability in these animals as a result of low numbers of gametes produced
may be offset by the phenomenon of sperm displacement by second matings [see Parker,
1970; Ward and Carrel, 19791. In the mammals the high number of sperm produced may
be significant in ensuring maximum genetic variability.
THE ACQUISITION BY THE OOCYTE OF THE ABILITY TO RESPOND TO THE
SPERMATOZOON
The oocyte acquires the ability to interact with the spermatozoon and to give rise
to a zygote during the process of maturation. This was aptly described by Delage [1901]
as cytoplasmic maturation. It should be noted that cytoplasmic maturation does not necessarily coincide with the completion of meiosis;however, in those eggs that are fertdized
normally before the completion of meiosis; decondensation of the sperm chromatin and
pronuclear fusion have to await the ejection of the second polar body.
The plasma membranes of sperm receptive oocytes have characteristic electrical
properties (Fig. 4). In starfish, sea urchins and amphibia the germinal vesicle stage oocyte
may be described as having a K' selective plasma membrane with a high resting potential
of -70 to -90 mV and a relatively low specific resistance. Following the breakdown of
the germinal vesicle (GVBD), the K' selective permeability is lost and there is a depolarization and decrease in conductance of the plasma membrane [for starfish: Moreau and Cheval,
1976; Miyazaki et al, 1975; Dale et al, 1979; for amphibia: Morrill and Watson, 1966; Wallace and Steinhardt, 1977; Morrill and Ziegler, 1980; for sea urchin: Dale and De Santis,
1981al. Starfish and amphibian oocytes are fertilized shortly after GVBD but before the
completion of meiosis, ie, in this low resting potential state. Other examples of sperm receptive oocytes demonstrating this low resting potential state [see also Hagiwara and Jaffe,
19791 are: mammalian oocytes at metaphase of the second meiotic division [Powers and
Tupper, 1974; Eusebi et al, 19791;ascidian oocytes arrested at metaphase of the first meiotic division [Dale et al, 1978'03; Urechis, an echiuroid worm, at diplotene-diakinesis of
first division [Could-Somero et al, 19791;and finally, fish eggs at second metaphase [Nuccitelli 19801. If starfish oocytes are left standing in sea water for several hours (ie, aged),
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Dale and Monroy
nA
1
0.5
mV
I
-80
1I
0
-0.5
-1
Fig. 4. Intracellular electrical recordings from oocytes of the starfish Astropecten aurantiacus, complete with jelly and follicle cells in natural sea water at 20”C, showing some changes in the electrical
properties of the oocyte plasma membrane following germinal vesicle breakdown (GVBD). Around
the time of GVBD the resting potential switches from about -80 mV to -20 mV (see inset, horizontal
bar represents 5 sec, vertical bar 10 mV). The I-V curve on the left is from an oocyte before GVBD,
that on the right from the same oocyte after GVBD (induced by
M 1-methyladenine). Note the increase in membrane resistance at rest, the shift in the resting potential and the change in curvature of
the I-V relationship following GVBD. Starfish oocytes are normally fertilized shortly after GVBD,
ie, while in this latter electrical state. [From Dale et al, 1979.1
they develop a new K’ permeability returning to a high potential of -70 to -90 mV [Miyazaki et al, 1975; Miyazaki and Hirai, 1979; Dale et al, in preparation].
The phenomenon of aging of marine invertebrate eggs is well known [see Harvey,
19561, although difficult to define. Eggs age, both in the ovary and following ovulation
(or removal from the ovary); however, in the latter situation the process is considerably
accelerated. The most elegant demonstration of this was provided by the experiments of
Borei [1948] who showed that the rate of oxygen consumption of sea urchin eggs removed
from the ovary rapidly and steadily declines. It is also worth remembering that underripe,
ripe, and overriGe eggs respond differently to the “hypertonicity test” [Runnstrom and
Monnk, 19451, which may be an indication of changes occurring in the microfilament system. [See Borei’s 1948 paper for an interesting discussion on maturation and aging of the
sea urchin egg]. Such rapidly occurring changes in the egg when removed from the ovary
may explain the observation that in mammals [see Gwatkin, 19771 and starfish [Fujimori
and Hirai, 19791 in vitro aging is paralleled by an increased incidence of polyspermy.
How Is Polyspermy Prevented?
159
Aging may also account for the discrepancy in the literature over the resting potential of sea urchm eggs. Several authors have reported the resting potential of sea urchin
sperm-receptive oocytes - w h c h being haploid may be considered eggs - to be in the
range -8 t o -30 mV [Steinhardt et al, 1971; Dale et al, 1978al ;in contrast, Jaffe and
Robinson [ 19781 and Chambers and De Armendi [ 19791 maintain that the resting potential approximates -70 to -90 mV. In recent work from our laboratory [Dale and De Santis 1981a], we have observed that eggs from suboptimal animals (underripe or in a state
of gamete regression) and eggs left standing in sea water for several hours may have high
resting potential of -70 t o -90 mV, whereas newly spawned eggs from mature animals
have low resting potentials. Two possibilities may be considered. First, and we feel the
most likely, the two resting potential levels reported in the literature are true measurements, but reflect different conditions of the sea urchin egg with regard t o age. That is,
the sea urchin sperm-receptive oocyte in its prime condition has a low resting potential
comparable to other sperm-receptive oocytes from different groups (see above) that increases with age (as in the starfish). Alternatively, following the depolarization at GVBD
[Dale and De Santis, 1980a1, sea urchin eggs from some species revert to a high resting
potential of -70 to -90 mV and the low resting potentials measured are the result of
leakage due to impalement.
In starfish, the optimum period for the fertilization of oocytes is between GVBD
and the formation of the first polar body [Delage, 19013 ;insemination after this period
results in an increase in the incidence of polyspermy and consequently abnormal development [Fujimori and Hirai, 19791. This has been interpreted in terms of the changed electrical properties of the oocyte plasma membrane [Miyazaki, 1979; Mijiazalu and Hirai,
19791. However, we do not entirely agree with this interpretation. The oocyte as a composite unit geared to interact with the spermatozoon at a precise moment in time will of
course undergo many changes during aging. For instance, the jelly layer will be dissolved
almost completely after several hours in sea water and the topographic distribution and the
function of sperm binding sites may well be altered. In the cytoplasm, synthetic processes
may be initiated precociously and there may be a partial dissolution of cortical granules.
In fact, in aged oocytes the initiation of fertilization membrane elevation appears to be
delayed by up to 60 seconds [compare Figs. 1 and 3 in Miyazaki and Hirai, 19791. A delay
in elevation of the fertilization membrane of sea urchins will increase the probability of polyspermy [see De Felice and Dale, 19791.
A similar situation exists with immature oocytes: when immature and mature oocytes
are inseminated with a sperm density that does not induce polyspermy in the latter, the
former are often polyspermic. This has been taken as circumstantial evidence that during
cytoplasmic maturation the oocyte develops “active” polyspermy-preventing mechanisms.
In a recent report [De Felice and Dale, 19791, it has been shown that the successful collision rate (a)is similar for immature oocytes and eggs. That is, at high sperm densities both
oocytes and eggs become polyspermic. However, in the latter case, spermatozoa successfully interact with the egg only for a period of about 10 seconds up to the initiation of
the cortical reaction. In immature oocytes, where there is no cortical reaction [Lonning,
19671, spermatozoa continue to enter the oocyte for several minutes [De Felice and Dale,
19791. Therefore, it is obvious that at lower sperm densities (and hence (Y lower than 1 t o
every 10 seconds) eggs are normally monospermic while oocytes are often polyspermic.
This is so not because the latter lack a polyspermy block, but rather, they are not triggered
into embryonic development by the entering spermatozoa. Under natural conditions, immature oocytes and aged oocytes normally would not be exposed to spermatozoa.
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Fig. 5 . Intracellular recording from an egg of Paracentrotus lividus during fertilization. The first detectable electrical event is the step-like depolarization accompanied by an increase in voltage noise.
The step event precedes the well-documented fertilization potential by about 13 seconds at 22°C.
Vertical bar represents 12 mV (upper trace), 1 mV (lower trace, ac corpled from 1 Hz); horizontal
bar represents 5 seconds and also the zero potential level. Sperm density was 103/ml. [From Dale et
al, 1978al.
On the basis of the observations discussed so far, we propose that the changes in the
electrical properties of the egg plasma membrane both in the course of maturation and
aging are a reflection of the physiological and structural reorganization occurring in the
egg as a whole. In the life history of the oocyte egg, maturation sets in motion a chain of
physiological and structural changes giving rise to a cell unit geared to interact with the
fertilizing spermatozoon at a particular moment in time. However, thn condition is a
transient one; should the egg not be fertilized, the continuation of the maturation processes
soon leads t o overripeness (and this is greatly accelerated in oocytes outside the ovary)
whereby the reactivity of the oocyte to the spermatozoon is altered. Among other things,
the probability of the egg becoming polyspermic is increased [see also Gwatkin, 19771.
POLYSPERMY PREVENTION AS A PART OF EGG ACTIVATION
The interaction of the spermatozoon with the egg triggers a series of events that
change the metabolically repressed egg into the zygote. While it may be correct to try and
dissect out the various events starting from the first interaction of the spermatozoon with
the egg, it is certainly erroneous to think of the changes in the egg plasma membrane as
sometlung independent of the cytoplasmic changes.
The so-called cortical reaction involves an extensive reorganization of the egg surface
and the vitelline coat. In the sea urchin egg, the most extensively studied, the vitelline
coat and the plasma membrane may be considered as forming a functional unit due to the
“vitelline posts” that connect one to the other [Kidd, 1978; Chandler and Heuser, 19801.
It has been shown that upon fertilization, the posts are broken [Chandler and Heuser,
How Is Polyspermy Prevented?
161
19801 possibly by a protease released from the cortical granules [Vacquier et al, 1973;
Carrol and Epel, 19751. This results in breakdown of the unit and an extensive alteration
of the organization of the egg surface and the vitelline coat. In the sea urchin egg the
major changes in the plasma membrane are due to the insertion of patches derived from
the membrane of the cortical granules. In the mouse oocyte it has been shown that the
lateral diffusion of proteins and lipids is strongly restricted after fertilization [Johnson
and Edidin, 19771.
Electrophysiological studies [Dale et al, 1978al have shown that the first detectable
event across the plasma membrane during sperm-egg interaction is a step-like depolarization of 1-2 mV accompanied by an increase in voltage noise and decrease in resistance
(Fig. 5 ) . By analogy with similar events in other biological membranes, this observation
has been interpreted as the introduction (or activation) of nonspecific ionic channels into
the egg plasma membrane. Between 10 and 15 seconds later, the cortical reaction causes
dramatic changes in the egg surface resulting in the slow overshooting fertilization potential (FP) [Dale and De Santis 1981al . There are also some changes in the actin filaments
located just beneath the plasma membrane that may be important in the reorganization
of the egg, including its surface, that are also related to activation [Spudich and Spudich,
1979; Wang and Taylor, 19791. The former authors have speculated that the G-actin,
sequestered on the inner surface of the unfertilized egg plasma membrane, may be induced
to polymerize upon fertilization; this is analogous to the situation described for the acrosome reaction in Thyone spermatozoa [Tilney, 19761. T h s change in the cortical microfilament system may well occur during the first few seconds of sperm-egg interaction. In
fact, when fertilization is carried out in the presence of cytochalasin (B or D), the normal
electrical events occurring at the egg plasma membrane are delayed [Dale and De Santis,
1981bl. A change in form of subcortical actin may be related to the release of Ca2+ and be
instrumental in the propagation of an “activation wave.” The Ca2+release is so far the
earliest detected change in the egg cytoplasm upon fertilization. There is evidence that
both in the fish [Ridgway et al, 1977; Gilkey et al, 19781 and in the sea urchin [Steinhardt et al, 19771 egg the surface cytoplasmic layers are the site of the Ca2+release.
Turning now to the vitelline coat the most relevant change at fertilization is the
loss of sperm-binding ability. This appears to be due to its interaction with a protease
released from the cortical granules [Vacquier et al, 1973; Carrol and Epel, 19751. The
connection between cortical granule exocytosis and the fertilization-dependent changes of
the vitelline coat has been known for a long time [see Monroy, 19731. The question is,
however, whether these changes can be considered as the primary mechanisms that prevent multiple sperm-egg fusions. In most species of sea urchin the events between the
attachment of the spermatozoon and the cortical and cytoplasmic changes follow one
another at such a rapid rate that it is very difficult to be sure of their temporal sequence.
In this connection, the observations of Ginsburg [1963b] are of particular relevance. In
Strongylocentrotus droebachiensis she found that the “polyspermy block” coincided temporally with the time of propagation of the breakdown of the cortical granules. The protease released from the egg only reaches a detectable concentration in the sea water 30
seconds following insemination [Vacquier et al, 19731;however, at the egg surface its
concentration may be quite high much earlier. Finally, it has recently been observed [Coburn et al, 19791 that hydrogen peroxide - a potent sperm inactivator - is released by
sea urchin eggs upon fertilization. Fertilization in the presence of catalase results in 100%
polyspermy. Also soybean trypsin inhibitor - which is known to induce polyspermy [Vacquier et al, 19721 - prevents the release of hydrogen peroxide, for example, in white blood
cells [Goldstein et al, 19793.
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All of the changes occurring in the egg, as a result of its activation by the fertilizing
spermatozoon, both physiological and structural will inhbit the progression of a second
spermatozoon. However, we have argued in the previous sections that under natural conditions the successful collision rate for mammals, echinoderms, ascidians, anurans, fish, and
insects (previously classified as animals exhibiting membrane-located polyspermy-blocking
mechanisms [see Rothschild, 19541) is extremely low and this is achieved in part by the
organization of the egg and its investments, and in part by the absolute sperm-egg ratio
at the site of fertilization. The role of activation changes in limiting multiple sperm-egg
fusions under natural conditions remains an open question for further study; in particular,
the spawning behavior of animals that practice external fertilization.
Finally, in this section let us devote a few words to the location and timing of proposed “active” polyspermy blocking mechanisms. Several authors have suggested that a
“late” plasma membrane block exists that comes into effect 10-1 5 minutes after fertilization in sea urchins [Schatten, 19781 and after several hours in mammalian eggs [Barros
and Yanagimachi, 1972; Wolf, 1978; see also Yanagimachi, 19781. However, if the fertilization membrane and the zona reaction prevent the entry of supernumerary spermatozoa,
what is the advantage of a second stage “block”; especially as it takes much longer to develop than the first stage “block”? For instance, in the hamster the zona reaction is effective after 15 minutes, whereas the vitelline reaction takes 2-3.5 hours to develop [Barros
and Yanagimachi, 19721; hence, if after 15 minutes a second spermatozoon managed to
penetrate the zona pellucida, it would also probably enter the egg.
In mammals, it is commonly thought that polyspermy-blocking mechanisms exist
either at the egg membrane (eg, in the rabbit [Braden et al, 1954; Yanagimachi, 1978]),
at the egg investments - the zona reaction - (eg, in the sheep and pig [see Austin, 1965]),
or a combination of both (eg, in the mouse and rat [Austin, 19651). However, some of
the experimental techniques, from which such ideas have arisen, are equivocable. For instance, an egg denuded of its zona pellucida of one species of mammal may become polyspermic upon insemination, while a denuded egg from a second species may not. One conclusion often drawn from such an experiment is that in the first species the zona pellucida
is responsible for polyspermy prevention, while in the second it is the egg membrane. When
eggs are denuded of their investments, the process of sperm-egg interaction will be altered,
in that the spermatozoon will not encounter a normal sequence of trigger events. Whether
fertilization occurs at all probably will depend upon a percentage of the spermatozoa undergoing a spontaneous (or certainly unprogrammed) acrosome reaction close to the egg membrane (ie, “tricked” into an interaction). Hence it is equally plausible in the above example
that it is difficult to “trick” the spermatozoa of the latter species into reacting with the
nude egg, whereas in the first species it is much easier. The technique of denuding eggs
may be of use in comparative studies only if the conditions are strictly comparable, ie,
sperm density and activity, method of removing the outer layers, and surface area of eggs.
In fact, such a situation as the one described above has been found in some Mediterranean
Ascidians deprived of their chorions.
Nude Ciona eggs become polyspermic with relatively low concentrations of spermatozoa, whereas in order to fertilize nude eggs of Phallusia, high sperm densities are required
and polyspermy is rare (G. Ortolani, personal communication).
THE FAST PARTIAL BLOCK HYPOTHESIS
If we make the assumption that the successful collision rate is quite high and greater
than the time for initiation of the cortical reaction and consequently its complete propaga-
How Is Polyspermy Prevented?
163
Fig. 6. (a) Phase-contrast micrograph of a Psammechinus egg inseminated at 3.5 X lo' spermlml. Three
sperm nuclei are indicated by arrows; altogether nine sperm nuclei were found in this egg. Egg diameter
is about 100 pm. @) The fertilization potential recorded during insemination of the egg in (a). Vertical
bar, 10 mV; horizontal bar, 5 sec and zero potential. (c) The shoulder of the fertilization potential at
higher magnification. The upper trace is dc-coupled (vertical bar, 1 mV). The lower trace is ac-coupled
at 1 Hz (vertical bar, 0.4 mV). The horizontal bar represents 1 sec for both traces. At least 5 step-depolarizations are seen. Each step depolarization signifies a successful sperm-egg interaction and consequently sperm entry. [From De Felice and Dale, 1979.1
tion, then to ensure monospermy, a faster acting block would also be required. Rothschild
and Swann [1951, 19521, on the basis that the rate of refertilization'of eggs is much lower
than the rate of initial fertilization, suggested that a rapid acting partial block existed that
reduced the probability of a successful reaction following the first [Rothschild and Swann,
19521 .By the nature of their refertilization experiments, it was not possible to measure
164
Dale and Monroy
initial fertilization rates at sperm densities hgher than 107/ml or refertilization rates lower
than lo* sperm/ml. The discontinuity of their data points is more obvious when displayed
on a linear graph of successful collision rate versus sperm density (see Fig. 4.) in De Felica
and Dale, 1979). In addition, their refertilization rate is perforce an underestimate of the
successful collision rate as it is calculated from the rate of increase of polyspermic eggs and
not the actual number of spermatozoa per egg. Finally, sperm-sperm interactions at higher
sperm densities (ie, as used per refertilization) may also contribute to the lower rate they
obtained.
Jaffe [1976] suggested that a fast polyspermic block existed that was electrically
mediated. That is the fertilizing spermatozoon causes a rapid overshooting depolarization
across the egg plasma membrane that renders the egg unreceptive to a second spermatozoon.
f i s response was only observed in a minority of eggs. Eggs were discarded that did not
adhere to plastic petri dishes or that displayed low resting potentials. Of the remainder,
only one-third (eight eggs in total) displayed this overshooting depolarization. Although
these eggs were indeed monospermic, there was no evidence that they were challenged by
a second spermatozoon before the cortical reaction was initiated. To further test this
hypothesis, we would expect the fraction of monospermic eggs to remain constant even at
hgher sperm densities. Of the 13 eggs that did not overshoot zero, six were monospermic
and seven were polyspermic.
More recently, the fast electrical block hypothesis has been applied to frogs [Cross
and Elinson, 19801, starfish [Miyazaki and Hirai, 19791, and the worm Urechis [GouldSomero et al, 19791. The major observation to support the hypothesis is that oocytes held
positive by current injection could not be fertilized [Jaffe, 19761. We agree with this observation, but not with the interpretation; the intricate steps involved in sperm-egg interaction will surely be interferred with by depolarizing an egg by current injection. Such a
treatment will alter not only the ionic constitution of the cytoplasm, but may well alter
the molecular organization of the plasma membrane (eg, the orientation and surface charge
of transmembrane structures. Furthermore, Cross and Elinson [ 19801 and Could-Somero
et a1 [1979] show that the amplitude of the fertilization potential depends upon the ionic
constitution of the environment. In a separate series of experiments, they show that in
various ion-substituted media the incidence of polyspermy increases. Extrapolating the
two series of experiments, the authors suggest that polyspermy is the result of the reduction in amplitude of the fertilization potential. The authors do not rule out convincingly
the possibility that polyspermy is either the result of toxicity of the ion replacement
molecule or simply due to removal of a major ion such as Na’ from the environment [CouldSomero et al, 19791. Na’ removal will disturb the morphological and physiological functioning of the egg, and the observed alteration in fertilization potential may essentially be
just a “by-product” of this change. It is known, for instance, that the metabolic activation
of the egg has a strict requirement for Na’ [Chambers, 19761.
Using over 150 eggs of the sea urchins Paracentrotus and Psammechinus, De Felice
and Dale [I9791 studied directly the successful collision rates of sperm with eggs over a
range of sperm densities. This electrophysiological method was based on the observation
that each sperm entry into an egg elicited a small step depolarizationof 1-2 mV (Fig. 6).
It was shown that the number of spermatozoa entering sea urchin eggs increased monotonically with sperm density [De Felice and Dale, 19791 ;consequently no support for a fast
electrically mediated block could be presented. This is in agreement with the results of
Byrd and Collins [1975] for Strongylocentrotus, where they conclude that there is no
How IS Polyspermy Prevented?
165
change in egg receptivity t o sperm for at least 12 seconds after the first spermatozoon-egg
fusion. Incidentally, this period is probably synonymous with the 13-second shoulder
period preceding the fertilization potential in the electrical records of De Felice and Dale
[19791 (see Fig. 6).
To summarize, the existence of a fast partial block remains controversial and its
solution awaits carefully designed experiments. Essentially, individual eggs will have to be
studied; the average time between exposure of the egg t o sperm and the first successful
spermatozoon collision should be compared with the average time between the first and
second successful spermatozoon collisions. A combination of microcinematographic and
electrophysiological techniques should realize t h s aim.
FINAL REMARKS
To conclude this discussion let us consider the paradoxical situation in the so-called
physiologically polyspermic animals (eg, some mollusks, birds, reptiles, sharks, and urodeles). Here several spermatozoa may enter an egg; however, following the fusion of one
male pronucleus with the female pronucleus, the supernumerary spermatozoa degenerate
and therefore do not interfere with the normal process of cleavage [see Rothschild, 19541.
In the urodele Triton if the supernumerary spermatozoa are isolated from the successfully
uniting male and female pronuclei in a portion of the egg cytoplasm, they do not degenerate [Spemann, 19141. These experiments might suggest that the female or the male pronucleus (or both), when proximal, produce a factor that causes the degeneration of supernumerary spermatozoa. It is interesting that this factor is only activated following the
interaction of the female with the first male pronucleus; ie, upon formation of the zygote
nucleus that may be considered as the actual beginning of embryonic development. Whether
it is released or synthesized at this time is of course not known [Fankhauser, 19251. However, the fact that in some animals supernumerary spermatozoa can enter the egg without
interfering with normal cleavage whde ruling out a membrane-located polyspermy block
accentuates the fundamental event in sexual reproduction - the assurance that only one
paternal and one maternal nucleus will fuse together.
In reflection, it is interesting to raise the question of how prokaryotes control the
amount of DNA transferred from donor t o recipient cells during conjugation. That such
control exists is suggested by the fact that, in the vast majority of cases, transfer of DNA
is limited to part of the bacterial chromosome, and even more so by the possibility of
multiple transfers, ie, from several donors to one recipient cell [Achtmann, 1977; Achtmann et al, 19781. This implies that at one point the processes of transfer must be interrupted by clipping the DNA at a very precise site and by separation of the conjugants.
We would like to venture the suggestion that the restriction enzymes may be intrumental
in t h s process, and in fact that this may be one of the major roles of these enzymes in the
bacterial cell. The safeguarding of constancy of DNA content in organisms should thus
have a very long phylogenetic history strictly related to the invention of sexuality.
ACKNOWLEDGMENTS
The work of our group has been supported by grants from the C.N.R., Project on
the Biology of Reproduction (to Alberto Monroy), the Royal Society and EMBO (to
Brian Dale).
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Dale and Monroy
We thank Drs. Rosaria De.Santis, Floriana Rosati, and Mario Stefanini for heIpful
suggestions and C . Metz for reading the manuscript.
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