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Sperm-egg interactionAlberto Monroy a , Floriana Rosati a & Brian Dale aa Stazione Zoologica di Napoli , Villa Comunale, 80121, Napoli, ItalyPublished online: 14 Sep 2009.

To cite this article: Alberto Monroy , Floriana Rosati & Brian Dale (1984) Sperm-egg interaction,Bolletino di zoologia, 51:1-2, 103-119

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Boll. Zool. 51: 103-119 (1984)

Sperm-egg interaction

ALBERT0 MONROY, FLORTANA ROSATI * BRIAN DALE Stazione Zoologica di Napoli, Villa Comunale, 80121 Napoli (Italy)

ABSTRACT

In this paper we discuss the events leading to the recognition and fusion of sperms and eggs. In the course of evolution a glycoprotein coat, the vitelline Coat, has differentiated around the egg. This coat carries the sperm receptors and is a product of the oocyte. The receptors are glycoprotein molecules; in most of the species studied fucose appears to be the most important component as regards receptor function. In most, though not in all, animals the interaction of the spermatozoa with the sperm receptors triggers the acrosome reaction. Some observa- tions further suggest that the vitelline coat may also serve to repress the metabolism of the un- fertilized egg.

Fusion of the spermatozoon with the egg plasma membrane does not require specific recep- tors. Interaction of the spermatozoon with the sperm receptors elicits profound changes in sperm metabolism that are described as “sperm activa- tion” which in fact precede the acrosome reac- tion. Prevention of polyspermic fertilization is a complex process: in some species it de- pends on changes in the molecular organiza- tion of the egg plasma membrane triggered by the interaction with the first spermatozoon. In naturally polyspermic eggs it depends on cyto- plasmic events that are set in motion once one sperm pronucleus has merged with the egg pronucleus.

On leave from the University of Siena.

1. INTRODUCTION

Up to a few years ago, in fact until the mammalian egg became experimentally accessible, at least 90’10 of our informa- tion on fertilization was based on research carried out on the sea urchin. This re- sulted in a heavy conditioning which led to the idea that what is true for the sea urchin is also true for all other animals. This extrapolation to all animals ignores the fact that in the course of evolution reproductive isolation may have arisen from changes in the mechanism of game- te encounter. Also insufficient attention has so far been paid to how long animals thought to be taxonomically closely re- lated have diverged in the course of evo- lution. Just to give an example, suffice here to mention that Asteroids and Echi- noids have been separated by about 500 million years. Although information ava- ilable is by no means complete, we start to zee how against a background of fun- damentally similar mechanisms specific events of gamete interaction have diver- ged.

The purpose of the present paper is to analyze the events of gamete encounter and fusion in different animal groups with the aim of tracing common principles and divergences. Indeed the study of diver- gences is not less interesting than that of the common principles for the understan- ding of the evolution of the strategies of gamete interaction.

Fertilization is the term given to in- clude all the events which lead to the fusion of two haploid genomes, that of the egg and that of the spermatozoon, be- longing to the same species.

These events in turn involve many com- plex interactions between the two game- tes which appear to be subject to conside- rable variation even in animals systema- tically closely related.

By analyzing these differences we have come to the conclusion that they are lar-

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A. MOSROY, F. ROSATI, B. DALE 104

gely variations on a theme; the theme being that of the control mechanisms Iea- ding to the fusion of two haploid genomes. The arguments which we will dixuss ha- ve, in large part, been presented in two recent articles from our laboratory (Dale & Monroy, 1981; Monroy & Rosati, 1983).

2. THE RECOGNITION OF GAMETES AND T H E INDUCTION OF T H E ACROSOhlE REACTION

We do not intend to enter here into the controversial subject of chemotaxis, for a discussion of which we refer to the work of R. Miller (review in press).

What we wish to discuss is the pre- sence on the surface of gametes of speci- fic molecular entities which play a role in the species-specific recognition between spermatozoa and eggs and which are cal- led receptors. The receptors have how- ever, a different location on the egg and on the spermatozoon. In the egg (except for those which lack a vitelline coat (v.c.) and which will be discussed later) the re- ceptors are components of the v .~ . which, however, though being morphologically extracellular, is a product of the oocyte (see Cotelli et al., 1981; Rosati et al., 1982). On the other hand in the sper- matozoa the receptors are components of the plasma membrane. As for the fusio- genic sites (i.e. for the molecular entities responsible for the sperm-egg fusion) in the spermatozoon they are components of the inner acrosomal membrane and hence they become exposed following the acro- some reaction; in the egg they are com- ponents of the plasma membrane (for a discussion see Rosati et al., 1983; Monroy & Rosati, 1983). In both cases they are “protected” structures that become expo- sed at the right moment (Epel, 1980). Here we wish to stress the point that whi- le the spermatozoon may be considered as a frriictional tinit, in that it contains all

.

the structures necessary for interaction with the egg, the egg in contrast is a com- posite structure in that the extracellular coats play an essential role in fertilization; and therefore it is the egg together with its coats that must be considered a func- tional unit. From the point of view of the interaction with the spermatozoon the V.C. is the most interesting of the extracel- lular envelopes as it contains the sperm receptors.

The species-specific recognition betwe- en receptors on the surface of the sper- matozoon and on the V.C. leads to the binding of the spermatozoon to the egg. This process has been studied in detail in the fertilization of the Ascidian, Ciorza iiztestitialis (Rosati & De Santis, 1978; De Santis el al., 1981; Pinto et al., 1982). In Cioiza the spermatozoa bind to tufts of fibrils emerging from the outer surface of the v.c.. When isolated V.C. (Fig. 2) are challenged with spermatozoa these attach only to its outer surface. This is interes- ting in view of the fact that similar tufts emerge also from the inner surface of the v.c.. However, while the outer tufts are positive to Ruthenium Red, Concanava- lin A and Fucose Binding Protein (FBP), the tufts of the inner side of the V.C. have no affinity for FBP. This observation ga- ve the first hint that fucose may play an important role in sperm-egg interaction and in fact might be a key component of the sperm receptors. This was confirmed by experiments which showed that L-fuco- se (but not D-fucose) is a competitive inhi- bitor of sperm attachment and of fertili- zation (Rosati & De Santis, 1980). The same has now been shown to be the case in the mammals (Huang et al., 1982). in the sea urchin (Glabe et al., 1982) and in the horseshoe crab (Barnum & Brown, 1983). More recent work has further shown that preparations of the fucosyl- glycoprotein components of the V.C. inhi- bit the sperm binding to the V.C. and fer- tilization; furthermore they trigger sperm

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SPERM-EGG INTERACTION 105

Fig. 1. D A section of an isolated vitelline coat of Cioiia intestindis stained with Ferritin- conjugated Fucose Binding Protein (FBP). FBP-positive tufts emerging from the outer surface of the coat (arrows). (From Pinto et ul., 1981).

Fig. ‘2. . Sperm of Ciona iirtestiriulis attached to the vitelline coat. Attachment is mediated by thin fibrils connecting the tip of the spermatozoon to a tuft of the vitelline coat. The black granules are stained by Ferritin-mnjugated Concanavalin A. (From De Santis et ul., 1980).

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106 A. AIOXROY, F. ROSATI, B. DALE

activation including the acrosome reaction (De Santis et al., 1983) (Fig. 3).

Ascidian eggs are surrounded by a layer of follicle cells attached to the outer sur- face of the V.C. (formerly called chorion) (Fig. 4); the spermatozoa must pass in the interstices between the follicle cells to ar- rive at the v.c.. Hence there are few sites on the V.C. which are accessible to the spermatozoa, i.e. those not covered by the follicle cells (De Santis et al., 1980). If follicle cells are removed thus exposing the whole surface of the v.c., then bet- ween 2500 and 3000 spermatozoa attach to each egg (Rosati & De Santis, 1980). Of these, however, only a minute propor- tion undergo the acrosome reaction and therefore are able to penetrate the V.C. (De Santis et al., 1980). We have put for- ward the hypothesis that in order for the attachment to be followed by the acro- some reaction there must be a “molecular match” between receptors on the sperma- tozoon and those on the egg (De Santis et al., 1980; Monroy & Rosati, 1983).

We assume a repertoire of receptors on the surface of the gametes arising from differential glycosylation of the protein moiety of the receptors (Monroy & Ro- sati, 1982, 1983). It is vell known that differential glycosylation is responsible for the microheterogeneity of integral gly- coproteins of cell membranes (Warren et al., 1982).

Also in the mammals the acrosome reaction is triggered when capacitated spermatozoa interact with the zona pel- lucida (Florman & Storey, 1982; Bleil & Wassarman, 1983); and in fact only sperm which have not undergone the acrosome reaction are able to attach to and pene- trate the zona pellucida (which is the equi- valent of the v.c.). Reacted spermatozoa are frequently observed in the cumulus oophorus; however these have been con- sidered to have undergone false reactions (Flormann & Storey, 1982). Sperm-egg interaction in the Echinoderms deserves a

more detailed discussion considering the role these animals have played in shaping up our ideas on fertilization.

In 1912 F.R. Lillie discovered that egg water of sea urchins, that is sea water in which eggs have been left standing for several hours (and therefore containing the jelly layer material in solution) agglu- tinated spermatozoa of the same species. Lillie called the agglutinating factor fer- tilizin, which he suggested played a role not only in the recognition of sperm and eggs, but also in the process of egg acti- vation at fertilization and in the defense against polyspermy (Lillie, 1919). Altho- ugh the theory of fertilizin is only of hi- storical interest today, there is no doubt that it has influenced our way of thinking for over half a century and infact until the present times. The real breakthrough in the problem of sperm-egg interaction was the discovery of the acrosome reac- tion by Jean C. Dan, in 1952. It was also discovered that the most potent inducer of this reaction is the material of the jelly coat. This has led to the assumption that the jelly coat is the physiological indiicer of the acrosome reaction. Hence, the se- quence of the events of sperm-egg interac- tion was described as follows: - the species-specific recognition bet-

ween spermatozoa and some compo- nent of the jelly coat;

- induction of the acrosome reaction by the jelly coat;

- penetration of the reacted spermato- zoon across the jelly layer and the V.C. and its eventual fusion with the egg.

A detailed and critical analysis oE data in the literature and on the basis of expe- rimental results from our laboratory has led to different conclusions (Monroy & Rosati, 1982, 19S3): namely that also in the sea urchin the V.C. is the site where the acrosome reaction takes place. First of all the properties of the jelly coat ma- terial in solution are not necessarily the

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SPERAI-EGG IXTERACTION 107 ___

Fig. 3. - Spermatozoa of Ciona intestinalis; negative staining. (a) Control; (b) smelling of the mitochondrion (m) which has also shifted tailwards and opening of the acrosome (arrow) (acrosome reaction) as a result of treatment with the sperm-receptor material from the vitelline coat. (From De Santis et uf., 1983).

’. same as when it is in situ; that is in so- lution reactive groups may be exposed which normally in situ are not (Dale & Monroy, 1981). Hagstrom (1959) arri- ved at the conclusion that the jelly coat inactivated the major portion of sperma- tozoa which passed through it, possibly by inducing a premature acrozome reac- tion. As a matter of fact reacted sperma- tozoa rapidly lose their fertilizing capa- city (Vacquier, 1979; Hinsey et al., 1980). Timourian et al. (1972) had presented da- ta suggesting that in the sea urchin only about 2 % of the spermatozoa in a sperm population is fertile, i.e. capable of suc- cessfully engaging in fertilization. To be “fertile” may simply mean to be able to go across the jelly coat and reach the sperm receptors on the V.C. in an unreacted con- dition.

Current work in our Laboratory sug- gests that also in the sea urchin sperma- tozoa bind to the V.C. in an unreacted condition.

Our interpretation thus agrees with that of Glabe et al. (1981) that “the fa- miliar electron microscopic image of sperm

adhering to the vitelline layer by the ex- tended acrosome process may not repre- sent an intermediate step in the process of gamete interaction, but rather the un- productive interaction of a typical unsuc- cessfuI sperm”. It is also worth mentioning that in Abalone the spermatozoa traverse the jelly coat in the unreacted condition and it is only after binding to the V.C. that they undergo the acrosome reaction (Le- wis et al., 1982).

We have hypothesized that spermato- zoa which are capable of fertilization are those which are able (by virtue of their surface organization) to pass through the jelly layer and reach the V.C. without un- dergoing the acrosome reaction. The acro- some reaction should be induced by the match between the receptors on the vi- telline coat and complementary structures on the surface of the spermatozoon.

An interesting discovery has been that of “bindin” (Vacquier & Moy, 1977), a protein found in the acrosome of several marine animals, in particular in echino- derms, which becomes exposed following the dehiscence of the acrosome during the

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~ - -- -- A. LIONROY, ._ F. ROSATI, B. DALE ._ _- 108

acrosome reaction and which species-spe- cifically binds the spermatozoon to the glycoprotein receptors of the egg surface.

I ts species specificity suggests that i t plays an important role in sperm-egg in- teraction. However, in view of the fact that it is released follozuiizg the dehiscen- ce of the acrosome, it may be thought not to intervene in the primary events of sperm egg recognition which precede the acrosome reaction but rather in establis- hing a firm attachment of the spermato- zoa to the V.C. following the dehiscence of the acrosome. I t would be interesting to know whether or not bindin-like pro- teins exist in animals other than the ma- rine species where they have been found (Moy & Vacquier, 1979, for a review).

Let us now consider sperm-egg interac- tion in the starfish. The starfish is in fact one of the two well documented ca- ses - the other is the holothurian, T h y one (Colmin & Colmin, 1956; Tihey et al., 1973), where the acrosome reaction is certainly induced upon the interaction of the spermatozoon with the surface of the jelly coat of the egg (Dale et al., 1981). I n these animals the acrosome reaction is spectacular because it involves the forma- tion of a extremely long acro:omal pro- cess (about 20 krm in the starfish and 95 I.tm in Thyone) which crosses the width of the jelly coat. However, in the majo- rity of cases the acrosomal process is emit- ted obliquely with respect to the egg sur- face and therefore fails to reach the egg. It is not known how the acrosomal pro- cess penetrates the vitelline coat or how the fusion with the plasma membrane of the egg comes about. I n the case of the starfish therefore it is clear that the jelly coat must contain molecules responsible for the induction of the acrosome reac- tion. An acrosome reaction - inducing gly- coprotein has indeed been purified from the jelly coat of a starfish (Uno & Hoshi, 1978; Ikedai & Hoshi, 1981 a, b). I s i t

possible to find a common denominator between this situation and that of the sea urchin on one hand and that of the ascidians and the mammals on the other? We believe it is. An important contri- bution to resolve this question has come from the study of the origin and differen- tiation of the egg envelopes and in parti- cular that of the vitelline coat. Until re- cently, it has been a widely held belief (see alzo Gwatkin, 1977) that the V.C. is a product of the follicle cells; even though this idea was not supported by any obser- vation or experiment. When it became evident that the sperm receptors mere an integral part of the v.c., it seemed stran- ge that the oocyte had delegated the pro- duction of structures of such fundamental importance to somatic cells. The work of Bleil and Wassarman (1980) on the mo- use and from our laboratory (Cotelli et al., 1981; Rosati et al., 1982) on Ciotza has shown that in these animals the gly- coprotein components of the V.C. (zona pellucida in the mammals), are synthesized by the oocyte (Fig. 5).

In the ascidians and mammals the V.C. is covered by the follicle cells (the cumu- lus oophorus in the mammals) whereas in the echinoderms, the egg is encased in a jelly coat. We shall not discuss here the case of Amphibian eggs in which the jelly envelope is a product of the oviduct and of which little is known with regards to its role in sperm-egg interaction (ex- cept that it is required for fertilization to occur). I n the starfish (Santella et al., 1983) during the growth phase the oocyte synthesizes and excretes a PAS-positive material which accumulates between the oocyte and the follicle cells. Later, this material differentiates into an inner fibril- lar layer between the microvilli which start to emerge in great number from the oocyte surface and will eventually beco- me the V.C. Externally, the material re- mains less dense and this becomes the jel- ly coat. There is no sign of secretory ac-

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SPERM-EGG INTEBACTIOS 109

Fig. .4. - (a) Scanning Ehi view of an egg of Cionrr infestinah: follicle cells attached to the vitelline coat; (b) a living egg showing the highly vacuolated follicle cells (fc); ch: chorion i.e. vitelline coat; tc: test cells in. the perivitelline space. (From De Santis et nl., 1980).

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110 A. hlONROY, F. ROSATI, B. DALE

Fig. 5. Autoradiograph of an oocyte of Cionu intestinalis at a late stage of maturation, 18 hours after the iniection of [3H]-fucose in the ovary. Silver grains are scattered all over the oocyte cytoplasm and very densely packed in the vitelline coat (arrow) while they are absent in the follicle cells (fc) and in the test cells (tc). (From Rosati et ul., 1981).

tivity in the follicle cells. Thus in the starfish not only the V.C. but also the jelly coat are products of the oocyte.

On the basis of this information let us now compare the situation in the starfish and in the sea urchin.

I n the starfish (1) both the jelly coat and the V.C. differentiate from a common precursor produced .by the oocyte; (2) the acrosome reaction is triggered upon the contact of the spermatozoon with the jelly coat and the latter in fact contains acro- some reaction-inducing molecules.

I n the sea zirchitr, (1) whether or not the jelly coat and the V.C. originate from the oocyte as in the starfish is not known; however, this is likely to be the case; (2) the acrosome reaction is triggered by jelly coat solutions but the evidence that this is also the case in vivo is rather uncertain. On the contrary there is evidence that the

,

V.C. is the site of the acrosome reaction. I n view of the evolutionary divergence

between Asteroids and Echinoids, we spe- culate that the Asteroids are representa- tive of a more primitive situation in which the acrosome reaction - inducing molecules mere incorporated in the jelly coat; the role of the V.C. in fertilization remains ob- scure. It has been suggested that it may contain the binding sites for complemen- tary sites on the acrosome process (San- tella et d., 1983). In the more advanced Echinoids, a fraction of the acrosome- reaction-inducing molecules still takes residence in the jelly coat thus confer- ring to it the ability to trigger the acro- some reaction under certain conditions (e. g. in solution); the bulk of these molecu- les, however, becomes bound to compo- nents of the V.C. the resulting complexes being the sperm receptors.

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111 SPERhI-EGG IXTERACTION .-

3. THE ORIGIN OF THE VITELLINE COAT AND ITS CHANGES IN THE COURSE OF EVOLUTION

\Ve think of the appearance of the V.C. and of the ensuing incorporation of the sperm receptors into it as an important evolutionary advance. The situation in unicellular eukaryotes and in some more primitive multicellular animals does in fact invite the speculation that in an an- cestral condition the receptors for gamete recognition and binding were components of the gamete plasma membrane.

In this connection we would like to mention a few systems which are parti- cularly interesting even though some of them do not belong to the animal king- dom.

In the unicellular alga, ChZui~zydo:nortas, the specificity of gtmete interaction de- pends on a glycoprotein associated with the plasma membrane. The flagella which are responsible for initiating gamete in- teraction are covered by a glycoprotein layer which, however, does not seem to play any role in conjugation (see Adair et ul., 1982, also for references to pre- vious work).

Also in Paramecirim the active material responsible for gamete interaction is an integral component of the plasma mem- brane (Ritamura & Hiwatashi, 1978).

Of great interest is the situation in Fitciis in which the egg has no extracel- lular coats at all: sperm attach directly to the plasma membrane. Plasma mem- brane preparations inhibit fertilization when acting on the spermatozoa (Bolmell et al., 1979, 1980) and the active frac- tion is a glycoprotein containing fucosyl and mannosyl residues (Bolwell et uZ., 1978), thus suggesting that the recep- tors are components of the egg plasma membrane.

Also the eggs of the Hydrozoans (0’ Rand & Miller, 1974; Noda & Kanai, 1981) and other Coelenterates (Honegger,

.

1983) lack a V.C. I n the Hydrozoans the “receptors” are components of the plas- ma membrane and in fact they appear concentrated in a small area, where the polar bodies are ejected (Freeman & Mil- ler, 1982).

The incorporation of the sperm recep- tors in the V.C. has at least two important functional advantages.

The first is that any molecule which is an integral component of the plasma membrane is bound to share its fluidity properties. In the case of the hor- mone receptors this has been shown to be an important condition (see Carraway- Carruthers & Carraway, review in press). On the contrary in the case of the sperm receptors it seems to be more advantageous for the receptors to be firmly anchored in a definite orientation. Secondly, as long as the receptors are components of the plasma membrane they are bound to be rare components as they have to share space with the other component molecu- les. By being transferred to an extracel- M a r coat their number may be increased by several orders of magnitude. On the other hand, irrespective of whether or not specific fusogenic sites for sperm-egg fusion exist fusion with the spermatozoon has remained a property of the egg plasma membrane. It may indeed be important for the molecules involved in fusion to keep their mobility. In several systems mobility of receptors is a prerequisite for the transduction of the stimulus (Carru- thers-Carraway & Carra\lvay, review in press). This may be the case in the egg if sperm-egg fusion is the trigger of the reactions leading to egg activation.

I n contrast with the situation of Hydro- zoans which may be representative of an ancestral condition, the lack of the V.C.

in the Teleosts is likely to be a secondary one. The spermatozoon arrives at the plasma membrane of the egg via the mi- cropylar canal (Fig. 6 ) and fuses with the egg. Although direct evidence is lacking,

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112 A. hlOSROY, F. ROSATI, B. DALE

Fig. 6 . 2 The micropyle (arrow) in the chorion (ch) of an unfertilized egg of Oryzius Zutipes (Teleosts). (Courtesy of Professor E. Nakano).

the micropyle seems to be the only site where species-specific gamete recognition should take place. Between the chorion and the surface of the egg we have obser- ved a gelatinous material that fills the micropyle and in fact extends outwards from the canal like a mushroom. This material may be analogous to the jelly coat of the echinoderms. Furthermore it is known that removal of the chorion results in polyspermic fertilization; dechorionated eggs are also accessible to fertilization by heterologous sperm. Finally, it should be noted that the spermatozoa of the teleosts lack an acrosome. Also in the Hydrozoans the spermatozoa lack a distinct acrosome: there are, however, small vesicles close to tip of the sperm that have been interpre- ted as pro-acrosomal structures.

A correlation has been suggested bet- ween the V.C. and the presence of the acrosome (Dale & Monroy, 1981).

There is too little known about fertili-

zation in Insects to discuss in this con- text. Also in the Insects the spermato- zoon reaches the egg via a micropyle; how- ever, given the mode of fertilization in these animals it is not surprising that the problem of species-specific recognition are different from those in fish which practi- ce external fertilization. Insect eggs have a so-called “vitelline membrane” whose origin and function in fertilization is un- known (see Perotti, 1974; Leopold, 1980).

4. Is THE VITELLINE COAT’S SOLE FUNC- TION TO CARRY THE SPERM RECEP- TORS?

In the oocytes in which the V.C. is tigh- tly apposed to the plasma membrane, such as those of the sea urchin and of the star- fish, fertilization i5 accompanied by its detachment from the egg surface and its conversion into the fertilization envelope. In the sea urchin egg this involves the

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SPEKXI-EGG INTERACTION 113

breakdown of the vitelline posts which connect the V.C. to the egg's plasma mem- brane (Kidd, 1968; Chandler & Heuser, 1980). These connections may function as transducers of stimuli originated upon the interaction of the spermatozoon with the V.C. to the egg plasma membrane and hence to the egg cytoplasm (Monroy & Rosati, 1983). It would be interesting to find out whether the vitelline posts are connected with the cytoskeleton of the egg. In the starfish oocyte, the V.C. is much thicker than in the sea urchin and is densely populated by microvilli emer- ging from the oocyte surface. The fibrils that make up the V.C. are tightly anchored to the plasma membrane of the microvilli (Santella et al., 1983). These connections loosen as an early response to the maturing hormone 1-methyladenine.

Mazia et al. (1973, succeeded in me- chanically peeling off the V.C. from unfer- tilized eggs of Lytechyriiis pictrrs and this resulted in a change of their membrane potential, in changes of the nuclei which entered the chromosome cycle, and in the elongation of the surface microvilli similar to what occurs in the ammonia-activated eggs. The Authors speculate that this ef- fect is due to the removal of "stoppers" which may block some ion channels. They propose that the V.C. plays the role of metabolic repressor of the egg; and hence its detachment from the egg surface should be a prerequisite for the metabolic de-re- pression of the egg at the time of fertili- zation.

5. SPERhl-EGG FUSION

Since gamete fusion occurs between the egg plasma membrane and the inner acrosomal membrane of the spermatozoon (either exposed as a result of the acroso- me reaction such as in mammals or exten- ded in an acrosomal process) the question is whether fusion requires specific fusio- genic structures in the plasma membranes

or contact between the two apposing mem- branes results in their destabilization thus leading to fusion. If fusiogenic sites are present a t all on the plasma membrane their specificity must be far lower than that of the receptors on the V.C. Indeed, denuded eggs are prone to be fertilized by heterologous spermatozoa and to be- come polyspermic (see Dale & Monroy, 1981).

Nevertheless in some animals sperm- egg fusion is restricted to specific areas of the egg surface thus suggesting a dif- ferential distribution of putative fusioge- nic sites. This is the case, for example, of the Ascidian and the Amphibian eggs. In the Ascidian egg, as observed by Con- lclin (1905), sperm-egg fusion is restricted to a sector of about 30" around the vegetal pole. Also following the removal of the V.C. sperm preferentially attach to the ve- getal pole (R. De Santis, personal com- munication) thus suggesting an organiza- tion of the plasma membrane that makes it receptive to spermatozoa. In the Anu- rans fusion occurs at the animal pole of the egg. Spermatozoa may be forced to penetrate the vegetal pole; however, once inside the egg they are unable to partici- pate in the processes leading to pronuclear fusion (Graham, 1966).

A most interesting case is that of Dis- coglossus in which sperm penetration oc- curs in a dimple at the animal pole which has an ultrastructural organization diffe- rent from that of the rest of the egg (Cam- panella, 1975). Also, the dimple is the only site of, the egg surface which binds FBP; binding disappears after fertilization (Denis-Donini & Campanella, 1977). Whether or not these fucosyl-containing glycoproteins are involved in sperm re- cognition and binding as in Cionu, is not known.

The recent discovery of Acethylcholine (Ach) receptors in the plasma membrane of the Xenopirs oocyte should also be mentioned (Kusano et ul., 1977, 1982).

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The concentration of the receptors is much greater in the animal than in the vegetal hemisphere and they are no longer detectable after fertilization. This finding acquires special interest in connection with the discovery of Ach receptors at the sperm surface (Nelson, 1976). It is temp- ting to speculate that these receptors play a role in sperm-egg interaction and in the activation of the egg. Another interesting observation is that receptors for a number of other transmitters, such as enkephalins, substance P, some amino acids are also present at the oocyte surface (Kusano et al., 1982).

Interestingly, except for the Ach recep- tors, that are present in every oocyte, the others are not. In other words not every oocyte is endowed with the whole spec- trum of receptors. This suggests that the oocyte plasma membrane except for some obligatory components, contains a wide spectrum of molecules whose physiological role remains still obscure.

6 . THE PREVENTION OF POLYSPERMIC

We consider the expression “polysper- my prevention” as inappropriate: the pro- blem is indeed Which are the mechanisms whereby the fusion of the egg nucleus with more than one sperm nucleus is pre- vented? I n many animals polyspermy is the norm (e.g. Sharks, Urodeles, Birds, etc.) In these cases the egg has deve- loped mechanisms which impede the fu- sion of more than one male nucleus with the female nucleus. In contrast, in other animals there are mechanisms which im- pede the fusion of the egg with more than one spermatozoon. I n these cases, and only in these cases, the expression “prevention of polyspermy ” is appropria- te. The subject has been reviewed in a previous article (Dale & Monroy, 1981).

The first question we should like to address concerns the role of the egg in-

FERTILIZATION

A. h fONROY, F. ROSATI , B. DALE

vestments in “protecting” the egg from being challenged by too great a number of spermatozoa. Thus far we have con- sidered the role of the egg investments in sperm recognition and binding and in triggering the acrosome reaction.

Ascidian eggs are covered by a layer of follicle cells which are fixed into the vitel- line coat; between which are passageways where the spermatozoa must pass to reach the egg surface. It is probable that the passageways can be varied in diameter by a system of microtubules at the bare of the follicle cells (De Santis et al., 1980). In a sense this egg may be considered to have a multiple system of micropyles. In ad- dition it has been shown that in ascidians the follicle cells phagocytize spermato- zoa (De Santis et al., 1980). Furthermo- re even under conditions in which the who- le surface of the V.C. is available for sperm attachment, only a very small percentage of the bound spermatozoa undergo the acrosome reaction (De Santis et al., 1980).

Florman & Storey (1982) have shown that spermatozoa which undergo the acro- some reaction whilst passing through the cumulus oophorus are effectively inacti- vated. This is of interest also to the si- tuation in the sea urchin. As we have already mentioned, Hagstrijm (1959) sug- gested that the majority of spermatozoa which pass through the sea urchin jelly layer undergo a premature acrosome reac- tion thereby being inactivated (see discus- sion in Section 3) .

We suggest that in all sperm popula- tions there is only a small percentage of spermatozoa which by virtue of the orga- nization of their plasma membrane, are not susceptible to being triggered into a premature acrosome reaction while passing the jelly layer thus reaching the V.C. in the unreacted condition. This, per se, will drastically reduce the number of sperma- tozoa and therefore the probability of sperm egg collisions. In the starfish and

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SPERM-EGG ISTERACTIOS 115 --

Thyone, as me have seen, irrespective of the large acrosomal process, the majority of processes traverse the jelly layer obli- quely and theretore do not make contact with the V.C.

We have cited these examples to de- monstrate that in animals which practice external fertilization, and therefore where the eggs may be exposed to a high den- sity of spermatozoa, the egg envelopes play an important role in reducing the number of collisions with spermatozoa. From this we can see that there is little sense in trying to calculate spepn-egg col- lision rates from the concentration of sperm in the environment. I n fact, the number of successful collisions depends, in addition to other factors, on the physio- logical condition of the spermatozoa at the moment they reach the egg surface which contains the receptors. I n addition it is very difficult to determine the concentra- tion of fertile spermatozoa in a mixture of semen from several animals. Everyone who works on fertilization of marine ani- mals has at one time or another come acrozs a male in which the spermatozoa appear infertile.

As already discussed, such spermato- zoa may be extremely susceptible to a premature acrosome reaction as they pass through the jelly layer thus becoming in- fertile.

I n some animals the female genital ap- paratus has evolved an elaborate system to control the number of spermatozoa which reach the egg: the most obvious case is that of the insects.

Nevertheless there is always the. possi- bility that more than one fertile sperma- tozoon arrives at the egg with the risk of polyspermic fertilization. Here we en- ter into a highly controversial field; that of whether a fast electrical block to polys- permy exists o r not. Such a block would have the function of drastically reducing the sperm receptivity of the egg plasma

,

membrane following the interaction of the first spermatozoon. Here we will only outline the problem; for further details see Dale & Monroy (19S1).

One of the first events of sperm-egg in- teraction is a depolarization of the egg plasma membrane; this mas first observed in the starfish (Tyler et al., 1956). This electrical change has been called the fer- tilization potential. Is this depolarization the factor which, by changing the condi- tion of the plasma membrane of the egg, prevents fusion with supernumerary sper- matozoa? The hypothesis of a fast elec- trical block is supported by several indi- rect experiments. The most convincing is perhaps the observation that when unferti- lized sea urchin eggs are artificially depo- larized by current injection to positive values they are not fertilized. When the current is turned off, and the potential returns to its original resting level, fertili- zation occurs (Jaffe, 1976). Interaction with a spermatozoon triggers a series of changes in the molecular organization of the egg plasma membrane, which starts at the point of sperm fusion and propa- gates around the egg surface. The first detectable change, which is in fact locali- zed to the region of sperm fusion, is the activation of large non-specific ion chan- nels and this change precedes the familiar activation potential by 10-13 seconds (Da- le et d., 1978).

This conductance change is the first indication of a precocious and significant change in the egg plasma membrane. The hypothesis has been forwarded that this conductance change is triggered by a fac- tor released from the acrosome of the spermatozoon (Dale et al., 1978). I n eggs in which the acrosome reaction is induced at a point distant from the plas- ma membrane, e.g. the starfish (Dale et al., 1981), this conductance change does not occur and this is further, albeit indi- rect, support for the hypothesis. The second electrical change the fertilization

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116 A. hlONROY, F. ROSATI, B. D.4LC

potential, coincides temporally in the sea urchin and starfish with (a) a very ob- vious re-organization of the egg plasma membrane, that is the insertion of patches of cortical granule membrane, and (b) the elevation of the V.C. and its transforma- tion into the fertilization membrane. This represents the permanent barrier to po- lyspermy. Nore or less at the same time, that is during the first minute following gamete fusion, HzOz is excreted from the egg (Coburn et al., 1979) which is a po- tent spermicide. Infact when eggs are inseminated in the presence of catalase there occurs a very high incidence of poly- spermy. We see the electrical changes in the egg at activation as an expression of the re-organization of the egg plasma mem- brane. Clamping the egg at positive po- tentials interferes with the organization of the e g i surface and could alter the re- ceptivity of fusogenic sites. I n relation to this it mould be interesting to see whc- ther the sperm which are attached to the clamped eggs have undergone the acroso- me reaction.

Of particular interest are eggs which are physiologically polyspermic. Unfortu- nately there is little detailed information on these eggs, perhaps the most studied are those of the urodele amphibians. I n these eggs, it is quite normal for 9-12 sper- matozoa to enter at fertilization; all the sperm nuclei form asters and migrate to- wards the female pronucleus. However, as soon as one male pronucleus fuses with the female pronucleus the other male nu- clei stop migrating and start degenerating (Fankhauser, 1925). If, however, the egg is constricted so that one half contains the fusing pronuclei and the other half the remaining male nuclei, then these latter do not degenerate (Spermann, 1914). This suggests that following the fusion of the two pronuclei a substance is produced which causes the degeneration of super- numerary spermatozoa.

7. THE ACTIVATION OF THE SPERMATO-

Finally we should like to mention so- me perspectives open by the use of phy- sical-chemical methods to the study of sperm-egg interaction. The approach has been made possible largely due to the nie- thod of obtaining “ghost eggs” in the Ascidians. Indeed, treatment of these eggs with glycerol results in the shedding of the follicle cells and cytolysis of the egg and of the test cells (Rosati & De Santis, 1978). The V.C. remains apparen- tly intact and in fact i t retains its ability to bind spermatozoa species-specifically; the bound spermatozoa do not undergo the acrosome reaction. By this method one can analyze sperm-binding per se, i.e. independently of events leading to the acrosome reaction. Sperm-egg fusion and egg response to fertilization are excluded. The thermodynamic study of sperm bin- ding to the ghost eggs has shown that the first event that follows binding is a meta- bolic activation of the spermatozoa. This is expressed as a sudden heat production not accompanied by oxygen consumption (Elia et al., 1983). Hence activation of the egg may be preceded by a change in the metabolism of spermatozoa which w e consider as part of the alterations the sper- matozoon undergoes upon binding to the egg and which we indicate as sperm acti- vation. The Colwins (1963) had already made the point that in order to acquire the ability to activate the egg the sperma- tozoon itself must be activated. Little is known about the processes of sperm acti- vation (see Lambert & Epel, 1979; Dc Santis et al., 1983; Elia et al., 1983), the study of which is likely to shed new light on the mechanisms of fertilization.

ZOON

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