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Studies on fertilization in the ascidians
Printed in Sweden Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014~827/78/1162-0419$02.00/0
Experimental Cell Research 116 (1978) 419-427
STUDIES
ON FERTILIZATION
IN THE ASCIDIANS
II. Lectin Binding to the Gametes of Ciona intestinalis FLORIANA
ROSATI,’ ROSARIA DE SANTIS and ALBERT0 Zoological
MONROY
Station, Naples, ltuly
SUMMARY In the present work we have compared the binding of fluorescein-conjugated lectins (concanavalin A (ConA), wheat germ agglutinin (WGA), fucose binding protein @BP) and soybean agglutinin (SBA)) to the sperm surface and to the egg and its envelopes of Ciona intestinalis. Only WGA is bound to the follicle cells: yet this lectin has no binding sites on the sperm surface. Both ConA and FBP are bound by the chorion, the oolemma and the sperm surface. However, while ConA reacts only with the sperm head, FBP is bound both to the head and to the flagellum. Experiments on the effect of ConA and FBP on the fertilization reaction have been carried out. The role of the lectin-binding sites that are shared by the surfaces of both gametes is discussed in connection with the nature of the sperm-binding sites.
Fertilization involves three main steps: (1) recognition; (2) binding; and (3) fusion of the male and female gamete. It is reasonable to assume that these processes depend on the complementarity of the molecular organization of the surface of the interacting gametes. However, while the spermatozoon is a naked cell, the egg is always surrounded by several envelopes which in most cases cannot be removed without recourse to chemical treatment which may damage the egg surface (oolemma). This is particularly true of those eggs, such as that of the sea urchin, in which the vitelline coat is tightly apposed to the oolemma. This makes it difficult to unequivocally answer the question as to the respective role(s) of the vitelline coat and of the oolemma (not to mention the additional egg coats such as the jelly coat or the follicle cells) in the three major processes eventually leading to gamete fusion, i.e. specific recognition, specific binding
and acrosome reaction. This is why we have addressed ourselves to the ascidian egg in which the egg envelopes-the test cells, the chorion and the follicle cells-can be cleanly removed mechanically, thus exposing an undamaged naked oolemma. Furthermore, the self-sterility phenomenon exhibited by these animals adds a new parameter that may help in the analysis of the conditions on which the sperm-egg interaction depends (see [ 111). In the first paper of this series it was shown that in the eggs of Ciona intestinalis of the Gulf of Naples, prevention of selffertilization, of interspecific fertilization and of polyspermy are properties of the chorion rather than of the oolemma. In fact, the chorion appears to be the carrier of the specific sperm receptors and the site at L On leave from the Institute of Zoology, University of Siena. Exp Cell Res 116 (1978)
420
Rosati, De Santis and Monroy
Table 1. Binding of some lectins to the egg and to the spermatozoon of Ciona intestinalis Lectin
Follicle cells
Chorion
Oolemma
Spermatozoon
ConA WGA FBP SBA
+ -
+ ++ -
++ -
+ (heads) + (heads & flagella) -
which the acrosome reaction takes place [I 11. On the other hand, how the oolemma participates in the final process of the sperm-egg association is still not well understood. The ability of lectins to specifically bind to certain carbohydrates may be a way to -obtain indirect information about the molecular components of the gamete membrane involved in the sperm-egg interaction. Indeed, there is an increasing body of evidence [2] pointing to the role of the carbohydrate side chains of the membrane glycoproteins in cell recognition. Our results show that the lectins we tested bind differentially to the surface of the spermatozoon and to the egg and its envelopes. In such a way, in fact, as to suggest that the binding sites may somehow be involved in the sperm-egg recognition and interaction. On the other hand, the question as to whether or not the lectin-binding sites on the surface of the chorion are essential components of the sperm receptors is still open. MATERIALS
AND METHODS
Sperm and eggs were collected from the gonoducts of of the Gulf of Naples. Glycerinated eggs were obtained as previously described [ll]. Fluoresceinisothiocyanate-conjugated (FITC) lectins were obtained from Miles-Yeda and were used at the same concentration throughout this work, except where specifically indicated. The following lectins were used: concanavalin A (ConA, used at a standard concentration of 150pglml); soybean agglutinin (SBA, at a concentration of 75 &g/ml); wheat germ agglutinin (WGA, at a concentration of 45 pg/ml); fucose binding
Ciona intestinalis
Exp Cell Res 116 /I9781
protein (FBP, at a concentration of 75 pg/ml). The competing sugars were added to the gamete suspension either prior to, or together with, the lectin. The material was observed and photographed using a Leitz Orthoplan Microscope equipped with fluorescent optics and using incident light from an Hg source. In the agglutination assays, the spermatozoa were treated with the non-fluorescent lectin, washed three times with sea water and observed with dark-field illumination as previously described [ 111. The protocol of the fertilization-inhibition experiments is described in the legend of table 2. Eggs were glycerol-treated and inseminated following treatment with ConA or FBP, and were prepared for electron microscopy as described [ 111.
RESULTS Lectin binding to the gametes Table 1 summarizes the results of the binding of the fluorescent lectins to the sperm and to the egg and its envelopes. The data in table 1 merit some comment. Regarding the spermatozoa, ConA is bound only to the heads. This is shown not only by the selective staining of the heads by the fluorescent lectin but also by their head-tohead agglutination. Indeed, agglutination appears to occur (at least at the resolution of the optic microscopy under dark-field illumination) by the tip of the spermatozoa which give rise to rosette-like formations (rig. 1). On the other hand, FBP is bound to the whole surface of the spermatozoon which becomes entirely fluorescent. In the agglutination experiments, the agglutinated spermatozoa form long threads resulting from the heads and flagella sticking to one another. Both in the experiments with
Lectin binding to Ciona gametes
421
Table 3. Percent of fertilization
Table 2. Percent of fertilized
eggs of Ciona intestinalis following treatment with ConA (150 pglml) or with FBP (75 pglml)
of eggs of Ciona intestinalis following treatment with ConA (150 hglml) or with FBP (150 pglml)
In each experiment two samples of 10 eggs each (in I ml of sea water) from two different animals (a, b) were inseminated with 5 ~1 of undiluted sperm from the same donor animal (different from the animals which had supplied the eggs). The eggs had been previously exposed to the lectin for 10 min and washed three times with sea water. Five minutes after the insemination, the eggs were washed to remove excess of sperm, and 1 h later they were checked for cleavage. In all the experiments, the controls showed 100% fertilization
Protocol of the experiment as in table 2
ConA
FBP
la
0
70
lb
0
90
2a 2b
100
Expt No.
Expt No.
ConA
FBP
la
10 0 loo 0 0
3b
0 0 100 0 0 10
4a 4b
50 loo
40
lb
2a 2b
3a
30
100
0 100
most completely abolished in the presence of n-acetylglucosamine (see [3, 6]), the i"b 60 90 100 100 fluorescence of the surface of the follicle 4a 100 100 cells is not altered by treatment with this 4b 0 100 sugar. Two possibilities were considered to 5a 70 80 account for this result. (1) That the binding 5b 100 0 of the lectin was due to sialic acid; (2) that 6a 100 90 6b 100 80 the lectin was pinocytized. The former al7a 100 50 ternative was discounted for by treatment 7b 0 0 with neuraminidase (from Clostridium per2 40 10 400 fringens, Boehringer, at 0.6 U/ml from 10 min to 12 h on both intact and glycerinated 9a 70 80 9b 20 90 eggs) which neither abolished nor decreased the fluorescence. This treatment results in the rapid detachment of the follicle cells ConA and those with FBP, the reaction is from the chorion (thus suggesting that sialic abolished in the presence of the competing acid may be involved in the attachment of sugar (at 0.1 M final concentration), i.e. (Y- the follicle cells to the chorion). The fallen methylmanno- or a-methylglucopyranoside, off follicle cells soon start undergoing dein the,case of ConA, and fucose in the case generation; yet, as long as they have a of FBP. healthy appearance their surface continues As to the eggs, the ConA, FBP and WGA to be fluorescent. The possibility of pinobinding differs for the follicle cells, for the cytosis was checked in experiments in chorion and for the oolemma. As shown in which the lectin binding was carried out on table 1, of the four lectins tested only WGA eggs fixed with 2% formaldehyde for 10 was bound to the follicle cells (fig. 2); this min. The binding of the lectin to the surface lectin also reacts with the chorion of both of the follicle cells was not counteracted by living and glycerol-treated eggs. However, the sugar while that to the chorion was (figs while the reaction with the chorion is al- 3, 4). The interpretation of the non-rever30
Exp Cell RPS 116 (1978~
E-V/I Cell Rcs 116 (197X)
Lectin binding to Ciona gametes
423
sibility of the WGA binding to the follicle Of the lectins used in the present expericells is at the moment obscure. However, ments only FBP is bound by the oolemma. it is worth mentioning that the agglutination A weak reaction of the oolemma with ConA of the Ehrlich ascites carcinoma cells by has also been indicated in table 1. This beConA is not reversed by the mannopyranocause of the previous observations that alside; and only about one-half of the mem- though fluorescent ConA did not stain the brane glycoproteins of these cells could be oolemma of the unfertilized egg [4], a low eluted by the specific sugar haptens from but significant binding could be detected ConA- or WGA-Sepharose columns [SJ. when 1251-ConAwas used [7]. The involvement of membrane components other than the glycoproteins (such as glyco- Effect of lectins on fertilization lipids, see [lo]) should hence be taken into The effect of several lectins on fertilization consideration. has been studied by a number of invesThe chorion of both living and glyceroltigators (see [ 1, 8, 91); the results show that treated eggs reacts strongly with ConA (fig. the various lectins have different effects on 5) and with FBP (fig. 6), and the reaction is different animal eggs. In the hope that exspecifically abolished by the competing periments of this kind could provide insugar. The same result was obtained with formation as to whether or not the lectin the chorion isolated from glycerol-treated binding sites at the surface of the Ciona eggs (figs 7 and 8). gametes are involved in the process of Glycerinated eggs were treated with non- sperm-egg interaction, the effect of ConA fluorescent ConA or FBP respectively, and and of FBP on fertilization was tested. Prelithen with the other fluorescent lectin; in minary experiments had shown that neither both cases the chorion became uniformly WGA (which has no binding sites at the fluorescent just as though it had been ex- sperm surface) nor SBA (which has no bindposed only to the fluorescent lectin. ing sites either on the sperm or on the egg) had an effect. The results of the experiments with ConA and with FBP are reFig. I. ConA-treated spermatozoa; head-to-head ag- ported in table 2. glutination. X950. The results show that the effect of the Fig. 2. Fluorescence of the follicle cells exposed to FITC-WGA. X650. two lectins on the fertilization response of Fig. 3. An egg partially deprived of its follicle cells by shaking. fixed with formaldehvde for IO min. and the eggs from different animals is quite diftreated with FITC-WGA. The chorion and a follicle ferent: it ranges from a complete block of cell (arrow) are fluorescent. X500. fertilization to no effect whatsoever. The Fig. 4. In this experiment the eggs were fixed with formaldehyde for IO min, washed in sea water and in- slight difference between the fertilizationcubated in I ml of 0.1 M n-acetylglucosamine for I5 min. FITC-WGA was then added. The micrograph inhibiting effect of ConA and FBP is not shows the fluorescence of a follicle cell (arrow). while statistically significant (ConA,.?=%, S.D.= the chorion is not fluorescent (double ahmvs)~‘x650. k40.8; FBP, X=65, S.D.=?38.3). HowFig. 5. Living egg treated with FITC-ConA. The follicle cells are present but only the chorion is brightly ever, in order to have a more direct comfluorescent. X650. parison between the two lectins and to enFig. 6. Fluorescence micrograph of a glycerol-treated sure that our experiments were done under egR after treatment with FITC-F’BP. X650. Fii. 7. Binding of FITC-ConA to a chorion isolated saturating conditions, several experiments from a glycerol-treated egg. X650. were carried out in which the FBP conFig. 8. Binding of FITC-FBP to the chorion isolated from glyceroltreated eggs. x6.50. centration was raised to that of ConA, i.e. E.x~ Cell Res I16 I 1978)
424
Rosati, De Santis and Monroy
Fig. 9. Attachment of spermatozoa to the chorion (ch) of a glycerol-treated egg incubated in ConA. Many tine particles can be seen between the chorion and the sperm head (II). x38 000.
Fig. 10. Glycerol-treated egg
incubated in PBP and inseminated with heterologous sperms. Randomly oriented spermatozoa attached to the chorion (ch); extraneous material is present on the chorion and between the chorion and the sperm plasma membrane. (h , nucleus; m, mitochondria,f, flagellum.)
to 150 pg/ml. The results of these experiments are reported in table 3. Although in the batch of animals used in this series of experiments, the effect of both lectins was greater than in the previous experiments, i.e. the fertilization-inhibitory effect was stronger, they essentially exhibited the same effect and the variability among the eggs from different animals was equally evident. Sperm attachment to the chorion of the lectin-treated eggs (after removal of the follicle cells either by shaking or by glycerol E.rr,
Cell
Res
116 (197RI
treatment) did not at first sight appear to be interfered with by the lectins. However, a single wash with sea water resulted in the complete removal of the attached spermatozoa. Also, eggs from which the follicle cells were removed either by shaking or by treatment with glycerol, were treated with ConA or with FBP, inseminated and immediately processed for electron microscopy. Fixation preserves the loosely attached spermatozoa, which otherwise, as mentioned before, would be removed by a single wash-
Lectin binding to Ciona gametes ing of the eggs with sea water. In the ConAtreated eggs, the spermatozoa were seen to be attached to the chorion by their tips (fig. 9); however, they did not display the regular radial orientation as in normal eggs (see fig. lob [ 111). In no case was an acrosome reaction detected. In the FBP-treated eggs, on the other hand, the spermatozoa became attached to the chorion by their whole surface (fig. 10); which is in accord with the observation that this lectin is bound to the whole sperm plasma membrane. Also extraneous material, not seen in the untreated eggs, appears to be deposited over the entire surface of the chorion.
DISCUSSION The experiments described in this paper show that the surface of the spermatozoon and the chorion share binding sites for ConA and for FBP. This finding is of interest in view of the observations described in the first paper of this series [ 1l] in which we presented evidence that the chorion is the carrier of the specific sperm receptors, and that it is upon its interaction with the chorion that the spermazoon undergoes the acrosome reaction. The analogy between the properties of the chorion and those of the vitelline membrane of the sea urchin egg and of the zona pellucida of the mammalian egg have also been pointed out [ 111. Very few ConA-binding sites are present on the oolemma of the unfertilized egg [7]. In this connection it is pertinent to mention that in the egg of the sea urchin, Strongylocentrotus purpuratus, high and low affinity binding sites for ConA have been found, the former being preferentially located on the vitelline coat [13]. On the other hand, the oolemma of the Ciona egg shares FBPbinding sites with the surface of the sper-
425
matozoon. However, while the ConAbinding sites are restricted to the sperm head, the FBP receptors extend over the entire sperm surface; i.e. they are present both on the head and on the flagellum. This observation may be of interest in connection with the process whereby the spermatozoon is incorporated by denuded eggs. In this case, the spermatozoa lie flat on the oolemma and are incorporated by a process which is reminiscent of phagocytosis [14]. No binding sites are shared by the sperm surface and the surface of the follicle cells. We would like to recall that in vivo observations have never shown binding of spermatozoa to the surface of the follicle cells [ 111. These observations encourage the view that the lectin-binding sites shared by the gamete surfaces may be involved in the specific recognition and binding reactions between spermatozoa and eggs. It is also known from the work of other laboratories [l, 8, 91 that the surface of the eggs of different animal species and also the surface of the different envelopes of the same species [12] exhibit different affinities for lectins, thus suggesting that the lectin binding may be a test (though admittedly an indirect one) of the specific organization of the membrane glycoproteins of the gametes. Yet, the question that still remains unanswered is to what extent the lectin-binding sites on the chorion are involved in the functional organization of the sperm receptors. The observations described in this paper show that fertilization can be interfered with by ConA and by FBP; both these lectins have binding sites on the sperm surface and on the chorion and on the oolemma. Attention is drawn to the finding that at the same concentration of either ConA or FBP the eggs of different animals exhibit a different fertilization response. Also in other species, for example in the hamster, it has been
426
Rosati, De Santis and Monroy
observed that at the same lectin concentra- result in a variability of the number of lection, the fertilization response varies greatly tin molecules actually reaching the chorion from animal to animal (see, for example, in the different eggs from the same animal fig. 10 of ref. [9]). We interpret this as a and may explain why the fertilization rereflection of a different organization of the sponse in the eggs from the same animal is lectin-binding sites on the chorion (or on the not an all-or-none phenomenon. zona pellucida) of the eggs from different From all this a picture begins to emerge animals. Two possibilities can be en- of the chorion surface as a complex molecvisaged. The first is that the lectin-binding ular mosaic of sperm-binding sites. The sites are essential components of the sperm question is: are all sperm-binding sites receptor sites and hence, when the mosaic sperm receptors as well, i.e. sites at which of the saccharides side chains of the glyco- sperm binding is followed by the acrosome proteins of the chorion is such that their reaction? interaction with one of the lectins results in In the first paper of this series [ 1I] it was the selective masking of the receptors that shown that in eggs from which the follicle fertilization is interfered with. The second cells had been removed by shaking, only is that an unspecific masking of the sperm some of the attached spermatozoa undergo receptors may be brought about by various acrosome reaction. Two main alternatives mechanisms, among which the haphazardly may be visualized to interpret this observacross-linking neighbouring binding sites on tion. First, that while spermatozoa can atthe chorion. This interpretation (which is tach anywhere at the surface of the chorion, more in line with the known mode of action there are only a few spots-which might be of the lectins) also implies that whether or indicated as not spots-at which, either benot the activities of the sperm receptors cause of their molecular configuration or will be obstructed by the lectins depends because they include some specific molecon their arrangement on the chorion and, ular component (an organization that is alhence, on whether or not the binding of the tered by the glycerol treatment), sperm lectins will result in a steric hindrance to binding is followed by the acrosome reactheir interaction with the spermatozoa. tion. The second alternative is that in order A factor whose role is difficult to evaluate for a successful sperm-egg interaction to ocis that of the follicle cells. Studies in prog- cur (i.e. binding followed by acrosome reacress [ 151show that the attachment of these tion and penetration of the spermatozoon cells to the chorion is not uniform; i.e. in through the chorion) there must be a some points they appear to bind more matching between the molecular organizaloosely than in others. Besides the evidence tion of the binding sites on the chorion and from the electron microscope, this is sug- that of the plasma membrane of the spergested by the fact that when the follicle cells matozoon. We know nothing of the variare mnechanically removed [ 1I], shreds of ability of the molecular organization of the cells always remain attached to the chorion. plasma membrane among spermatozoa but Furthermore, microtubules and microfilathere is no reason to believe that it is not ments are abundant in the contact areas be- just as great as that among eggs. This varitween adjacent follicle cells; this may con- ability may clearly play a role also in the trol the closing and opening of the channels case that the sperm receptors on the chorion between cells [15]. These conditions may are restricted to the hot spots. .%/I Cell Res I16 (1978)
Lectin binding to Ciona gametes We are indebted to Dr M. Burger for reading the manuscript and for his comments, to Dr S. Denis-Donini for helpful advice on the use of lectins, and to Mr G. Iamunno and Mr C. Gargiulo for their technical assistance. This work has been supported by a grant (no. 77.00349.85) of the CNR Project on the Biology of Reproduction.
REFERENCES 1. Aketa, K, Exp cell res 90 (1975) 56. 2. Cook, G M W & Stoddart, R W, Surface carbohydrates on the eukaryotic cell, pp. 257. Academic Press, London & New York (1974). 3. Goldstein, I J, Hammarstrom, S & Sundblad, G, Biochim biophys acta 405 (1975) 53. 4. Monroy, A, Ortolani, G. O’Dell, D & Millonig, G, Nature 242 (1973) 409. 5. Nachbar, M S, Oppenheim, J D & Aull, F, Biochim biophys acta 419 (1976) 512.
427
6. Nagata, Y & Burger, M M, J biol them 249 (1974) 3116. 7. O’Dell, D S, Ortolani, G & Monroy, A, Exp cell res 83 (1974) 408. 8. Oikawa, T, Nicolson, G L & Yanagimachi, R, Exp cell res 83 (1974) 239. 9. Oikawa, T, Yanagimachi, R & Nicholson, G L, J reprod fertil43 (1975) 137. 10. Read, B D, Demel, R A, Weigandt, H & Deenen, L L M, Biochim biophys acta 470 (1977) 325. II. Rosati, F & De Santis, R, Exp cell res 112 (1978) 111. 12. Stenuit, K, Genskens, M, Steiner-t, G & Tencer, R, Exp cell res 105(1977) 159. 13. Veron, M & Shapiro, B M, J biol them 252 (1977) 1286. 14. De Santis, R & Rosati, F. In preparation. 15. De Santis, R, Rosati. F & Jamunno, V. Submitted for publication. Received February 2 I, 1978 Revised version received May 9, 1978 Accepted May 12, 1978
E-Y/J CrUKrs
II6
(1978,
Experimental Cell Research 116 (1978) 419-427
STUDIES
ON FERTILIZATION
IN THE ASCIDIANS
II. Lectin Binding to the Gametes of Ciona intestinalis FLORIANA
ROSATI,’ ROSARIA DE SANTIS and ALBERT0 Zoological
MONROY
Station, Naples, ltuly
SUMMARY In the present work we have compared the binding of fluorescein-conjugated lectins (concanavalin A (ConA), wheat germ agglutinin (WGA), fucose binding protein @BP) and soybean agglutinin (SBA)) to the sperm surface and to the egg and its envelopes of Ciona intestinalis. Only WGA is bound to the follicle cells: yet this lectin has no binding sites on the sperm surface. Both ConA and FBP are bound by the chorion, the oolemma and the sperm surface. However, while ConA reacts only with the sperm head, FBP is bound both to the head and to the flagellum. Experiments on the effect of ConA and FBP on the fertilization reaction have been carried out. The role of the lectin-binding sites that are shared by the surfaces of both gametes is discussed in connection with the nature of the sperm-binding sites.
Fertilization involves three main steps: (1) recognition; (2) binding; and (3) fusion of the male and female gamete. It is reasonable to assume that these processes depend on the complementarity of the molecular organization of the surface of the interacting gametes. However, while the spermatozoon is a naked cell, the egg is always surrounded by several envelopes which in most cases cannot be removed without recourse to chemical treatment which may damage the egg surface (oolemma). This is particularly true of those eggs, such as that of the sea urchin, in which the vitelline coat is tightly apposed to the oolemma. This makes it difficult to unequivocally answer the question as to the respective role(s) of the vitelline coat and of the oolemma (not to mention the additional egg coats such as the jelly coat or the follicle cells) in the three major processes eventually leading to gamete fusion, i.e. specific recognition, specific binding
and acrosome reaction. This is why we have addressed ourselves to the ascidian egg in which the egg envelopes-the test cells, the chorion and the follicle cells-can be cleanly removed mechanically, thus exposing an undamaged naked oolemma. Furthermore, the self-sterility phenomenon exhibited by these animals adds a new parameter that may help in the analysis of the conditions on which the sperm-egg interaction depends (see [ 111). In the first paper of this series it was shown that in the eggs of Ciona intestinalis of the Gulf of Naples, prevention of selffertilization, of interspecific fertilization and of polyspermy are properties of the chorion rather than of the oolemma. In fact, the chorion appears to be the carrier of the specific sperm receptors and the site at L On leave from the Institute of Zoology, University of Siena. Exp Cell Res 116 (1978)
420
Rosati, De Santis and Monroy
Table 1. Binding of some lectins to the egg and to the spermatozoon of Ciona intestinalis Lectin
Follicle cells
Chorion
Oolemma
Spermatozoon
ConA WGA FBP SBA
+ -
+ ++ -
++ -
+ (heads) + (heads & flagella) -
which the acrosome reaction takes place [I 11. On the other hand, how the oolemma participates in the final process of the sperm-egg association is still not well understood. The ability of lectins to specifically bind to certain carbohydrates may be a way to -obtain indirect information about the molecular components of the gamete membrane involved in the sperm-egg interaction. Indeed, there is an increasing body of evidence [2] pointing to the role of the carbohydrate side chains of the membrane glycoproteins in cell recognition. Our results show that the lectins we tested bind differentially to the surface of the spermatozoon and to the egg and its envelopes. In such a way, in fact, as to suggest that the binding sites may somehow be involved in the sperm-egg recognition and interaction. On the other hand, the question as to whether or not the lectin-binding sites on the surface of the chorion are essential components of the sperm receptors is still open. MATERIALS
AND METHODS
Sperm and eggs were collected from the gonoducts of of the Gulf of Naples. Glycerinated eggs were obtained as previously described [ll]. Fluoresceinisothiocyanate-conjugated (FITC) lectins were obtained from Miles-Yeda and were used at the same concentration throughout this work, except where specifically indicated. The following lectins were used: concanavalin A (ConA, used at a standard concentration of 150pglml); soybean agglutinin (SBA, at a concentration of 75 &g/ml); wheat germ agglutinin (WGA, at a concentration of 45 pg/ml); fucose binding
Ciona intestinalis
Exp Cell Res 116 /I9781
protein (FBP, at a concentration of 75 pg/ml). The competing sugars were added to the gamete suspension either prior to, or together with, the lectin. The material was observed and photographed using a Leitz Orthoplan Microscope equipped with fluorescent optics and using incident light from an Hg source. In the agglutination assays, the spermatozoa were treated with the non-fluorescent lectin, washed three times with sea water and observed with dark-field illumination as previously described [ 111. The protocol of the fertilization-inhibition experiments is described in the legend of table 2. Eggs were glycerol-treated and inseminated following treatment with ConA or FBP, and were prepared for electron microscopy as described [ 111.
RESULTS Lectin binding to the gametes Table 1 summarizes the results of the binding of the fluorescent lectins to the sperm and to the egg and its envelopes. The data in table 1 merit some comment. Regarding the spermatozoa, ConA is bound only to the heads. This is shown not only by the selective staining of the heads by the fluorescent lectin but also by their head-tohead agglutination. Indeed, agglutination appears to occur (at least at the resolution of the optic microscopy under dark-field illumination) by the tip of the spermatozoa which give rise to rosette-like formations (rig. 1). On the other hand, FBP is bound to the whole surface of the spermatozoon which becomes entirely fluorescent. In the agglutination experiments, the agglutinated spermatozoa form long threads resulting from the heads and flagella sticking to one another. Both in the experiments with
Lectin binding to Ciona gametes
421
Table 3. Percent of fertilization
Table 2. Percent of fertilized
eggs of Ciona intestinalis following treatment with ConA (150 pglml) or with FBP (75 pglml)
of eggs of Ciona intestinalis following treatment with ConA (150 hglml) or with FBP (150 pglml)
In each experiment two samples of 10 eggs each (in I ml of sea water) from two different animals (a, b) were inseminated with 5 ~1 of undiluted sperm from the same donor animal (different from the animals which had supplied the eggs). The eggs had been previously exposed to the lectin for 10 min and washed three times with sea water. Five minutes after the insemination, the eggs were washed to remove excess of sperm, and 1 h later they were checked for cleavage. In all the experiments, the controls showed 100% fertilization
Protocol of the experiment as in table 2
ConA
FBP
la
0
70
lb
0
90
2a 2b
100
Expt No.
Expt No.
ConA
FBP
la
10 0 loo 0 0
3b
0 0 100 0 0 10
4a 4b
50 loo
40
lb
2a 2b
3a
30
100
0 100
most completely abolished in the presence of n-acetylglucosamine (see [3, 6]), the i"b 60 90 100 100 fluorescence of the surface of the follicle 4a 100 100 cells is not altered by treatment with this 4b 0 100 sugar. Two possibilities were considered to 5a 70 80 account for this result. (1) That the binding 5b 100 0 of the lectin was due to sialic acid; (2) that 6a 100 90 6b 100 80 the lectin was pinocytized. The former al7a 100 50 ternative was discounted for by treatment 7b 0 0 with neuraminidase (from Clostridium per2 40 10 400 fringens, Boehringer, at 0.6 U/ml from 10 min to 12 h on both intact and glycerinated 9a 70 80 9b 20 90 eggs) which neither abolished nor decreased the fluorescence. This treatment results in the rapid detachment of the follicle cells ConA and those with FBP, the reaction is from the chorion (thus suggesting that sialic abolished in the presence of the competing acid may be involved in the attachment of sugar (at 0.1 M final concentration), i.e. (Y- the follicle cells to the chorion). The fallen methylmanno- or a-methylglucopyranoside, off follicle cells soon start undergoing dein the,case of ConA, and fucose in the case generation; yet, as long as they have a of FBP. healthy appearance their surface continues As to the eggs, the ConA, FBP and WGA to be fluorescent. The possibility of pinobinding differs for the follicle cells, for the cytosis was checked in experiments in chorion and for the oolemma. As shown in which the lectin binding was carried out on table 1, of the four lectins tested only WGA eggs fixed with 2% formaldehyde for 10 was bound to the follicle cells (fig. 2); this min. The binding of the lectin to the surface lectin also reacts with the chorion of both of the follicle cells was not counteracted by living and glycerol-treated eggs. However, the sugar while that to the chorion was (figs while the reaction with the chorion is al- 3, 4). The interpretation of the non-rever30
Exp Cell RPS 116 (1978~
E-V/I Cell Rcs 116 (197X)
Lectin binding to Ciona gametes
423
sibility of the WGA binding to the follicle Of the lectins used in the present expericells is at the moment obscure. However, ments only FBP is bound by the oolemma. it is worth mentioning that the agglutination A weak reaction of the oolemma with ConA of the Ehrlich ascites carcinoma cells by has also been indicated in table 1. This beConA is not reversed by the mannopyranocause of the previous observations that alside; and only about one-half of the mem- though fluorescent ConA did not stain the brane glycoproteins of these cells could be oolemma of the unfertilized egg [4], a low eluted by the specific sugar haptens from but significant binding could be detected ConA- or WGA-Sepharose columns [SJ. when 1251-ConAwas used [7]. The involvement of membrane components other than the glycoproteins (such as glyco- Effect of lectins on fertilization lipids, see [lo]) should hence be taken into The effect of several lectins on fertilization consideration. has been studied by a number of invesThe chorion of both living and glyceroltigators (see [ 1, 8, 91); the results show that treated eggs reacts strongly with ConA (fig. the various lectins have different effects on 5) and with FBP (fig. 6), and the reaction is different animal eggs. In the hope that exspecifically abolished by the competing periments of this kind could provide insugar. The same result was obtained with formation as to whether or not the lectin the chorion isolated from glycerol-treated binding sites at the surface of the Ciona eggs (figs 7 and 8). gametes are involved in the process of Glycerinated eggs were treated with non- sperm-egg interaction, the effect of ConA fluorescent ConA or FBP respectively, and and of FBP on fertilization was tested. Prelithen with the other fluorescent lectin; in minary experiments had shown that neither both cases the chorion became uniformly WGA (which has no binding sites at the fluorescent just as though it had been ex- sperm surface) nor SBA (which has no bindposed only to the fluorescent lectin. ing sites either on the sperm or on the egg) had an effect. The results of the experiments with ConA and with FBP are reFig. I. ConA-treated spermatozoa; head-to-head ag- ported in table 2. glutination. X950. The results show that the effect of the Fig. 2. Fluorescence of the follicle cells exposed to FITC-WGA. X650. two lectins on the fertilization response of Fig. 3. An egg partially deprived of its follicle cells by shaking. fixed with formaldehvde for IO min. and the eggs from different animals is quite diftreated with FITC-WGA. The chorion and a follicle ferent: it ranges from a complete block of cell (arrow) are fluorescent. X500. fertilization to no effect whatsoever. The Fig. 4. In this experiment the eggs were fixed with formaldehyde for IO min, washed in sea water and in- slight difference between the fertilizationcubated in I ml of 0.1 M n-acetylglucosamine for I5 min. FITC-WGA was then added. The micrograph inhibiting effect of ConA and FBP is not shows the fluorescence of a follicle cell (arrow). while statistically significant (ConA,.?=%, S.D.= the chorion is not fluorescent (double ahmvs)~‘x650. k40.8; FBP, X=65, S.D.=?38.3). HowFig. 5. Living egg treated with FITC-ConA. The follicle cells are present but only the chorion is brightly ever, in order to have a more direct comfluorescent. X650. parison between the two lectins and to enFig. 6. Fluorescence micrograph of a glycerol-treated sure that our experiments were done under egR after treatment with FITC-F’BP. X650. Fii. 7. Binding of FITC-ConA to a chorion isolated saturating conditions, several experiments from a glycerol-treated egg. X650. were carried out in which the FBP conFig. 8. Binding of FITC-FBP to the chorion isolated from glyceroltreated eggs. x6.50. centration was raised to that of ConA, i.e. E.x~ Cell Res I16 I 1978)
424
Rosati, De Santis and Monroy
Fig. 9. Attachment of spermatozoa to the chorion (ch) of a glycerol-treated egg incubated in ConA. Many tine particles can be seen between the chorion and the sperm head (II). x38 000.
Fig. 10. Glycerol-treated egg
incubated in PBP and inseminated with heterologous sperms. Randomly oriented spermatozoa attached to the chorion (ch); extraneous material is present on the chorion and between the chorion and the sperm plasma membrane. (h , nucleus; m, mitochondria,f, flagellum.)
to 150 pg/ml. The results of these experiments are reported in table 3. Although in the batch of animals used in this series of experiments, the effect of both lectins was greater than in the previous experiments, i.e. the fertilization-inhibitory effect was stronger, they essentially exhibited the same effect and the variability among the eggs from different animals was equally evident. Sperm attachment to the chorion of the lectin-treated eggs (after removal of the follicle cells either by shaking or by glycerol E.rr,
Cell
Res
116 (197RI
treatment) did not at first sight appear to be interfered with by the lectins. However, a single wash with sea water resulted in the complete removal of the attached spermatozoa. Also, eggs from which the follicle cells were removed either by shaking or by treatment with glycerol, were treated with ConA or with FBP, inseminated and immediately processed for electron microscopy. Fixation preserves the loosely attached spermatozoa, which otherwise, as mentioned before, would be removed by a single wash-
Lectin binding to Ciona gametes ing of the eggs with sea water. In the ConAtreated eggs, the spermatozoa were seen to be attached to the chorion by their tips (fig. 9); however, they did not display the regular radial orientation as in normal eggs (see fig. lob [ 111). In no case was an acrosome reaction detected. In the FBP-treated eggs, on the other hand, the spermatozoa became attached to the chorion by their whole surface (fig. 10); which is in accord with the observation that this lectin is bound to the whole sperm plasma membrane. Also extraneous material, not seen in the untreated eggs, appears to be deposited over the entire surface of the chorion.
DISCUSSION The experiments described in this paper show that the surface of the spermatozoon and the chorion share binding sites for ConA and for FBP. This finding is of interest in view of the observations described in the first paper of this series [ 1l] in which we presented evidence that the chorion is the carrier of the specific sperm receptors, and that it is upon its interaction with the chorion that the spermazoon undergoes the acrosome reaction. The analogy between the properties of the chorion and those of the vitelline membrane of the sea urchin egg and of the zona pellucida of the mammalian egg have also been pointed out [ 111. Very few ConA-binding sites are present on the oolemma of the unfertilized egg [7]. In this connection it is pertinent to mention that in the egg of the sea urchin, Strongylocentrotus purpuratus, high and low affinity binding sites for ConA have been found, the former being preferentially located on the vitelline coat [13]. On the other hand, the oolemma of the Ciona egg shares FBPbinding sites with the surface of the sper-
425
matozoon. However, while the ConAbinding sites are restricted to the sperm head, the FBP receptors extend over the entire sperm surface; i.e. they are present both on the head and on the flagellum. This observation may be of interest in connection with the process whereby the spermatozoon is incorporated by denuded eggs. In this case, the spermatozoa lie flat on the oolemma and are incorporated by a process which is reminiscent of phagocytosis [14]. No binding sites are shared by the sperm surface and the surface of the follicle cells. We would like to recall that in vivo observations have never shown binding of spermatozoa to the surface of the follicle cells [ 111. These observations encourage the view that the lectin-binding sites shared by the gamete surfaces may be involved in the specific recognition and binding reactions between spermatozoa and eggs. It is also known from the work of other laboratories [l, 8, 91 that the surface of the eggs of different animal species and also the surface of the different envelopes of the same species [12] exhibit different affinities for lectins, thus suggesting that the lectin binding may be a test (though admittedly an indirect one) of the specific organization of the membrane glycoproteins of the gametes. Yet, the question that still remains unanswered is to what extent the lectin-binding sites on the chorion are involved in the functional organization of the sperm receptors. The observations described in this paper show that fertilization can be interfered with by ConA and by FBP; both these lectins have binding sites on the sperm surface and on the chorion and on the oolemma. Attention is drawn to the finding that at the same concentration of either ConA or FBP the eggs of different animals exhibit a different fertilization response. Also in other species, for example in the hamster, it has been
426
Rosati, De Santis and Monroy
observed that at the same lectin concentra- result in a variability of the number of lection, the fertilization response varies greatly tin molecules actually reaching the chorion from animal to animal (see, for example, in the different eggs from the same animal fig. 10 of ref. [9]). We interpret this as a and may explain why the fertilization rereflection of a different organization of the sponse in the eggs from the same animal is lectin-binding sites on the chorion (or on the not an all-or-none phenomenon. zona pellucida) of the eggs from different From all this a picture begins to emerge animals. Two possibilities can be en- of the chorion surface as a complex molecvisaged. The first is that the lectin-binding ular mosaic of sperm-binding sites. The sites are essential components of the sperm question is: are all sperm-binding sites receptor sites and hence, when the mosaic sperm receptors as well, i.e. sites at which of the saccharides side chains of the glyco- sperm binding is followed by the acrosome proteins of the chorion is such that their reaction? interaction with one of the lectins results in In the first paper of this series [ 1I] it was the selective masking of the receptors that shown that in eggs from which the follicle fertilization is interfered with. The second cells had been removed by shaking, only is that an unspecific masking of the sperm some of the attached spermatozoa undergo receptors may be brought about by various acrosome reaction. Two main alternatives mechanisms, among which the haphazardly may be visualized to interpret this observacross-linking neighbouring binding sites on tion. First, that while spermatozoa can atthe chorion. This interpretation (which is tach anywhere at the surface of the chorion, more in line with the known mode of action there are only a few spots-which might be of the lectins) also implies that whether or indicated as not spots-at which, either benot the activities of the sperm receptors cause of their molecular configuration or will be obstructed by the lectins depends because they include some specific molecon their arrangement on the chorion and, ular component (an organization that is alhence, on whether or not the binding of the tered by the glycerol treatment), sperm lectins will result in a steric hindrance to binding is followed by the acrosome reactheir interaction with the spermatozoa. tion. The second alternative is that in order A factor whose role is difficult to evaluate for a successful sperm-egg interaction to ocis that of the follicle cells. Studies in prog- cur (i.e. binding followed by acrosome reacress [ 151show that the attachment of these tion and penetration of the spermatozoon cells to the chorion is not uniform; i.e. in through the chorion) there must be a some points they appear to bind more matching between the molecular organizaloosely than in others. Besides the evidence tion of the binding sites on the chorion and from the electron microscope, this is sug- that of the plasma membrane of the spergested by the fact that when the follicle cells matozoon. We know nothing of the variare mnechanically removed [ 1I], shreds of ability of the molecular organization of the cells always remain attached to the chorion. plasma membrane among spermatozoa but Furthermore, microtubules and microfilathere is no reason to believe that it is not ments are abundant in the contact areas be- just as great as that among eggs. This varitween adjacent follicle cells; this may con- ability may clearly play a role also in the trol the closing and opening of the channels case that the sperm receptors on the chorion between cells [15]. These conditions may are restricted to the hot spots. .%/I Cell Res I16 (1978)
Lectin binding to Ciona gametes We are indebted to Dr M. Burger for reading the manuscript and for his comments, to Dr S. Denis-Donini for helpful advice on the use of lectins, and to Mr G. Iamunno and Mr C. Gargiulo for their technical assistance. This work has been supported by a grant (no. 77.00349.85) of the CNR Project on the Biology of Reproduction.
REFERENCES 1. Aketa, K, Exp cell res 90 (1975) 56. 2. Cook, G M W & Stoddart, R W, Surface carbohydrates on the eukaryotic cell, pp. 257. Academic Press, London & New York (1974). 3. Goldstein, I J, Hammarstrom, S & Sundblad, G, Biochim biophys acta 405 (1975) 53. 4. Monroy, A, Ortolani, G. O’Dell, D & Millonig, G, Nature 242 (1973) 409. 5. Nachbar, M S, Oppenheim, J D & Aull, F, Biochim biophys acta 419 (1976) 512.
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6. Nagata, Y & Burger, M M, J biol them 249 (1974) 3116. 7. O’Dell, D S, Ortolani, G & Monroy, A, Exp cell res 83 (1974) 408. 8. Oikawa, T, Nicolson, G L & Yanagimachi, R, Exp cell res 83 (1974) 239. 9. Oikawa, T, Yanagimachi, R & Nicholson, G L, J reprod fertil43 (1975) 137. 10. Read, B D, Demel, R A, Weigandt, H & Deenen, L L M, Biochim biophys acta 470 (1977) 325. II. Rosati, F & De Santis, R, Exp cell res 112 (1978) 111. 12. Stenuit, K, Genskens, M, Steiner-t, G & Tencer, R, Exp cell res 105(1977) 159. 13. Veron, M & Shapiro, B M, J biol them 252 (1977) 1286. 14. De Santis, R & Rosati, F. In preparation. 15. De Santis, R, Rosati. F & Jamunno, V. Submitted for publication. Received February 2 I, 1978 Revised version received May 9, 1978 Accepted May 12, 1978
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(1978,