DEVELOPMENTAL
BIOLOGY
146,416-422 (1991)
Glycoproteins from Bufo arenarum Vitelline Envelope with Fertility-Impairing Effect on Homologous Spermatozoa J.N. VALZ-GIANINET,* *Departamento
de Biologia de1 Desurrollo-INSIBIO Bioquimicas y Furmnceuticas,
E.J. DELPINO,*
(CONICET-UNT), UrGversidad Nacionul Accepted
When spermatozoa from Bufo arenarum are homologous oocytes, they lose their fertilizing mannoside residues from them results in activity effect. Sepharose-concanavalin A columns were column. The fertility-impairing effect observed fect.
t$) 1991 Academic
CABADA,+~'
Tucumcin (.hOOO), R. Argentina; und TArea Biologia, Facultad de Rosario, Suipacha 531, Rosario (.OOO) R. Argentina
April
de Ciencias
19, 1991
incubated with molecules extracted from the vitelline envelopes of capacity. Those molecules are glycoproteins, and the elimination of loss, while digestion of the proteic moiety did not alter their biological used to purify the glycoproteins, since the active fraction binds to the does not seem to be mediated by an acrosome reaction-inducing ef-
Press, Inc.
INTRODUCTION
Although fertilization involves a complex series of events, it is evident from a number of studies that the specific recognition between gametes is mediated by surface-located macromolecules. Recent studies on sperm-oocyte interactions in several species led to the conclusion that important events occur when spermatozoa arrive at the zona pellucida of mammals (Gwatkin, 1976; Gwatkin et al., 1976; Bleil and Wassarman, 1980a,b; Gwatkin 1978) or the vitelline envelope of Ascidians (Rosati and de Santis, 1980; de Santis et al., 1980) and sea urchins (Glabe and Vacquier, 19’78; Shapiro and Eddy, 1980; Kinsey and Lennarz, 1981). Current evidence indicates that, in these animals at least, a sperm recognition activity involving glycoproteins is present, although the exact chemical composition of the molecules has not yet been elucidated. It has been suggested that gamete recognition in the toad Bufo urenarum might be based in the association between lectin-binding sites in the vitelline envelope and the complementary binding molecules on the sperm surface (Raisman et al., 1977; Cabada et ab, 1978; de1 Pino and Cabada, 1987). From both gametes, molecules bearing gamete-recognizing properties have been proposed to exist on the basis of either their ability to cause species-specific agglutination of spermatozoa or to inhibit fertilization when oocytes are treated with them before insemination. In this paper we report the presence of glycoproteins extractable from the vitelline envelope with the 1 Member of the Research Career (CONICET). ‘To whom reprint requests and correspondence dressed.
0012-1606/91$3.00 Copyright All rights
AND M.O.
‘CI 1991 by Academic Press, Inc. of reproduction in any form reserved.
should
be ad-
416
property of impairing gous spermatozoa. MATERIALS
the fertilizing
AND
capacity of homolo-
METHODS
Animals. Sexually mature specimens of B. arenarum were collected in the neighborhoods of the cities San Miguel de Tucumbn and Rosario and kept in a moist chamber at 15°C until used. Gametes. B. arenarum oocytes were obtained by intraperitoneal injection of homologous hypophysis suspension (Houssay et al., 1929). Injected females were kept at 21°C and oviposition began 12 hr afterwards. At this moment, animals were pithed and oocyte strings removed from ovisacs. Removal of jelly coats was accomplished as follows: oocyte strings were hydrated by immersion in 10% Ringer solution for 10 min prior to treatment with 1% sodium thyoglycolate (pH 8.0) solution. Dejellied oocytes were thoroughly rinsed with Ringer solution and kept in it until used. B. arenarum sperm suspensions were obtained by gently disrupting the testes with a glass homogenizer in cold 10% Ringer solution containing 10 mM Tris-HCl buffer, pH 7.6 (hereafter, 10% Ringer-Tris solution). After removing tissue debris by gauze filtration, spermatozoa were washed three times by centrifugation at 200~ for 15 min each, at 4°C with cold Ringer-Tris solution. Sperm concentration was estimated by counting an aliquot in a hemocytometer. Egg water was prepared according to Barbieri and de1 Pino (1975) and stored at -20°C until used. Vitelline envelope isolation. Dejellied oocytes were homogenized in Ringer-Tris (calcium free) solution in a Potter-Elvehjem homogenizer. Vitelline envelopes
VALZ-GIANINET,
417
DEL PINO, AND CABADA
WV
were recovered by filtering the homogenate through a double sheet of 30-mesh nylon screen, and were finally washed with cold Ringer-Tris (calcium free) solution. All procedures were carried out at 0-4°C. VE suspensions were stored at the same temperature, or freeze dried, until used. Vitelline und fertilization envelopes extraction. Extraction of soluble molecules from the VE was carried out by incubating a suspension of 2000-4000 isolated VEs in 2 ml of 10% Ringer-Tris for 30 min in a water bath at 60°C. The resulting suspensions were cooled at room temperature and centrifuged at 3000g to discard the rest of undissolved VEs, and the supernatant was stored at -20°C until used. Extracts of fertilization envelopes (FE) were prepared from dejellied eggs after fertilization by incubating FEs in 0.1 N of NaOH for 10 min. After the rest of undisolved FEs were separated by centrifugation, the supernatant was dialized against 10% Ringer-Tris overnight at 4°C. Enzymatic treatments. The effect of oc-mannosidase and trypsin on solubilized VE was examined by incubating the fractions with 1 pg/ml of cu-mannosidase or 50 pg/ml of trypsin in 1 ml of buffered medium for 30 min at 22°C. Fertilization assay. The ability of the different extracts to inhibit fertilization was estimated by an cn vib0 fertilization assay: 100 /*l of a sperm suspension (lo7 sperm/ml) in Ringer-Tris solution was incubated with different amounts of solubilized VEs or FEs for 30 min at 20°C. Treated sperm were collected by centrifugation at 1000~ for 5 min at 4°C. The supernatant was discarded and the pellet suspended with cold 10% Ringer-Tris. The suspension was washed once more before being used. Batches of 30-40 dejellied oocytes in a mixture of egg water and 10% Ringer-Tris (1:l) were inseminated with aliquots of control and pretreated sperm suspensions, at a final concentration of lo6 cells/ml. Oocytes were considered to be fertilized when subsequent cleavages were normal. Lectins. The purified plant lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), and the chromatographically pure saccharides (a-methyl-D-mannoside, N-acetyl galactosamine, and N-acetyl glucosamine) were purchased from Sigma Chemical Co. (St. Louis, MO). Phytohemagglutinin P (PHA-P) was from DIFCO Laboratories.
3 Abbreviations used: VE, vitelline envelope; FE, fertilization envelope; Con A, concanavalin A; SBA, soybean agglutinin; WGA, wheat germ agglutinin; PHA-P, phytohemagglutinin-P; SDS, sodium dodecyl sulfate; PAGE, polpacrylamide gel electrophoresis.
Coupling of lectins to Sepharose. Lectins were coupled to Sepharose 4B by a modification of the cyanogen-bromide procedure of Cuatrecasas (1970). Briefly, 10 to 20 ml of Sepharose 4B was activated with 2-4 g of CNBr, at pH 9.0 for 10 min. Sepharose was then washed with 1000 ml of cold 0.1 M NaHCO, on a Buchner funnel, and the moist gel cake was transferred to a beaker containing 60-80 ml of 0.9% NaCl, 0.01 M NaHCO,, 50-120 mg of the lectin, and 0.1 M of the haptene saccharide. The pH was adjusted to 7.4 with HCl and the mixture gently stirred overnight at 4°C. The lectin-Sepharose complex was finally poured into a glass column and washed with 500 ml of 0.9% NaCl, 0.01 MNaHCO,, followed by 300 ml of Ringer-Tris. Through this procedure, 120 mg of Con A, 50 mg of PHA-P, and 90 mg of WGA were coupled to Sepharose 4B with an efficiency of approximately 90%. A&nity chromatography. The VE extracts were dialized overnight against Ringer-Tris containing 1 mM MgCl, and 1 mM MnCl, and applied to a 1 X 10 cm column of lectin-Sepharose 4B. The column was thoroughly washed with the same solution until the absorbance of the eluate at 280 nm was below 0.015 OD. The bound glycoproteins were eluted from the column with a 0.1 M solution of the corresponding saccharide (TVmethyl-D-mannoside for Con A, N-acetyl-D-galactosamine for PHA-P, and N-acetyl-D-glucosamine for WGA), until the absorbance of the fractions was below 0.015 OD. Treatment with borohydride. Treatment of VE extracts with borohydride were carried out according to Kornfeld and Kornfeld (1970). The extracts were incubated in 0.5 M borohydride and dissolved in 0.2 NNaOH for 72 hr at 37°C under an atmosphere of nitrogen and continuous stirring. After incubation, the samples were neutralized with HCl. Electrophoresis. SDS-PAGE of VE extracts was performed according to Laemmli (1970) with 7.5% running gels. Proteins were stained with Coomassie brilliant blue R-250 and carbohydrates with periodic acid-Schiff according to Fairbanks et ul. (1971). Electron rrcicroscovy. Aliquots of sperm suspensions were fixed in 2% glutaraldehyde in Ringer-Tris for 20 min at 4°C. Spermatozoa were pelleted by centrifugation and washed three times with phosphate buffer (pH 7.2), and postfixed in 1% osmium tetroxide. After treatment with 1% uranyl acetate for 30 min at 4”C, pellets were dehydrated in ethanol and acetone and embedded in Epon-Araldite resin. Observations were carried out with a Zeiss 109 electron microscope. RESlJLTS
Eflect of VE and FE extracts on the fertilizing capacity of B. arenarum sperma,toxoa. The effect of the extracts
418
DEVELOPMENTAL TABLE
EFFECT
OF INCUBATING OF VE
Gamete treated Oocytes
Spermatozoa
Bufo AND
BIOLOGY
1
urenarurn
GAMETES FE IN FERTILIZATION
WITH
EXTRACTS
Inhibition fertilization
Incubation solution Ringer-Tris VE extract FE extract
o-+0 6.3 f 0.9 Ok0
Ringer-Tris VE extract FE extract
Ok0 80.7 k 2.3 Ok0
VOLUME
146,
1991
a 2
5 60-
of (%)
Note. 10’ sperm/ml were incubated in each medium at 20°C for 30 min. VE or FE extracts were at a final concentration of 50 pg/ml in the media. Inseminations were carried out with lo6 sperm/ml in the media. Results are expressed as mean f SEM (n = 3).
from both VE and FE on the fertilization process was examined by incubating either oocytes or spermatozoa with them before insemination. As observed in Table 1, when spermatozoa were treated with the molecules extracted from VEs, prior to being used to inseminate oocyte strings, fertilization was inhibited in about 80%. The same treatment applied to oocytes did not alter the fertilization rate. When either gamete was treated with molecules extracted from FE, no fertility inhibition was observed. Treatments applied to sperm suspensions were carried out with different concentrations of VE and FE extracts. In this case, a concentration-response effect was observed for VE-treated spermatozoa, while no effect with any concentration was observed when spermatozoa were incubated in FE extracts (Fig. 1). When low concentrations of VE extracts were used, the effect observed depended linearly on the incubation period (Fig. 2). The molecules extracted from VEs are glycoproteins, and in different preparations they differ in the ratio of carbohydrate/protein, ranging between 7 and 14%.
0
10 Incubatlcn
20 30 Period (minutes)
FIG. 2. Time-dependence of inhibition of fertilization by solubilized VE: lo7 sperm/ml were incubated with 30 pg/ml of VE extracts in the medium at 20°C for 30 min. Fertilization assays were performed with lo6 sperm/ml in the media. Data represent the mean -t SEM (n = 3).
These different preparations differ also in their capacity to inhibit spermatozoa fertility. In Fig. 3, the positive correlation between the carbohydrate content of the preparations and fertility inhibition capacity is clear. Incubation of spermatozoa with either mannosidase or trypsin, in the same conditions used here, did not alter their fertilizing capacity. Furthermore, the inhibitory activity of VE extracts was eliminated by treatment with cu-mannosidase, an exoglycosidase that removes terminal mannose residues from hexose polymers. On the contrary, the activity of these extracts was not altered by incubation with trypsin (Table 2). Similar results were observed when VE extracts were digested with other proteases. Isolation of sperm-interacting molecules from VE by afinity chromatography. VE extracts were subjected to
affinity chromatography with columns of Con A, WGA, or PHA-P coupled to Sepharose. In all cases most of the protein applied to the column was not retained by it. In the case of Con A, the fraction
Carbohydrate
Protein
Added
(pg/ml)
FIG. 1. Inhibition of fertilization by solubilized VE and FE: lo7 sperm/ml were incubated in each medium at 20°C for 30 min. Fertilization assays were carried out with lo6 sperm/ml in the media. Data represent mean + SEM (12 = 3). (o), VE extracts; (A), FE extracts.
Content
(%I
FIG. 3. Inhibition of fertilization by solubilized VE with different carbohydrate content. Spermatozoa incubations and fertilization assays were performed as described in the legend to Fig. 2. Carbohydrate content of VE solutions was established as
carbohydrate
content
=
carbohydrates proteins
(pg/ml) (pg/ml)
x 1oo.
VALZ-GIANINET,
EFFECT
DEL PINO, AND CABADA
TABLE 2 OF ENZYMATIC TREATMENTS ON THE INHIBITION OF FERTILIZATION BY SOLUBILIZED VEs Inhibition fertilization
Treatment
of (%)
C4
100 m 5
E O3 E 8 N 02
50
=:
None Mannosidase (1 pg:/ml) Trypsin (50 pg/ml) Note lo7 sperm/ml were tracted VE) at 20°C for 30 were performed with lo6 pressed as mean ? SEM (n
incubated min, after sperm/ml = 3).
69.7 k 4.4 3.3 k 0.9 65.7 + 2.7 in the media (50 pg/ml of exdigestions. Fertilization assays in the media. Results are ex-
excluded from the column had no effect on sperm fertility. On the contrary, the glycoproteins bound to the column inhibited sperm fertility: sperm incubation with ‘7 yg/ml of the product was effective in reducing fertility to 50% (Fig. 4). The same degree of reduction in fertility with the original product is observed when spermatozoa are incubated at least with 30 yg/ml (see Fig. 1). When testing the biological effect of the bound fraction of either WGA or PHA-P-Sepharose 4B conjugates, no fertilizing-inhibitory effect was found (Fig. 4). In these cases, the inhibitory effect was present in the void fraction. In the case of Con A bound fraction, the inhibitory activity was destroyed, as in the crude preparation, by incubation with a-mannosidase, but not with trypsin. In addition, when oocytes were incubated with the bound fraction, as was observed with the crude preparation, they did not modify their fertilizability. Since Con A itself is very effective in inhibiting fertilization even at low concentrations (Cabada et al., 1978;
5 Protem
10 Added
15 (ug/ml)
FIG. 4. Inhibition of fertilization by lectin-affinity-isolated molecules. Spermatozoa incubations and fertilization assays were carried out as described in the legend to Fig. 1. Solubilized VE were submitted to affinity chromatography in immobilized Con A (0), WGA (o), and PHA-P (A). The lectin-bound fractions were eluted from the columns with the appropriate sugar haptene (see Materials and Methods). Data represent mean ? SEM (n = 3).
5 I g 03 ^, >
0 I 0 IO
:o
20
20
30
FrockIon
40
n:
FIG. 5. Effect of treatment with borohydride on the fertility-inhihitory activity of VE extracts. Untreated extracts from VEs (3 mg) (A) or after treatment with borohydride (3.2 mg) (B) were loaded onto a Sephadex-Con A and eluted with 0.1 M of n-methyl-D-mannoside (fraction 17 onwards). The fractions (1 ml) were pooled as indicated for each peak (dashed bars). Spermatozoa were incubated in 50 pg of protein/ml of each pool before being used to inseminate oocyte strings (conditions as in the legend to Fig. 2). The bars indicate the fertilization inhibition (mean f SEM; 11 = 4), referenced to controls, i.e., spermatozoa incubated in Ringer-Tris, 0.1 M a-methyl-D-mannoside (97.4 i 6.9% fertilization). When spermatozoa were incubated in untreated crude extracts of VE, fertilization was 40.7 -+ 7.6%
de1Pino and Cabada, 1987), considerable care was taken to prevent the leakage of Con A from the columns. For this reason, the Con A-Sepharose column was extensively washed before use until the washings of the column did not agglutinate spermatozoa (de1 Pino et al., 1981). It was also checked that 0.1 Ma-methyl-D-mannoside (used to elute the molecules that bind to the column) did not significantly reduce fertilization rates when present in the sperm-incubation media. Role of the glycosidic moiety. The treatment of glycoproteins with borohydride and NaOH separates the Oglycosidic residues. After alkaline borohydride treatment, samples were loaded onto a Sepharose-Con A column, and the bound and void fractions separated. Untreated extracts from VE were also run. The fractions collected were pooled as indicated in Fig. 5. Spermatozoa were incubated in aliquots of each pool before being used to inseminate oocyte strings. The results
420
DEVELOPMENTAL
BIOLOGY
summarized in Fig. 5 indicate that, as was already quoted, the bound fraction of the untreated samples (A) (containing 65% of total proteins), contained most of the fertility-impairing activity, if compared with the void fraction (43.3 + 6.9% vs 18.5 f 5.2% of fertility inhibition). In the experiments with borohydratetreated samples (B), it is observed that most of the material is in the void fraction (76% OD BO,,-absorbing of total proteins). This result indicates that the borohydrate treatment separates a considerable part of the glycosidic component from the glycoprotein that binds to concanavalin A, thus not permiting the corresponding proteic component to bind the column. However, the fertility-impairing effect of the bound fraction not only does not decrease, but increases, if the total fertility-impairing effect observed in both bound peaks is considered (70% of total effect in experiment A vs 81% in experiment B). Together, these results and those of trypsin treatment indicate that the effect of inhibiting fertilization can be ascribed to the glycosidic moiety of the glycoprotein extracted from the VE. This type of experiment does not indicate that O-linked oligosaccharides are the only ones involved in the effect observed, since those N-linked glycosidic residues resistant to fl elimination might also be present in the bound fraction. Preliminary results of experiments with labeling extracts from VEs with lz51 indicate that least some of the glycoproteins can be labeled and actually bind to spermatozoa (Valz-Gianinet; de1 Pino and Cabada, unpublished results). pattern of VE extracts. The results obElectrophoretic tained by SDS-PAGE of the molecules bound to Con A-Sepharose are shown in Fig. 6. All bands stained for both proteins and glucids. Six peaks were consistently found in several runs. Their molecular weights (in Daltons) are: (I) 101,000, (II) 97,000, (III) 90,000, (IV) 71,000, (V) 41,500, and (VI) 33,500. Electron microscopy of spermaElectron microscopy. tozoa incubated in extracts of VEs or saline solutions indicate that no observable alterations can be ascribed to the treatment. We scored about 60 treated spermatozoa, and a similar number of controls, in three different assays. All of them presented the acrosome intact. This result suggests that acrosome breakdown is not involved in the mechanism of inhibition of sperm fertilizing capacity.
DISCUSSION
The process of fertilization consists of a complex sequence of events which involves the species-specific fusion of the plasma membranes of sperm and oocyte. Ga-
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146, 1991
.* -220
I lY+ I
-67 -60
-36
FIG. 6. Electrophoretic pattern of VE extracts. Photography of the electrophoresis of solubilized VEs in SDS-PAGE (7.5%), after staining with Coomassie brilliant blue R-250. Molecular weight markers are indicated in kDa.
metes must be able to recognize each other as a requisite for their fusion. At a molecular level, this process seems to be mediated by molecules at the surface of both gametes that must specifically interact to render a successful contact. During its approximation to the oocyte, amphibian spermatozoa must traverse the jelly coats covering the oocytes and penetrate the VE before it can fuse with the plasma membrane of the oocyte. The vitelline envelope of amphibian oocytes and analogous structures in other animals seem to be an important site of interaction between both gametes (Florman and Wassarman, 1985; Rosati, 1985; Macek and Shur, 1988) and participate in the block to polyspermy (Grey et ah, 1974; Wolf, 1974; Wyrick et al., 1974; Grey et al., 1976; Wolf et ah, 1976; Greve and Hedrick, 19’78;Gerton and Hedrick, 1986; Cabada et al., 1978). These properties of the VE can be explained by the presence on its surface of sites for recognition and binding for homologous spermatozoa. In this work we present evidence that spermatozoa lose their fertilizing capacity when incubated in the presence of molecules extracted from the VE, before being used to inseminate oocytes. This process is concentration and time-dependent. The fertility-inhibitory property was not observed when sperm were incubated with extracts of FE, obtained from activated oocytes. This result suggests that the modifications suffered by the VE due to products released from the cortical granules during the process of oocyte activation hinder and/ or modify the sites of interaction of the VE with spermatozoa.
VALZ-GIANINET, DEL PINO, AND CABADA
Some preliminary experiments working with ‘251-labeled molecules from VE suggest that the glycoproteins bind to spermatozoa. The molecules bearing the fertility-impairing activity are glycoproteins since they bind to Sepharose-Con A columns. The results of the experiments of /3-elimination and trypsin and a-mannosidase digestions indicate that the glycosidic residues are the responsible for the effect observed. Binding to Con A does not necessarily indicate that the mannoside residues are the ones responsible for the effect observed since the glycosidic residues involved in the fertility-impairing effect could not be the same as those involved in binding to Con A, However, since digestion with cu-mannosidaseeliminates the inhibitory effect, it seems that the mannoside residues (or others stereochemically related) are indeed involved in the effect. The mechanism through which spermatozoa lose their fertility is different from that inducing a premature acrosome reaction as was observed when spermatozoa were incubated with lipidic vesicles (Cabada et al., 1984; Diaz Fontdevila et al., 1988). A possible explanation for the phenomena described is that the substances released from the VE bind to the spermatozoa occupying the binding or recognition sites on the sperm surface, which cannot interact any longer with similar molecules present in the VE of the oocyte to be fertilized, rendering an infertile spermatozoa. In this connection, and at least for this part of the process of fertilization, amphibians are similar to mammals (Shur, 1989), sea urchins (Rossignol et al., 1984), and ascidians (de Santis and Pinto, 1987) in which the sperm-receptor activity is located in the zona pellucida and vitelline coat, respectively. This work was supported by grants from CONICET (PID No. 3055500/88), Fundacihn Antorchas, and TWAS (No. BC 898-127) awarded to M.O.C. REFERENCES BARBIERI, F. D., and DEL PINO, E. J. (1975). Jelly coats and diffusible factor in anuran fertilization. Arch. Bid. 86, 311-321. BLEIL, J. M., and WASSARMAN, P. M. (1980a). Mammalian sperm-egg interactions: Identification of glycoproteins in mouse egg zona pellucida possessing receptor activity for sperm. Cell 20, 8’73-882. BLEIL, J. M., and WASSARMAN, P. M. (1980b). Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte zona pellicida. Dev. Biol. 76, 15-20. CABADA, M. O., MARIANO, M. I., and RAISMAN, J. S. (1978). Effect of trypsin inhibitors and concanavalin A on the fertilization of Bufo urenm-um coelomic oocytes. J. Exp Zool. 204, 409-416. CABADA, M. O., BLOJ, B., ORTIZ, L. P., DE, VALZ-GIANINET, J. N., and DfAZ FONTDEVILA, M. F. (1984). Effect of phosphatidylcholine on acrosome breakdown and fertilizing capacity of amphibian spermatozoa. Dw. Growth Differ. 26, 515-523. CUATRECASAS,P. (1970). Protein purification by affinity chromatogra-
Sperm Fertility
in Awphitriaws
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phy. Derivatizations of agarose and polyacrylamide and agarose beads. J. Bid. C’hem. 245, 3059-3065. DEL PINO, E. J., CABADA, M. O., and FONIO, M. C. (1981). Lectin-mediated sperm agglutination of B?lfb urprlnr~lm and Lcptotloct!/l/r.s cha~wnsis spermatozoa. Ga tnrtu Rec. 4, 97-104. DEL PINO, E. J., and CABADA, M. 0. (1987). Lectin-binding sites in the vitelline envelope of Bafo orewurum oocytes. Role in fertilization. Ga metr Res. 17, 333-342. DE SANTIS, R., JAMUNNO, G., and ROSATI, F. (1980). A study of the chorion and the follicle cells in relation to sperm-egg interaction in the Ascidian Ciorrc~ i~tf&inalis. &I,. Rio2. 74, 490-499. DE SANTIS, R., and PINTO, M. R. (1987). Isolation and partial characterization of a glycoprotein complex with sperm-receptor activity from Cio?/u intestincrlis ovary. net:. Grodh Di&r. 29, 617-625. D~AZ FONTDEVILA, M. F., BLOJ, B., and CABADA, M. 0. (1988). Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity on amphihian sprrmatozoa. Gumf~tc RPS. 21, 59-70. FAIRBANKS, G., STECK, T. L., and WALLACH, D. L. H. (1971). Elertrophoretic analysis of the major polypeptides of the human rrythrocyte membrane. Biochemistr!/ 10, 2606-2619. FLORMAN, H. M., and WASSARMANN, P. M. (1985). O-linked oligosaccharides of mouse egg ZP3 accounts for its sperm receptor activity. (‘t,// 41, 313-324. GERTON, G. L., and HEDRICK, J. L. (19X6). The vitelline envelope to fertilization envelope conversion in eggs of Xen0lnr.s lorvis. Ikt~. Riot. 116, l-7. GLABE, C. G., and VACQUIER, D. V. (1978). Egg surface glycoprotein receptor for sea urchin sperm bindin. Proc. h’trtt. Artrd. Sci. USA 75, 881-885. GREY, R. D., WOLF, D. P., and HEDRICK, J. L. (1974). Formation and structure of the fertilization envelope in X(YCO~>US /(IEI,~s.nc,l: Bid. 36.44-61. GREY, R. D., WORKING, P. K., and HEDRICK, J. L. (1976). Evidence that the fertilization envelope blocks sperm entry in eggs of Xe~col~~.s lun>is: Interaction of sperm with isolated envelopes. nrr,. Bid. 54, 52-60. GREVE, C. L., and HEDRICK, J. L. (1978). An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of Xenopas tawis. Gamete Rex 1, 13-18. GWATKIN, R. B. L. (1976). Fertilization. 1~! “The Cell Surface in Animal Embryo: Genesis and Development” (G. Poste and G. L. Nicholson, Eds.), p. l-54. Elsevier/North Holland Biomedical Press, Amsterdam. GWATKIN, R. B. L. (1978). Cell-cell interactions in mammalian fertilization. 2~ “Birth Defects: Original Articles Series” Vol. 14 f2), pp. 363-376. The National Foundation. GWATKIN, R. B. L., CARTER, H. W., and PATTERSON, H. (1976). Association of mammalian sperm with the cumulus cells and the zona pellucida, studied by SEM. ITL “Scanning Electron Microscopy.” pp. 379-384. ITT Research Institute, Chicago. HOUSSAY, B. A., GIUSTI, L., and LASCANO GONZALEZ, J. M. (1929). Implantation d’hypophyse et stimulation des fonctions sexuelles du Crapaud. C. R. S’ecrwx?Sot. Bid. 102, 864-866. KINSEY, W. H., and LENNARZ, W. J. (1981). Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits spermegg binding and fertilization. .I. Cell Biol. 91, 325-331. KORNFELD, R., and KORNFELD, S. (1970). The structure of phytohemagglutinin receptor site from human erythrocytes. J. Bid. C’hwx 245,2536-2545. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. AVature 227,680-685.
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MACEK, M. B., and SHUR, B. D. (1988). Protein-carbohydrate complementarity in mammalian gamete recognition. Gamete Res. 19,1-l?‘. RAISMAN, J. S., MARIANO, M. I., and CABADA, M. 0. (1977). Effect of trypsin inhibitors and concanavalin A on Bufo arenarum fertilization. Dee Growth Differ. 19, 119-123. ROSATI, F. (1985). Sperm-egg interactions in Ascidians. In. “Biology of Fertilization” (C. B. Metz and A. Monroy, Eds.), Vol. 2, pp. 361-388. Academic Press, New York. ROSATI, F., and DE SANTIS, R. (1980). Role of the surface carbohydrates in sperm-egg interaction in Ciou~cr. intestinulis. Nature 283, 762-764. ROSSIGNOL, D. P., EARLES, B. J., and LENNARZ, W. J. (1984). Characterization of the sperm receptor on the surface of eggs of Stroqolyocentrotzcs yurpurahs. De?: Biol. 104, 308321. SHAPIRO, B. M., and EDDY, B. M. (1980). When sperm meets egg: Bio-
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chemical mechanism of gamete interaction. ht. Reu. CytoL 66,257302. SHUR, B. D. (1989). Galactosyltransferase as a recognition molecule during fertilization and development. In “The Molecular Biology of Fertilization” (H. Schatten and G. Schatten Eds.), pp. 38-72. Academic Press, New York. WOLF, D. P. (1974). The cortical granule reaction in living eggs of the toad Xenopus lwevis. Dev. BioL 36, 62-71. WOLF, D. P. (1976). Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus 1uwi.s. Biochemistry 15, 3671-3678. WYRICK, R. E., NISHIHARA, T., and HEDRICK, J. L. (1974). Agglutination of jelly coats and cortical granules components and the block to polyspermy in the Amphibian Xenopus laevis. Proc. Natl. Acad. Sci. USA 71,2067-2071.
BIOLOGY
146,416-422 (1991)
Glycoproteins from Bufo arenarum Vitelline Envelope with Fertility-Impairing Effect on Homologous Spermatozoa J.N. VALZ-GIANINET,* *Departamento
de Biologia de1 Desurrollo-INSIBIO Bioquimicas y Furmnceuticas,
E.J. DELPINO,*
(CONICET-UNT), UrGversidad Nacionul Accepted
When spermatozoa from Bufo arenarum are homologous oocytes, they lose their fertilizing mannoside residues from them results in activity effect. Sepharose-concanavalin A columns were column. The fertility-impairing effect observed fect.
t$) 1991 Academic
CABADA,+~'
Tucumcin (.hOOO), R. Argentina; und TArea Biologia, Facultad de Rosario, Suipacha 531, Rosario (.OOO) R. Argentina
April
de Ciencias
19, 1991
incubated with molecules extracted from the vitelline envelopes of capacity. Those molecules are glycoproteins, and the elimination of loss, while digestion of the proteic moiety did not alter their biological used to purify the glycoproteins, since the active fraction binds to the does not seem to be mediated by an acrosome reaction-inducing ef-
Press, Inc.
INTRODUCTION
Although fertilization involves a complex series of events, it is evident from a number of studies that the specific recognition between gametes is mediated by surface-located macromolecules. Recent studies on sperm-oocyte interactions in several species led to the conclusion that important events occur when spermatozoa arrive at the zona pellucida of mammals (Gwatkin, 1976; Gwatkin et al., 1976; Bleil and Wassarman, 1980a,b; Gwatkin 1978) or the vitelline envelope of Ascidians (Rosati and de Santis, 1980; de Santis et al., 1980) and sea urchins (Glabe and Vacquier, 19’78; Shapiro and Eddy, 1980; Kinsey and Lennarz, 1981). Current evidence indicates that, in these animals at least, a sperm recognition activity involving glycoproteins is present, although the exact chemical composition of the molecules has not yet been elucidated. It has been suggested that gamete recognition in the toad Bufo urenarum might be based in the association between lectin-binding sites in the vitelline envelope and the complementary binding molecules on the sperm surface (Raisman et al., 1977; Cabada et ab, 1978; de1 Pino and Cabada, 1987). From both gametes, molecules bearing gamete-recognizing properties have been proposed to exist on the basis of either their ability to cause species-specific agglutination of spermatozoa or to inhibit fertilization when oocytes are treated with them before insemination. In this paper we report the presence of glycoproteins extractable from the vitelline envelope with the 1 Member of the Research Career (CONICET). ‘To whom reprint requests and correspondence dressed.
0012-1606/91$3.00 Copyright All rights
AND M.O.
‘CI 1991 by Academic Press, Inc. of reproduction in any form reserved.
should
be ad-
416
property of impairing gous spermatozoa. MATERIALS
the fertilizing
AND
capacity of homolo-
METHODS
Animals. Sexually mature specimens of B. arenarum were collected in the neighborhoods of the cities San Miguel de Tucumbn and Rosario and kept in a moist chamber at 15°C until used. Gametes. B. arenarum oocytes were obtained by intraperitoneal injection of homologous hypophysis suspension (Houssay et al., 1929). Injected females were kept at 21°C and oviposition began 12 hr afterwards. At this moment, animals were pithed and oocyte strings removed from ovisacs. Removal of jelly coats was accomplished as follows: oocyte strings were hydrated by immersion in 10% Ringer solution for 10 min prior to treatment with 1% sodium thyoglycolate (pH 8.0) solution. Dejellied oocytes were thoroughly rinsed with Ringer solution and kept in it until used. B. arenarum sperm suspensions were obtained by gently disrupting the testes with a glass homogenizer in cold 10% Ringer solution containing 10 mM Tris-HCl buffer, pH 7.6 (hereafter, 10% Ringer-Tris solution). After removing tissue debris by gauze filtration, spermatozoa were washed three times by centrifugation at 200~ for 15 min each, at 4°C with cold Ringer-Tris solution. Sperm concentration was estimated by counting an aliquot in a hemocytometer. Egg water was prepared according to Barbieri and de1 Pino (1975) and stored at -20°C until used. Vitelline envelope isolation. Dejellied oocytes were homogenized in Ringer-Tris (calcium free) solution in a Potter-Elvehjem homogenizer. Vitelline envelopes
VALZ-GIANINET,
417
DEL PINO, AND CABADA
WV
were recovered by filtering the homogenate through a double sheet of 30-mesh nylon screen, and were finally washed with cold Ringer-Tris (calcium free) solution. All procedures were carried out at 0-4°C. VE suspensions were stored at the same temperature, or freeze dried, until used. Vitelline und fertilization envelopes extraction. Extraction of soluble molecules from the VE was carried out by incubating a suspension of 2000-4000 isolated VEs in 2 ml of 10% Ringer-Tris for 30 min in a water bath at 60°C. The resulting suspensions were cooled at room temperature and centrifuged at 3000g to discard the rest of undissolved VEs, and the supernatant was stored at -20°C until used. Extracts of fertilization envelopes (FE) were prepared from dejellied eggs after fertilization by incubating FEs in 0.1 N of NaOH for 10 min. After the rest of undisolved FEs were separated by centrifugation, the supernatant was dialized against 10% Ringer-Tris overnight at 4°C. Enzymatic treatments. The effect of oc-mannosidase and trypsin on solubilized VE was examined by incubating the fractions with 1 pg/ml of cu-mannosidase or 50 pg/ml of trypsin in 1 ml of buffered medium for 30 min at 22°C. Fertilization assay. The ability of the different extracts to inhibit fertilization was estimated by an cn vib0 fertilization assay: 100 /*l of a sperm suspension (lo7 sperm/ml) in Ringer-Tris solution was incubated with different amounts of solubilized VEs or FEs for 30 min at 20°C. Treated sperm were collected by centrifugation at 1000~ for 5 min at 4°C. The supernatant was discarded and the pellet suspended with cold 10% Ringer-Tris. The suspension was washed once more before being used. Batches of 30-40 dejellied oocytes in a mixture of egg water and 10% Ringer-Tris (1:l) were inseminated with aliquots of control and pretreated sperm suspensions, at a final concentration of lo6 cells/ml. Oocytes were considered to be fertilized when subsequent cleavages were normal. Lectins. The purified plant lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), and the chromatographically pure saccharides (a-methyl-D-mannoside, N-acetyl galactosamine, and N-acetyl glucosamine) were purchased from Sigma Chemical Co. (St. Louis, MO). Phytohemagglutinin P (PHA-P) was from DIFCO Laboratories.
3 Abbreviations used: VE, vitelline envelope; FE, fertilization envelope; Con A, concanavalin A; SBA, soybean agglutinin; WGA, wheat germ agglutinin; PHA-P, phytohemagglutinin-P; SDS, sodium dodecyl sulfate; PAGE, polpacrylamide gel electrophoresis.
Coupling of lectins to Sepharose. Lectins were coupled to Sepharose 4B by a modification of the cyanogen-bromide procedure of Cuatrecasas (1970). Briefly, 10 to 20 ml of Sepharose 4B was activated with 2-4 g of CNBr, at pH 9.0 for 10 min. Sepharose was then washed with 1000 ml of cold 0.1 M NaHCO, on a Buchner funnel, and the moist gel cake was transferred to a beaker containing 60-80 ml of 0.9% NaCl, 0.01 M NaHCO,, 50-120 mg of the lectin, and 0.1 M of the haptene saccharide. The pH was adjusted to 7.4 with HCl and the mixture gently stirred overnight at 4°C. The lectin-Sepharose complex was finally poured into a glass column and washed with 500 ml of 0.9% NaCl, 0.01 MNaHCO,, followed by 300 ml of Ringer-Tris. Through this procedure, 120 mg of Con A, 50 mg of PHA-P, and 90 mg of WGA were coupled to Sepharose 4B with an efficiency of approximately 90%. A&nity chromatography. The VE extracts were dialized overnight against Ringer-Tris containing 1 mM MgCl, and 1 mM MnCl, and applied to a 1 X 10 cm column of lectin-Sepharose 4B. The column was thoroughly washed with the same solution until the absorbance of the eluate at 280 nm was below 0.015 OD. The bound glycoproteins were eluted from the column with a 0.1 M solution of the corresponding saccharide (TVmethyl-D-mannoside for Con A, N-acetyl-D-galactosamine for PHA-P, and N-acetyl-D-glucosamine for WGA), until the absorbance of the fractions was below 0.015 OD. Treatment with borohydride. Treatment of VE extracts with borohydride were carried out according to Kornfeld and Kornfeld (1970). The extracts were incubated in 0.5 M borohydride and dissolved in 0.2 NNaOH for 72 hr at 37°C under an atmosphere of nitrogen and continuous stirring. After incubation, the samples were neutralized with HCl. Electrophoresis. SDS-PAGE of VE extracts was performed according to Laemmli (1970) with 7.5% running gels. Proteins were stained with Coomassie brilliant blue R-250 and carbohydrates with periodic acid-Schiff according to Fairbanks et ul. (1971). Electron rrcicroscovy. Aliquots of sperm suspensions were fixed in 2% glutaraldehyde in Ringer-Tris for 20 min at 4°C. Spermatozoa were pelleted by centrifugation and washed three times with phosphate buffer (pH 7.2), and postfixed in 1% osmium tetroxide. After treatment with 1% uranyl acetate for 30 min at 4”C, pellets were dehydrated in ethanol and acetone and embedded in Epon-Araldite resin. Observations were carried out with a Zeiss 109 electron microscope. RESlJLTS
Eflect of VE and FE extracts on the fertilizing capacity of B. arenarum sperma,toxoa. The effect of the extracts
418
DEVELOPMENTAL TABLE
EFFECT
OF INCUBATING OF VE
Gamete treated Oocytes
Spermatozoa
Bufo AND
BIOLOGY
1
urenarurn
GAMETES FE IN FERTILIZATION
WITH
EXTRACTS
Inhibition fertilization
Incubation solution Ringer-Tris VE extract FE extract
o-+0 6.3 f 0.9 Ok0
Ringer-Tris VE extract FE extract
Ok0 80.7 k 2.3 Ok0
VOLUME
146,
1991
a 2
5 60-
of (%)
Note. 10’ sperm/ml were incubated in each medium at 20°C for 30 min. VE or FE extracts were at a final concentration of 50 pg/ml in the media. Inseminations were carried out with lo6 sperm/ml in the media. Results are expressed as mean f SEM (n = 3).
from both VE and FE on the fertilization process was examined by incubating either oocytes or spermatozoa with them before insemination. As observed in Table 1, when spermatozoa were treated with the molecules extracted from VEs, prior to being used to inseminate oocyte strings, fertilization was inhibited in about 80%. The same treatment applied to oocytes did not alter the fertilization rate. When either gamete was treated with molecules extracted from FE, no fertility inhibition was observed. Treatments applied to sperm suspensions were carried out with different concentrations of VE and FE extracts. In this case, a concentration-response effect was observed for VE-treated spermatozoa, while no effect with any concentration was observed when spermatozoa were incubated in FE extracts (Fig. 1). When low concentrations of VE extracts were used, the effect observed depended linearly on the incubation period (Fig. 2). The molecules extracted from VEs are glycoproteins, and in different preparations they differ in the ratio of carbohydrate/protein, ranging between 7 and 14%.
0
10 Incubatlcn
20 30 Period (minutes)
FIG. 2. Time-dependence of inhibition of fertilization by solubilized VE: lo7 sperm/ml were incubated with 30 pg/ml of VE extracts in the medium at 20°C for 30 min. Fertilization assays were performed with lo6 sperm/ml in the media. Data represent the mean -t SEM (n = 3).
These different preparations differ also in their capacity to inhibit spermatozoa fertility. In Fig. 3, the positive correlation between the carbohydrate content of the preparations and fertility inhibition capacity is clear. Incubation of spermatozoa with either mannosidase or trypsin, in the same conditions used here, did not alter their fertilizing capacity. Furthermore, the inhibitory activity of VE extracts was eliminated by treatment with cu-mannosidase, an exoglycosidase that removes terminal mannose residues from hexose polymers. On the contrary, the activity of these extracts was not altered by incubation with trypsin (Table 2). Similar results were observed when VE extracts were digested with other proteases. Isolation of sperm-interacting molecules from VE by afinity chromatography. VE extracts were subjected to
affinity chromatography with columns of Con A, WGA, or PHA-P coupled to Sepharose. In all cases most of the protein applied to the column was not retained by it. In the case of Con A, the fraction
Carbohydrate
Protein
Added
(pg/ml)
FIG. 1. Inhibition of fertilization by solubilized VE and FE: lo7 sperm/ml were incubated in each medium at 20°C for 30 min. Fertilization assays were carried out with lo6 sperm/ml in the media. Data represent mean + SEM (12 = 3). (o), VE extracts; (A), FE extracts.
Content
(%I
FIG. 3. Inhibition of fertilization by solubilized VE with different carbohydrate content. Spermatozoa incubations and fertilization assays were performed as described in the legend to Fig. 2. Carbohydrate content of VE solutions was established as
carbohydrate
content
=
carbohydrates proteins
(pg/ml) (pg/ml)
x 1oo.
VALZ-GIANINET,
EFFECT
DEL PINO, AND CABADA
TABLE 2 OF ENZYMATIC TREATMENTS ON THE INHIBITION OF FERTILIZATION BY SOLUBILIZED VEs Inhibition fertilization
Treatment
of (%)
C4
100 m 5
E O3 E 8 N 02
50
=:
None Mannosidase (1 pg:/ml) Trypsin (50 pg/ml) Note lo7 sperm/ml were tracted VE) at 20°C for 30 were performed with lo6 pressed as mean ? SEM (n
incubated min, after sperm/ml = 3).
69.7 k 4.4 3.3 k 0.9 65.7 + 2.7 in the media (50 pg/ml of exdigestions. Fertilization assays in the media. Results are ex-
excluded from the column had no effect on sperm fertility. On the contrary, the glycoproteins bound to the column inhibited sperm fertility: sperm incubation with ‘7 yg/ml of the product was effective in reducing fertility to 50% (Fig. 4). The same degree of reduction in fertility with the original product is observed when spermatozoa are incubated at least with 30 yg/ml (see Fig. 1). When testing the biological effect of the bound fraction of either WGA or PHA-P-Sepharose 4B conjugates, no fertilizing-inhibitory effect was found (Fig. 4). In these cases, the inhibitory effect was present in the void fraction. In the case of Con A bound fraction, the inhibitory activity was destroyed, as in the crude preparation, by incubation with a-mannosidase, but not with trypsin. In addition, when oocytes were incubated with the bound fraction, as was observed with the crude preparation, they did not modify their fertilizability. Since Con A itself is very effective in inhibiting fertilization even at low concentrations (Cabada et al., 1978;
5 Protem
10 Added
15 (ug/ml)
FIG. 4. Inhibition of fertilization by lectin-affinity-isolated molecules. Spermatozoa incubations and fertilization assays were carried out as described in the legend to Fig. 1. Solubilized VE were submitted to affinity chromatography in immobilized Con A (0), WGA (o), and PHA-P (A). The lectin-bound fractions were eluted from the columns with the appropriate sugar haptene (see Materials and Methods). Data represent mean ? SEM (n = 3).
5 I g 03 ^, >
0 I 0 IO
:o
20
20
30
FrockIon
40
n:
FIG. 5. Effect of treatment with borohydride on the fertility-inhihitory activity of VE extracts. Untreated extracts from VEs (3 mg) (A) or after treatment with borohydride (3.2 mg) (B) were loaded onto a Sephadex-Con A and eluted with 0.1 M of n-methyl-D-mannoside (fraction 17 onwards). The fractions (1 ml) were pooled as indicated for each peak (dashed bars). Spermatozoa were incubated in 50 pg of protein/ml of each pool before being used to inseminate oocyte strings (conditions as in the legend to Fig. 2). The bars indicate the fertilization inhibition (mean f SEM; 11 = 4), referenced to controls, i.e., spermatozoa incubated in Ringer-Tris, 0.1 M a-methyl-D-mannoside (97.4 i 6.9% fertilization). When spermatozoa were incubated in untreated crude extracts of VE, fertilization was 40.7 -+ 7.6%
de1Pino and Cabada, 1987), considerable care was taken to prevent the leakage of Con A from the columns. For this reason, the Con A-Sepharose column was extensively washed before use until the washings of the column did not agglutinate spermatozoa (de1 Pino et al., 1981). It was also checked that 0.1 Ma-methyl-D-mannoside (used to elute the molecules that bind to the column) did not significantly reduce fertilization rates when present in the sperm-incubation media. Role of the glycosidic moiety. The treatment of glycoproteins with borohydride and NaOH separates the Oglycosidic residues. After alkaline borohydride treatment, samples were loaded onto a Sepharose-Con A column, and the bound and void fractions separated. Untreated extracts from VE were also run. The fractions collected were pooled as indicated in Fig. 5. Spermatozoa were incubated in aliquots of each pool before being used to inseminate oocyte strings. The results
420
DEVELOPMENTAL
BIOLOGY
summarized in Fig. 5 indicate that, as was already quoted, the bound fraction of the untreated samples (A) (containing 65% of total proteins), contained most of the fertility-impairing activity, if compared with the void fraction (43.3 + 6.9% vs 18.5 f 5.2% of fertility inhibition). In the experiments with borohydratetreated samples (B), it is observed that most of the material is in the void fraction (76% OD BO,,-absorbing of total proteins). This result indicates that the borohydrate treatment separates a considerable part of the glycosidic component from the glycoprotein that binds to concanavalin A, thus not permiting the corresponding proteic component to bind the column. However, the fertility-impairing effect of the bound fraction not only does not decrease, but increases, if the total fertility-impairing effect observed in both bound peaks is considered (70% of total effect in experiment A vs 81% in experiment B). Together, these results and those of trypsin treatment indicate that the effect of inhibiting fertilization can be ascribed to the glycosidic moiety of the glycoprotein extracted from the VE. This type of experiment does not indicate that O-linked oligosaccharides are the only ones involved in the effect observed, since those N-linked glycosidic residues resistant to fl elimination might also be present in the bound fraction. Preliminary results of experiments with labeling extracts from VEs with lz51 indicate that least some of the glycoproteins can be labeled and actually bind to spermatozoa (Valz-Gianinet; de1 Pino and Cabada, unpublished results). pattern of VE extracts. The results obElectrophoretic tained by SDS-PAGE of the molecules bound to Con A-Sepharose are shown in Fig. 6. All bands stained for both proteins and glucids. Six peaks were consistently found in several runs. Their molecular weights (in Daltons) are: (I) 101,000, (II) 97,000, (III) 90,000, (IV) 71,000, (V) 41,500, and (VI) 33,500. Electron microscopy of spermaElectron microscopy. tozoa incubated in extracts of VEs or saline solutions indicate that no observable alterations can be ascribed to the treatment. We scored about 60 treated spermatozoa, and a similar number of controls, in three different assays. All of them presented the acrosome intact. This result suggests that acrosome breakdown is not involved in the mechanism of inhibition of sperm fertilizing capacity.
DISCUSSION
The process of fertilization consists of a complex sequence of events which involves the species-specific fusion of the plasma membranes of sperm and oocyte. Ga-
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.* -220
I lY+ I
-67 -60
-36
FIG. 6. Electrophoretic pattern of VE extracts. Photography of the electrophoresis of solubilized VEs in SDS-PAGE (7.5%), after staining with Coomassie brilliant blue R-250. Molecular weight markers are indicated in kDa.
metes must be able to recognize each other as a requisite for their fusion. At a molecular level, this process seems to be mediated by molecules at the surface of both gametes that must specifically interact to render a successful contact. During its approximation to the oocyte, amphibian spermatozoa must traverse the jelly coats covering the oocytes and penetrate the VE before it can fuse with the plasma membrane of the oocyte. The vitelline envelope of amphibian oocytes and analogous structures in other animals seem to be an important site of interaction between both gametes (Florman and Wassarman, 1985; Rosati, 1985; Macek and Shur, 1988) and participate in the block to polyspermy (Grey et ah, 1974; Wolf, 1974; Wyrick et al., 1974; Grey et al., 1976; Wolf et ah, 1976; Greve and Hedrick, 19’78;Gerton and Hedrick, 1986; Cabada et al., 1978). These properties of the VE can be explained by the presence on its surface of sites for recognition and binding for homologous spermatozoa. In this work we present evidence that spermatozoa lose their fertilizing capacity when incubated in the presence of molecules extracted from the VE, before being used to inseminate oocytes. This process is concentration and time-dependent. The fertility-inhibitory property was not observed when sperm were incubated with extracts of FE, obtained from activated oocytes. This result suggests that the modifications suffered by the VE due to products released from the cortical granules during the process of oocyte activation hinder and/ or modify the sites of interaction of the VE with spermatozoa.
VALZ-GIANINET, DEL PINO, AND CABADA
Some preliminary experiments working with ‘251-labeled molecules from VE suggest that the glycoproteins bind to spermatozoa. The molecules bearing the fertility-impairing activity are glycoproteins since they bind to Sepharose-Con A columns. The results of the experiments of /3-elimination and trypsin and a-mannosidase digestions indicate that the glycosidic residues are the responsible for the effect observed. Binding to Con A does not necessarily indicate that the mannoside residues are the ones responsible for the effect observed since the glycosidic residues involved in the fertility-impairing effect could not be the same as those involved in binding to Con A, However, since digestion with cu-mannosidaseeliminates the inhibitory effect, it seems that the mannoside residues (or others stereochemically related) are indeed involved in the effect. The mechanism through which spermatozoa lose their fertility is different from that inducing a premature acrosome reaction as was observed when spermatozoa were incubated with lipidic vesicles (Cabada et al., 1984; Diaz Fontdevila et al., 1988). A possible explanation for the phenomena described is that the substances released from the VE bind to the spermatozoa occupying the binding or recognition sites on the sperm surface, which cannot interact any longer with similar molecules present in the VE of the oocyte to be fertilized, rendering an infertile spermatozoa. In this connection, and at least for this part of the process of fertilization, amphibians are similar to mammals (Shur, 1989), sea urchins (Rossignol et al., 1984), and ascidians (de Santis and Pinto, 1987) in which the sperm-receptor activity is located in the zona pellucida and vitelline coat, respectively. This work was supported by grants from CONICET (PID No. 3055500/88), Fundacihn Antorchas, and TWAS (No. BC 898-127) awarded to M.O.C. REFERENCES BARBIERI, F. D., and DEL PINO, E. J. (1975). Jelly coats and diffusible factor in anuran fertilization. Arch. Bid. 86, 311-321. BLEIL, J. M., and WASSARMAN, P. M. (1980a). Mammalian sperm-egg interactions: Identification of glycoproteins in mouse egg zona pellucida possessing receptor activity for sperm. Cell 20, 8’73-882. BLEIL, J. M., and WASSARMAN, P. M. (1980b). Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte zona pellicida. Dev. Biol. 76, 15-20. CABADA, M. O., MARIANO, M. I., and RAISMAN, J. S. (1978). Effect of trypsin inhibitors and concanavalin A on the fertilization of Bufo urenm-um coelomic oocytes. J. Exp Zool. 204, 409-416. CABADA, M. O., BLOJ, B., ORTIZ, L. P., DE, VALZ-GIANINET, J. N., and DfAZ FONTDEVILA, M. F. (1984). Effect of phosphatidylcholine on acrosome breakdown and fertilizing capacity of amphibian spermatozoa. Dw. Growth Differ. 26, 515-523. CUATRECASAS,P. (1970). Protein purification by affinity chromatogra-
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in Awphitriaws
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phy. Derivatizations of agarose and polyacrylamide and agarose beads. J. Bid. C’hem. 245, 3059-3065. DEL PINO, E. J., CABADA, M. O., and FONIO, M. C. (1981). Lectin-mediated sperm agglutination of B?lfb urprlnr~lm and Lcptotloct!/l/r.s cha~wnsis spermatozoa. Ga tnrtu Rec. 4, 97-104. DEL PINO, E. J., and CABADA, M. 0. (1987). Lectin-binding sites in the vitelline envelope of Bafo orewurum oocytes. Role in fertilization. Ga metr Res. 17, 333-342. DE SANTIS, R., JAMUNNO, G., and ROSATI, F. (1980). A study of the chorion and the follicle cells in relation to sperm-egg interaction in the Ascidian Ciorrc~ i~tf&inalis. &I,. Rio2. 74, 490-499. DE SANTIS, R., and PINTO, M. R. (1987). Isolation and partial characterization of a glycoprotein complex with sperm-receptor activity from Cio?/u intestincrlis ovary. net:. Grodh Di&r. 29, 617-625. D~AZ FONTDEVILA, M. F., BLOJ, B., and CABADA, M. 0. (1988). Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity on amphihian sprrmatozoa. Gumf~tc RPS. 21, 59-70. FAIRBANKS, G., STECK, T. L., and WALLACH, D. L. H. (1971). Elertrophoretic analysis of the major polypeptides of the human rrythrocyte membrane. Biochemistr!/ 10, 2606-2619. FLORMAN, H. M., and WASSARMANN, P. M. (1985). O-linked oligosaccharides of mouse egg ZP3 accounts for its sperm receptor activity. (‘t,// 41, 313-324. GERTON, G. L., and HEDRICK, J. L. (19X6). The vitelline envelope to fertilization envelope conversion in eggs of Xen0lnr.s lorvis. Ikt~. Riot. 116, l-7. GLABE, C. G., and VACQUIER, D. V. (1978). Egg surface glycoprotein receptor for sea urchin sperm bindin. Proc. h’trtt. Artrd. Sci. USA 75, 881-885. GREY, R. D., WOLF, D. P., and HEDRICK, J. L. (1974). Formation and structure of the fertilization envelope in X(YCO~>US /(IEI,~s.nc,l: Bid. 36.44-61. GREY, R. D., WORKING, P. K., and HEDRICK, J. L. (1976). Evidence that the fertilization envelope blocks sperm entry in eggs of Xe~col~~.s lun>is: Interaction of sperm with isolated envelopes. nrr,. Bid. 54, 52-60. GREVE, C. L., and HEDRICK, J. L. (1978). An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of Xenopas tawis. Gamete Rex 1, 13-18. GWATKIN, R. B. L. (1976). Fertilization. 1~! “The Cell Surface in Animal Embryo: Genesis and Development” (G. Poste and G. L. Nicholson, Eds.), p. l-54. Elsevier/North Holland Biomedical Press, Amsterdam. GWATKIN, R. B. L. (1978). Cell-cell interactions in mammalian fertilization. 2~ “Birth Defects: Original Articles Series” Vol. 14 f2), pp. 363-376. The National Foundation. GWATKIN, R. B. L., CARTER, H. W., and PATTERSON, H. (1976). Association of mammalian sperm with the cumulus cells and the zona pellucida, studied by SEM. ITL “Scanning Electron Microscopy.” pp. 379-384. ITT Research Institute, Chicago. HOUSSAY, B. A., GIUSTI, L., and LASCANO GONZALEZ, J. M. (1929). Implantation d’hypophyse et stimulation des fonctions sexuelles du Crapaud. C. R. S’ecrwx?Sot. Bid. 102, 864-866. KINSEY, W. H., and LENNARZ, W. J. (1981). Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits spermegg binding and fertilization. .I. Cell Biol. 91, 325-331. KORNFELD, R., and KORNFELD, S. (1970). The structure of phytohemagglutinin receptor site from human erythrocytes. J. Bid. C’hwx 245,2536-2545. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. AVature 227,680-685.
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MACEK, M. B., and SHUR, B. D. (1988). Protein-carbohydrate complementarity in mammalian gamete recognition. Gamete Res. 19,1-l?‘. RAISMAN, J. S., MARIANO, M. I., and CABADA, M. 0. (1977). Effect of trypsin inhibitors and concanavalin A on Bufo arenarum fertilization. Dee Growth Differ. 19, 119-123. ROSATI, F. (1985). Sperm-egg interactions in Ascidians. In. “Biology of Fertilization” (C. B. Metz and A. Monroy, Eds.), Vol. 2, pp. 361-388. Academic Press, New York. ROSATI, F., and DE SANTIS, R. (1980). Role of the surface carbohydrates in sperm-egg interaction in Ciou~cr. intestinulis. Nature 283, 762-764. ROSSIGNOL, D. P., EARLES, B. J., and LENNARZ, W. J. (1984). Characterization of the sperm receptor on the surface of eggs of Stroqolyocentrotzcs yurpurahs. De?: Biol. 104, 308321. SHAPIRO, B. M., and EDDY, B. M. (1980). When sperm meets egg: Bio-
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chemical mechanism of gamete interaction. ht. Reu. CytoL 66,257302. SHUR, B. D. (1989). Galactosyltransferase as a recognition molecule during fertilization and development. In “The Molecular Biology of Fertilization” (H. Schatten and G. Schatten Eds.), pp. 38-72. Academic Press, New York. WOLF, D. P. (1974). The cortical granule reaction in living eggs of the toad Xenopus lwevis. Dev. BioL 36, 62-71. WOLF, D. P. (1976). Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus 1uwi.s. Biochemistry 15, 3671-3678. WYRICK, R. E., NISHIHARA, T., and HEDRICK, J. L. (1974). Agglutination of jelly coats and cortical granules components and the block to polyspermy in the Amphibian Xenopus laevis. Proc. Natl. Acad. Sci. USA 71,2067-2071.
