Fertilization in Fucus serratus is directly proportional to the number of sperm added, saturating at approximately 250 sperm per egg with an apparent Km of 120 sperm per egg. The effect of a range of lectins on fertilization has been tested. Preincubation of gametes with Con A and fucose-binding protein (FBP) inhibited fertilization. At low concentrations this was by specifically binding to eggs; at high concentrations pretreatment of either gametes inhibited fertilization probably due to cytotoxicity. Fertilization was not inhibited by simple sugar haptens, but polysaccharides containing fucosyl or mannosyl residues (yeast mannan, fucoidan, ascophyllan) inhibited fertilization by binding to sperm. Pretreatment of eggs with α-fucosidase or α-mannosidase was effective in inhibiting fertilization. All the results indirectly demonstrate that fertilization in Fucus serratus is based on an association between fucosyl- and mannosyl-containing ligands on the egg surface and specific carbohydrate-binding receptors on the sperm surface.

The initial binding of sperm to egg surfaces represents one of a chain of interactions culminating in fertilization. In green plants the structural and physiological processes of gamete fusion have been studied mainly in isogamous species of green algae, particularly Chlamydomonas (Wiese, 1974; Snell, 1976a, b;Wiese, Goodenough & Goodenough, 1977). To date, studies on oogamous fertilization in plants have been limited largely to microscopic observations on the processes of sperm penetration (e.g. Friedmann, 1961, 1962; Manton, 1969; Brawley, Wetherbee & Quatrano, 1976; Callow, Evans, Bolwell & Callow, 1978) and there is little evidence concerning the mechanisms of gamete recognition and binding. The form of oogamous fertilization presented by the brown fucoid algae promises to be particularly useful in investigations of the molecular basis of fertilization in a single species. The fucoid algae are common on rocky coastlines. Dioecious species occur and male and female plants may be readily distinguished. The plants can be transferred to the laboratory and large quantities of viable gametes may be obtained at will from stored material up to 10 days old. When subjected to appropriate washing procedures, released eggs present a naked plasmalemma, free from the envelopes or jelly coats which surround the eggs of those animal species such as sea urchins (Ishihara, Oguri & Taniguchi, 1973) and crabs (Brown, 1976) frequently used as model systems. Furthermore, studies in this laboratory have established a quick, quantitative assay for fertilization, and have shown that fertilization in the fucoids is highly species-specific (Bolwell, Callow, Callow & Evans, 1977).

As in other instances of cell recognition (Callow, 1977) it seems probable that the initial binding and recognition of gametes is mediated by an association between specific complementary macromolecules or ‘cognitive elements’ located on the gamete surfaces involving some aspect of saccharide binding. Several workers have examined the role of specific saccharide residues in gamete recognition by using plant lectins (specific carbohydrate-binding proteins, Lis & Sharon, 1973; Callow, 1976) to inhibit fertilization in mammals (Oikawa, Nicolson & Yanagimachi, 1974), sea urchins (Howe & Metz, 1972; Aketa, 1975; Schmell, Earles, Breaux & Lennarz, 1977), green algae (Wiese & Shoemaker, 1970) and protozoa (Frisch, Lerkovitz & Loyter, 1977). In the present paper results of experiments using several plant lectins, polysaccharides and carbohydrases are presented which broadly support the hypothesis that recognition between gametes in the brown seaweed Fucus serratus involves an association between specific carbohydrate-containing ligands and carbohydrate-binding receptors located on the egg and sperm surfaces respectively.

Fertile plants of F. serratus were collected from several localities on the N.E. coast of Yorkshire and from Anglesey, N. Wales and stored moist at 4 °C for up to to days. Gametes were released as required and washed in the case of eggs to remove any adhering mucilage (Callow, Coughlan & Evans, 1978).

Fertilization was measured quantitatively by staining the polysaccharide cell wall secreted immediately after fertilization using the fluorescent brightener Calcofluor white ST (Cyanamid) as previously described (Bolwell et al. 1977; Callow et al. 1978).

The effects of the following lectins on fertilization were examined: Concanavalin A (Con A, Sigma), fucose-binding protein from Tetragonolobolus purpureus (FBP, Miles), Ricin 120 from Ricinus communis (RC12O, Miles), Phytohaemagglutinin B from Phaseolus vulgaris (PHA, Calbiochem), soya bean agglutinin (SBA, a gift of Dr D. Bowles) and wheat germ agglutinin (WGA prepared from wheat germ by the method of Allen, Neuberger & Sharon, 1973). Dimeric maleyl-Con A was prepared as described by Young (1974). Two types of experiment were conducted. Lectins were either added to eggs at the same time as sperm in standard assays (5000 eggs plus enough sperm to give 50–60 % fertilization in untreated controls after 5 min at 22 °C), or, alternatively, either 5000 eggs or 4 × 107 sperm were preincubated with various lectin concentrations for 10 min at 22 °C in a total volume of 2 cm’. Pretreated eggs were allowed to settle and washed twice with Millipore-filtered seawater before adding untreated sperm. Pretreated sperm were collected by centrifugation at IO∞ g for 5 min. The supernatant was removed before resuspending the sperm in seawater and adding to untreated eggs in the standard assay. Controls were subjected to the same procedures.

The effects of simple sugars and polysaccharides on fertilization were determined in the standard assay by preincubating gametes for 10 min in the appropriate compound, or by adding the test compound at the same time as gametes were mixed. In pretreatments, eggs were washed as described for the lectins. However, in the case of sperm, it having been initially determined that the various polysaccharides had no effect in egg pretreatments, pretreated sperm were simply diluted at least 40-fold rather than centrifuging down and washing.

To test the effects of various hydrolytic enzymes on fertilization 5000 eggs or 4 × 107 sperm were pretreated separately with the enzymes in 2 cm3 seawater adjusted to the pH optima of the enzymes with HC1, in no case less than pH 5·0, for 30 min at 22 °C. The composition and pH of each enzyme treatment is given in Table 2, p. 26. α- and β-glucosidases, α- and β-galactosidases, α-mannosidase, α-L-fucosidase, β-N-Ac-glucosaminidase, β-glucuronidase, neuraminidase, hyaluronidase, pectinase and trypsin were all purchased from Sigma and protease and pronase from Calbiochem. Carbohydrases effective in inhibiting fertilization were tested for possible protease contamination (Hatton & Regoeczi, 1976) using I125-labelled insulin B chain as substrate by the method of Kenny (1977) and found to be free of protease at levels used in our treatments. Neuraminidase had insufficient protease activity (Sigma data) to cause inhibition of fertilization.

Table 2.

Effects of enzymes on fertilization

Effects of enzymes on fertilization
Effects of enzymes on fertilization

Eggs treated with enzymes were allowed to settle, and then washed extensively with seawater before adding sperm. Pretreated sperm were diluted 40-fold with seawater which reactivated sperm which had temporarily lost their motility at low pH. At this dilution, not enough extraneous enzyme could reach the eggs to cause significant inhibition over 5 min in those cases where eggs were sensitive to a given enzyme in preincubations.

In all cases where the effects of lectins, sugars, polysaccharides, and enzymes on fertilization were being assessed, observed inhibition is expressed as % inhibition compared with control assays, in which gametes were subjected to identical procedures before mixing together in a standard assay designed to give 50–60 % fertilization in 5 min at 22 °C. All values reported are the means of duplicate determinations with an average variation between duplicates of ± 3 %. Each experiment reported was repeated a number of times and typical data are presented.

The rate of fertilization was directly proportional to the relative concentration of sperm, saturating at approximately 250 sperm egg−1 (Fig. IA). Such saturation kinetics have been taken to indicate the presence of a finite number of sperm-binding sites on egg surfaces (Vaquier & Payne, 1973), although in the present case it must be noted that fertilization and not sperm-binding itself was being measured. A double reciprocal plot of the initial rate of fertilization against sperm concentration gave an apparent Km of 120 sperm egg−1 (Fig. 1B). It must be recalled, however, that kinetic parameters used for enzyme reactions are only apparent ones when applied to a multistep process like fertilization and must be interpreted with caution. Apparent Km values are useful however, in checking the integrity of gametes and the efficiency of fertilization in heterologous situations and to take into account seasonal variation in gamete viability.

Fig. 1.

The kinetics of fertilization. All assays contained 5000 eggs in 2 cm3 Millipore-filtered seawater, shaken with various amounts of sperm at 22 °C. Sperm concentrations were estimated spectrophotometrically at 645 nm and were linear between 0·025-0·25 absorbance units (106-5·5 × 106 sperm cm−4). Sperm were immobilized with 20 mm’ of 0·2 % 1, in 2 % KI, before staining the eggs with Calcofluor and estimating fertilization by fluorescence microscopy. All values are the means of duplicate determinations with an average variation between duplicates of ± 3 %. A, % fertilization after 5 min, plotted against sperm numbers per egg; ß, double reciprocal plot of (1/% fertilization 5 min−1) × 10 against (1/sperm egg−1) × 103; c, time course of fertilization at half-saturating sperm concentration.

Fig. 1.

The kinetics of fertilization. All assays contained 5000 eggs in 2 cm3 Millipore-filtered seawater, shaken with various amounts of sperm at 22 °C. Sperm concentrations were estimated spectrophotometrically at 645 nm and were linear between 0·025-0·25 absorbance units (106-5·5 × 106 sperm cm−4). Sperm were immobilized with 20 mm’ of 0·2 % 1, in 2 % KI, before staining the eggs with Calcofluor and estimating fertilization by fluorescence microscopy. All values are the means of duplicate determinations with an average variation between duplicates of ± 3 %. A, % fertilization after 5 min, plotted against sperm numbers per egg; ß, double reciprocal plot of (1/% fertilization 5 min−1) × 10 against (1/sperm egg−1) × 103; c, time course of fertilization at half-saturating sperm concentration.

At half-saturating levels of sperm, fertilization was rapid, linear up to 5 min and complete within 15 min (Fig. 1c). Subsequent experiments involving various treatments were therefore carried out under conditions where control incubations gave 50–60% fertilization after 5 min, i.e. where the rate of fertilization was linear and the sperm concentration non-saturating. It was considered essential to measure initial rates of fertilization since differences in rates as affected by lectins for example, need not necessarily be reflected in the final degree of fertilization.

Although lectins have been used in inhibition studies to identify specific carbohydrate residues involved in gamete recognition (Wiese & Shoemaker, 1970; Howe & Metz, 1972; Aketa, 1975; Oikawa et al. 1974; Løvlie & Bryhni, 1976; Schmell et al. 1977) their effects have not always been measured quantitatively or on the basis of a rate inhibition. In view of a possible variety of effects induced by lectins the kinetic approach was adopted here to permit a distinction between unilateral blocking of gamete recognition sites and non-specific, or cytotoxic inhibition of fertilization.

Wheat germ agglutinin (WGA, binding specifically to oligosaccharides containing N-acetyl-glucosamine) had no effect on fertilization at concentrations up to 10−4g cm−3 (Fig. 2 A). Phytohaemagglutinin B (PHA, specific for W-acetyl-galactosamine), however, acted on both eggs and sperm in preincubation experiments, stimulating fertilization below 10−4 g cm−3 (Fig. 2B). However, soyabean agglutinin (SBA), which has a saccharide specificity similar to that of PHA, neither stimulated nor inhibited fertilization.

Fig. 2.

The effects of lectins on fertilization. Standard assays contained 5000 eggs with identical amounts of sperm to those which gave 50—60% fertilization in untreated controls, after 5-min incubation at 22 °C. A, effects of RC120 (•) and WGA (○); lectins were added to eggs at the same time as sperm, B-D, effects of PHA (B), Con A (c) and FBP (D); pretreatments of eggs (•) and sperm (○),E,F, double reciprocal plots of Con A (E) and FBP (F) inhibition; lectins were added to 5000 eggs at the same time as varying concentrations of sperm, under standard assay conditions; ®, no addition of lectin; O, 10−4 g cm−3lectin; ◐, 10−4 g cm−3 lectin.

Fig. 2.

The effects of lectins on fertilization. Standard assays contained 5000 eggs with identical amounts of sperm to those which gave 50—60% fertilization in untreated controls, after 5-min incubation at 22 °C. A, effects of RC120 (•) and WGA (○); lectins were added to eggs at the same time as sperm, B-D, effects of PHA (B), Con A (c) and FBP (D); pretreatments of eggs (•) and sperm (○),E,F, double reciprocal plots of Con A (E) and FBP (F) inhibition; lectins were added to 5000 eggs at the same time as varying concentrations of sperm, under standard assay conditions; ®, no addition of lectin; O, 10−4 g cm−3lectin; ◐, 10−4 g cm−3 lectin.

Three lectins, RC120, Con A and FBP inhibited fertilization. However, whereas the β-D-galactose-specific RCl20 only produced marked inhibition at relatively high concentration (10−4 g cm−3, Fig. 2A), both Con A and FBP were inhibitory at much lower concentrations in both preincubation experiments (Fig. 2C, D) and when added at the same time as gametes were mixed (Fig. 2E, F). Preincubation experiments with Con A and FBP demonstrated a biphasic inhibition; below 10−8 g cm−3 both lectins acted specifically on eggs but above this concentration preincubation of both gametes inhibited fertilization (Fig. 2C, D). The actual magnitude of inhibition observed was strongly time-dependent. Short time-course experiments on eggs preincubated with 10−4 or 10−8 g cm−3 Con A showed that fertilization was strongly inhibited during the first 2 min, after which the inhibition gradually decreased (Fig. 3) at a rate which presumably reflected the rate of dissociation of bound Con A molecules from the egg surface and their replacement with sperm.

Fig. 3.

Short time-course of fertilization of eggs preincubated with Con A. O, control; ©, preincubation with 10−6 g cm−3 Con A; ◐, preincubation with 10−4 g cm−3 Con A.

Fig. 3.

Short time-course of fertilization of eggs preincubated with Con A. O, control; ©, preincubation with 10−6 g cm−3 Con A; ◐, preincubation with 10−4 g cm−3 Con A.

When treating cells with lectins it is particularly important to distinguish between effects resulting from specific binding to surface-localized carbohydrate receptors, the non-specific, gross disruptive effects on membranes caused by high concentrations of lectins and toxic effaects resulting from the uptake of the lectin by endocytosis (Grabel & Farnsworth, 1977). Fucus eggs pretreated with 10−6 and 10−4 g cm−3 Con A were washed with the hapten sugar, α-methyl mannoside (10 mM) before adding sperm. In the former case, the inhibitory effect of the lectin was completely abolished by this sugar, indicating that at this lectin concentration, inhibition of fertilization was due to a reversible binding of this lectin to mannose- or glucose-containing ligands on the egg surface. However, inhibition when eggs were pretreated with 101 g cm−3 Con A could be only partially reversed, indicating that the higher lectin concentrations were causing some form of cytotoxic effect. At higher concentrations of Con A eggs undergo morphological changes, particularly a blebbing of the plasma membrane, and sperm lose motility. At 10−β g cm−3 Con A there was no effect on sperm motility. The Ricinus lectin RC120 induced similar morphological changes at 10−4 g cm−3. Concentrations which did not induce any apparent morphological changes (below 106 g cm−3) were not inhibitory (Fig. 2A). Inhibition of fertilization by 10−6 g cm−3 FBP was fully reversed by 10 mM α-L-fucose.

Double reciprocal plots (Fig. 2E, F) of the rate of fertilization over a range of sperm concentrations showed that when io−6 g cm−3 Con A or FBP were added to fertilization assays at the same time as gametes were mixed, the inhibition obtained was competitive in nature (i.e. the apparent Kin was increased), whilst at 10−4 g cm−3 Con A, a ‘mixed’ type of inhibition was obtained. At the lower concentrations of lectin, fertilization eventually reached control levels (after 20—30 min), confirming that the inhibitory effects of low concentrations of these lectins result from limitations in the rate of fertilization.

The effects of Con A were examined in more detail. Con A exists in a temperature- and pH-dependent dimer-tetramer equilibrium. Temperatures below 15 °C favour reversible dissociation of the tetramer into a dimeric, divalent form (Huet, Lonchampt, Huet & Bernadac, 1974). The importance of valency in the biological effects of Con A may therefore be assessed by varying the temperature. Alternatively, temperature- and pH-stable dimeric, carboxylated derivatives of Con A, viz. succinyl-acetyl- and maleyl-Con A may be prepared. These modifications do not affect the carbohydrate-binding specificity or affinity of the lectin (Gunther et al. 1973; Young, 1974) but the dimeric derivatives no longer have the ability to modulate cell surface receptor distribution (Reeke et al. 1975).

At 4 °C preincubation of eggs with 10−4 g cm−3 Con A had no subsequent effect on their capacity for fertilization. Dimeric, maleyl-Con A, prepared as described by Young (1974) had no effect on fertilization at a range of concentrations, although it had bound to the egg surfaces, as demonstrated by a competition experiment with native Con A. Native Con A, added to eggs at 10−6 g cm−3 at the same time as sperm and incubated at 22 °C, inhibited fertilization by 28%. However, if the eggs were given a 10-min preincubation with maleyl-Con A, then incubated with native Con A, fertilization was no longer inhibited, indicating that the maleyl-Con A had bound to the eggs in a manner which subsequently restricted access of the native Con A molecule. Preincubation of eggs with maleyl-Con A also prevented inhibition by 10−6g cm−3 FBP, indicating that the receptors for the 2 different lectins may be in close proximity to each other.

Further, indirect evidence relating to the saccharide specificity of Fucus fertilization was obtained by examining the effects of putative simple sugar and polysaccharide haptens, and various carbohydrases.

Fertilization was not inhibited by simple sugar haptens (Table 1). A role for glucosyl residues in recognition is probably eliminated, since glucans containing β(1→3), α(1→4) and α(1→6) linkages had no effect. However, polysaccharides containing predominantly α(1→2) linked fucosyl residues (fucoidan (Percival & McDowell, 1967), ascophyllan (Percival, 1968, 1971)) and variously linked mannosyl residues (yeast mannan (Sentandreu & Northcote, 1968)) were potent inhibitors of fertilization when added at the same time as gametes were mixed. They had no effect when preincubated with eggs alone, but did inhibit when sperm were pretreated.

Table 1.

Effects of sugars and polysaccharides on fertilization

Effects of sugars and polysaccharides on fertilization
Effects of sugars and polysaccharides on fertilization

Although these results are consistent with the presence of fucose- and mannosebinding receptors on the sperm surface recognizing complementary carbohydrate ligands on egg surfaces, additional sugar specificity is not precluded since these polysaccharides are heterogeneous. The similar inhibitory effects obtained with a xylan (Cambrian Chemicals) (Table 1) supports this possibility, since xylosyl residues are also found in fucoidan and ascophyllan (Percival, 1971).

The neccessity for fucose- and mannose-containing ligands in fertilization has been further demonstrated by the effects of specific glycan hydrolase pretreatments of gametes (Table 2). When sufficiently high amounts of various glycan hydrolases were added to completely hydrolyse possible substrates presented by the gametes, several inhibited fertilization in preincubations. However, when 0·01 1.u. of enzyme were added, only α-D-mannosidase, α-L-fucosidase and neuraminidase were effective, and then only when eggs were pretreated. The significance of neuraminidase action is not clear.

Several workers (Howe & Metz, 1972; Aketa, 1975; Schmell et al. 1977) have reported that Con A affects fertilization in sea urchins. In Arbacia punctulata, Con A at 3 × 10−5 to 1 × 10−4 g cm−3 inhibited fertilization by 50-100% through binding to eggs (Howe & Metz, 1972; Schmell et al. 1977). Dimeric Con A derivatives prepared by papain digestion (Howe & Metz, 1972) or succinylation (Schmell et al. 1977) still bound to egg surfaces but did not inhibit fertilization. Although Arbacia sperm bound Con A, this had no apparent effect on fertilization (Howe & Metz, 1972). In contrast, it has also been reported that both native and succinyl Con A inhibit fertilization in the sea urchin Anthocidaris crassispina by binding specifically to sperm, and that in another species, Hemicentrotus pulcherrimus neither sperm nor eggs were affected by Con A (Aketa, 1975).

The inhibitory effects of Con A on fertilization in F. serratus reported here are very similar to those demonstrated for Arbacia but differ in magnitude. In Arbacia fertilization was totally inhibited by pre-incubating eggs with 10−4 g cm−3 Con A, but was unaffected by 10−4 g cm−3. In Fucus, Con A is effective at very much lower concentrations, down to 10−8 g cm−3, but 100% inhibition of fertilization was not observed in the standard 5-min assays. However, in Fucus the short time-course experiment demonstrated that the degree of inhibition was strongly time-dependent, 100% inhibition being obtained up to 2 min after adding sperm, the degree of inhibition then gradually decreasing at a rate which presumably reflects the rate of dissociation of bound Con A molecules from the egg surfaces and their replacement by sperm.

The majority of the biological effects of plant lectins on cells are generally understood to result from the binding of the lectins to surface oligosaccharides. The specific, competitive effects of Con A and FBP reported here are consistent with the hypothesis that gamete recognition in F. serratus is based upon some aspect of saccharide binding and that α-mannose-like and α-L-fucose-like residues on the egg surface are important in this process. The further indirect evidence resulting from experiments with polysaccharides and glycan hydrolases broadly supports this conclusion. A number of possible mechanisms may be advanced to explain the inhibitory effects of Con A and FBP. The simplest explanation would be that these lectins bind to specific saccharide residues forming part of the sperm receptors, thus effectively ‘masking’ these receptors from the sperm, preventing binding. An alternative, simple explanation would be that the lectins bind to surface molecules which are not part of the sperm receptors on the egg surface, the bound lectin sterically inhibiting the contact of the complementary egg-binding component on the sperm surface. This would be a non-specific effect. Its existence is perhaps supported by the non-inhibitory effects of the less bulky dimeric Con A or dimeric maleyl-Con A.

A less likely possibility is that the inhibitory lectins enter the cell by endocytosis (Grabel & Farnsworth, 1977) where they interfere with post-fertilization wall release. Hence, fertilized eggs would not be detected by the assay technique adopted. This alternative explanation is not considered to be likely, however, since at low concentrations, Con A and FBP inhibition were readily reversed by the simple sugar haptens, and furthermore, the glycan hydrolase and polysaccharide treatments are more consistent with surface-localized events.

A more complex explanation of the lectin effects, open to experimentation, stems from a comparison with the effects of lectins on the mobility of surface receptors in certain animal cell systems. Con A may exhibit 2 types of activity with respect to the mobility of receptors within the lymphocyte plasma membrane (Reeke et al. 1975). It may inhibit receptor mobility, presumably through cross-linking adjacent receptors, or it may promote the formation of caps, i.e. receptors previously distributed in a homogeneous manner are caused to aggregate in distinct areas of the cell surface. Dimeric Con A derivatives, with their reduced valency, do not possess either of these activities. In lymphocytes, anti-immunoglobulin-induced cap formation is inhibited by native Con A at 37 but not at 4 °C. However, this inhibitory effect at 37 °C is suppressed if cells are incubated with colchicine, suggesting that inhibition of receptor mobility by Con A is correlated with some change in the properties of the colchicine-binding microtubule-containing cytoskeleton.

It might be tentatively suggested therefore, that sperm-binding in Fucus requires a local confluence of many sperm receptors in the membrane. A similar suggestion has recently been made for sea-urchin fertilization (Schmell et al. 1977). Native Con A, with its greater ability to cross-link receptors compared with dimeric forms, may prevent this migration and thus inhibit fertilization. Experiments are currently being performed to test this, and other hypotheses.

The authors wish to thank the Science Research Council and International Paints Marine Coatings for financial support to G.P.B. and M.E.C. respectively, Dr J. M. Dow for valuable discussions, Dr E. Percival for the gift of ascophyllan, Dr D. Bowles for the gift of SBA and Dr A. J. Kenny for advice and facilities for protease estimations.

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