J. Cell Set. 36, 19-30 (1979)
Printed in Great Britain © Company of Biologists Limited
FERTILIZATION IN
19
BROWN ALGAE.
II. EVIDENCE FOR LECTIN-SENSITIVE
COMPLEMENTARY RECEPTORS INVOLVED
IN GAMETE RECOGNITION IN
FUCUSSERRATUS
G. P. BOLWELL, J. A. CALLOW, MAUREEN E. CALLOW AND
L. V. EVANS
Department of Plant Sciences, University of Leeds, Leeds LS2 <)JT, U.K.
SUMMARY
Fertilization in Fttcus 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
a-fucosidase or a-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.
INTRODUCTION
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
20
G. P. Bolwell, J. A. Callow, M. E. Callow and L. V. Evans
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.
MATERIALS AND METHODS
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 10 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 comrnunis (RCi2O, 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 x io7 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 1000 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.
Gamete recognition in brown algae
21
To test the effects of various hydrolytic enzymes on fertilization 5000 eggs or 4 x 10' 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 50, for 30 min at 22 °C. The composition and
pH of each enzyme treatment is given in Table 2, p. 26. a- and /?-glucosidases, a- and /?galactosidases, a-mannosidase, a-L-fucosidase, /J-A^Ac-glucosaminidase, /?-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 Im-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.
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.
RESULTS
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. IB). 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.
At half-saturating levels of sperm, fertilization was rapid, linear up to 5 min and
complete within 15 min (Fig. ic). 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; Lovlie & 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
22
G. P. Bolwell, J. A. Callow, M. E. Callow and L. V. Evans
approach was adopted here to permit a distinction between unilateral blocking of
gamete recognition sites and non-specific, or cytotoxic inhibition of fertilization.
100
=
100
r
50
f
1 2
3
4
Sperm egg" 1 , X 10~ 2
5
l_
i
-10
-5
I
0
+5
'/sperm e g g " ' , X 10 3
50
J
+10
10
20
Time, min
Fig. i. The kinetics of fertilization. All assays contained 5000 eggs in 2 cm* Milliporefiltered seawater, shaken with various amounts of sperm at 22 °C. Sperm concentrations were estimated specrrophotometrically at 645 ran and were linear between
0-025-0-25 absorbance units (io8-5-5 x 10* sperm cm"*). Sperm were immobilized
with 20 mm* of 0-2% I, 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; fl, double reciprocal
plot of (1/% fertilization 5 min"1) x 10 against (i/sperm egg"1) x io3; c, time
course of fertilization at half-saturating sperm concentration.
Wheat germ agglutinin (WGA, binding specifically to oligosaccharides containing
iV-acetyl-glucosamine) had no effect on fertilization at concentrations up to io~ 4 g
cm"3 (Fig. 2 A). Phytohaemagglutinin B (PHA, specific for N-acetyl-galactosamine),
however, acted on both eggs and sperm in preincubation experiments, stimulating
fertilization below io" 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.
Three lectins, RC la) , Con A and FBP inhibited fertilization. However, whereas the
/?-D-galactose-specific RC120 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 io" 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
io" 4 or io" 6 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.
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
Gamete recognition in brown algae
25
|
12-5 -
1
1
2
log ( (lectin], g e m " 3 X10")
2
3
log | [ P H A ] , g c m * 3 X10 8 )
100r
0
1
2
3
4
log ([Con A ] , g e m ' 3 X 10 8 )
-10
0
1 /sperm e g g " ' , X 10 3
0
1
2
3
log ( [ F B P l . g c r r T ^ X 10 s )
4
+ 10
1/sperm e g g " ' , X 103
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 RCJJ 0 ( • ) and WGA (O);
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 (O)-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"' g cm-* lectin; C, io"4 g cm"3 lectin.
of lectins and toxic effects resulting from the uptake of the lectin by endocytosis
(Grabel & Farnsworth, 1977). Fucus eggs pretreated with io" 6 and io" 4 g cm"8
Con A were washed with the hapten sugar, a-methyl mannoside (10 HIM), 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
24
G. P. Bolwell, J. A. Callow, M. E. Callow and L. V. Evans
fertilization was due to a reversible binding of this lectin to mannose- or glucosecontaining ligands on the egg surface. However, inhibition when eggs were pretreated
with io" 4 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 io~6 g cm"3 Con A there was no effect
on sperm motility. The Ricinus lectin RC120 induced similar morphological changes
at io" 4 g cm"3. Concentrations which did not induce any apparent morphological
changes (below io~6 g cm"3) were not inhibitory (Fig. 2A). Inhibition of fertilization
by io" 6 g cm"3 FBP was fully reversed by io min a-L-fucose.
Fig. 3. Short time-course of fertilization of eggs preincubated with Con A. O, control;
C, preincubation with io~* g cm"3 Con A; # , preincubation with io" 1 g cm"3 Con A.
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 Km was increased), whilst at io" 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 temperatureand 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 carbohydratebinding 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).
Gamete recognition in brown algae
25
3
At 4 °C preincubation of eggs with io~* g cm" 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 io" 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 io" 6 g
cm"3 FBP, indicating that the receptors for the 2 different lectins may be in close
proximity to each other.
Table 1. Effects of sugars and polysaccharides on fertilization
Inhibition of fertilization, %
t
Treatment
Pretreated
eggs
Pretreated
sperm
Simultaneous
addition
a-D-glucose
^
a-D-methyl mannoside
a-D-galactose
a-D-fucose
a-L-fucose
0
0
0
iV-acetyl-D-glucosamine
JV-acetyl-D-galactosamine
D-xylose
D-arabinose
a-L-rhamnose
D-cellobiose
/
0
28
Yeast mannan
33
0
S8
Ascophyllan
53
0
29
Fucoidan
45
0
40
30
Xylan
—
—
0
Galactan
0
0
0
Laminaran
0
0
0
Dextran
0
0
0
Amylose
3
Simple sugars (o-oi M) or polysaccharides (io~* g cm- ) were preincubated with gametes for
10 min or added at the same time as gametes were mixed. Having initially determined that the
various polysaccharides had no effect in egg pretreatments, pretreated sperm were simply
diluted at least 40-fold rather than centrifuging them down and washing. Assays contained
5000 eggs and enough sperm to give 50-60 % fertilization in controls. All values are the means
of duplicates. Variation between duplicates was ± 3 %.
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
26
G. P. Bolwell, J. A. Callow, M. E. Callow and L. V. Evans
glucosyl residues in recognition is probably eliminated, since glucans containing
/?(i->-3), a(i->4) and <z(i->6) linkages had no effect. However, polysaccharides
containing predominantly a(i -»• 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 2. Effects of enzymes on fertilization
Treatment
Inhibition of
fertilization
1
Enzyme
pH
a-glucosidase
6-8
/?-glucosidase
a-galactosidasc
/9-galactosidase
a-manno8idase
a-L-fucosidase
/?-iV-acetylglucosaminidase
/9-glucuronidase
Neuraminidase
5°
5-°
7-2
5°
6-5
7-0
5-°
no. of I.U. pretreated pretreated
added
eggs
sperm
o-i
0
o-oi
0
0
2-0
0
0
60
10
o-i
0
o-oi
0
8
2-0
0
0
o-i
9i
0
o-oi
o-oi
64
63
0
o-i
i-o
o-i
0
0
0
0
0
61
72
o-oi
24
3-0 (NFU) 0
0
0
Hyaluronidase
5-°
o-i
0
0
Pectinase
5-o
7-6
0
Trypsin (100 fig/cm3)
45
Protease (Calbiochem, 100 /Jg/cm*) 7-6
83
79
100
100
Pronase (Calbiochem, 100 /tg/cm3) 7-6
5000 eggs or 4 x io7 sperm were pretreated with enzymes (all Sigma unless stated otherwise)
in 2 cm' seawater adjusted with HC1 to the pH optima given above at 22 °C for 30 min. Eggs
were allowed to settle and washed extensively with seawater before adding sperm. Sperm were
diluted 40-fold with seawater which reactivated sperm which had temporarily lost their motility at low pH. This dilution did not add sufficient extraneous enzyme to eggs to cause significant inhibition over 5 min in those cases where eggs were sensitive to a given enzyme in
preincubations. Controls were subjected to identical procedures and assays for fertilization
were carried out as described in Fig. 2.
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
Gamete recognition in brown algae
27
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 o-oi i.u. of enzyme were
added, only a-D-mannosidase, a-L-fucosidase and neuraminidase were effective, and
then only when eggs were pretreated. The significance of neuraminidase action is
not clear.
DISCUSSION
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 x io~8 to 1 x 10^ 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 io~* g cm-3 Con A, but was
unaffected by io" 6 g cm"3. In Fucus, Con A is effective at very much lower concentrations, down to io" 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 a-mannose-like and a-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
28
G. P. Bolwell, J. A. Callow, M. E. Callow and L. V. Evans
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 colchicinebinding 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.
REFERENCES
AKETA, K. (1975). Physiological studies on the sperm surface component responsible for spermegg bonding in sea urchin fertilization. II. Effect of concanavalin A on fertilization capacity
of sperm. Expl Cell Res. 90, 50-62.
ALLEN, A. K., NEUBERGEK, A. & SHARON, N. (1973). The purification, composition and specificity of wheat-germ agglutinin. Biochem. J. 131, 155-162.
BOLWELL, G. P., CALLOW, J. A., CALLOW, M. E. & EVANS, L. V. (1977). Cross-fertilization in
fucoid seaweeds. Nature, Lond. 268, 626—627.
BRAWLEY, S. H., WETHERBEE, R. & QUATRANO, R. S. (1976). Fine-structural studies of the
gamete and embryo of Fucus vesiculosus L. (Phaeophyta) I. Fertilization and pronuclear
fusion. J. Cell Set. 20, 233-254.
BROWN A. A. (1976). SEM and other observations of sperm fertilization reactions in
Limulus polyphemus L. (Merostomata: Xiphosura). J. Cell Set. 22, 547-562.
Gamete recognition in brown algae
29
J. A. (1976). Plant lectins. In Commentaries in Plant Science (ed. H. Smith), pp. 221233. Oxford: Pergamon.
CALLOW, J. A. (1977). Recognition, resistance and the role of plant lectins in host-parasite
interactions. In Advances in Botanical Research (ed. R. D. Preston & H. W. Woolhouse),
pp. 1-49. London, New York, San Francisco: Academic Press.
CALLOW, M. E., COUCHLAN, S. J. & EVANS, L. V. (1978). The role of the Golgi in polysaccharide sulphation in Fucus zygotes. J. Cell Sci. 32, 337-356.
CALLOW, M. E., EVANS, L. V., BOLWELL, G. P. & CALLOW, J. A. (1978). Fertilization in brown
algae. I. SEM and other observations on Fucus serratus. J. Cell Sci. 32, 45-54.
FRIEDMANN, I. (1961). Cinemicrography of spermatozoids and fertilization in Fucales. Bull.
Res. Coun. Israel 10 D, 73-83.
FRIEDMANN, I. (1962). Cell membrane fusion and the fertilization mechanism in plants and
animals. Science, N.Y. 136, 711-712.
FRISCH, A., LERKOVITZ, H. & LOYTER, A. (1977). Inhibition of conjugation in Tetrahymena
pyriformis by concanavalin A. Expl Cell Res. 106, 293-301.
GRABEL, L. B. & FARNSWORTH, P. A. (1977). The endocytosis of concanavalin A by Dictyostelium discoideum cells. Expl Cell Res. 105, 285-289.
GUNTHER, G. R., WANG, J. L., YAHARA, I., CUNNINGHAM, B. A. & EDELMAN, G. M. (1973).
Concanavalin A derivatives with altered biological activities. Proc. natn. Acad. Sci. U.S.A.
CALLOW,
70,
1012-1016.
M. W. C. & REGOECZI, E. (1976). The proteolytic nature of commercial samples of
galactose oxidase, purification of the enzyme by a simple affinity method. Biochim. biophys.
Ada 438, 339-346.
HOWE, C. W. S. & METZ, C. B. (1972). Multivalent and univalent concanavalin A as probes
for studying sperm-egg interactions. Biol. Bull. mar. biol. Lab., Woods Hole 143, 465.
HUET, Ch., LONCHAMPT, M., HUET, M. & BERNADAC, A. (1974). Temperature effects on the
concanavalin A molecule and on concanavalin A binding. Biochim. biophys. Acta 365, 28-39.
ISHIHARA, K., OCURI, K. & TANIGUCHI, H. (1973). Isolation and characterization of fucose
sulphate from jelly coat glycoproteins of sea urchin eggs. Biochim. biophys. Acta 320,
628-634.
KENNY, A. J. (1977). In Proteinases in Mammalian Cells and Tissues (ed. A. J. Barrett), pp. 436439. Amsterdam, New York, Oxford: North-Holland Publishing.
Lis, H. & SHARON, N. (1973). The biochemistry of plant lectins (phytohemagglutinins).
A. Rev. Biochem. 42, 541-574.
LevLiE, A. & BRYHNI, E. (1976). Signal for cell fusion. Nature, Lond. 263, 779-781.
MANTON, I. (1969). Plant spermatozoids. In Comparative Spermatology, Proc. 1st int. Symp.
(ed. B. Baccetti), pp. 143-158. Rome: Accademia Nazionale dei Lincei.
OIKAWA, T., NICOLSON, G. L. & YANAGIMACHI, R. (1974). Inhibition of hamster fertilization
by phytoagglutinins. Expl Cell Res. 83, 239-246.
PERCIVAL, E. (1968). Glucuronoxylofucan, a cell-wall component of Ascophyllum nodosum. I.
Carbohyd. Res. 7, 272-283.
PBRCIVAL, E. (1971). Glucuronoxylofucan, a cell-wall component of Ascophyllum nodosum.
II. Methylation. Carbohyd. Res. 17, 121-126.
PERCIVAL, E. & MCDOWELL, R. H. (1967). The Chemistry and Enzymology of Marine Algal
Polysaccharides. New York, London: Academic Press.
HATTON,
REEKE, G. N., BECKER, J. W., CUNNINGHAM, B. A., WANG, J. L., YAHARA, I. & EDELMAN, G. M.
(i975)- Structure and function of concanavalin A. In Concanavalin A (ed. T. K. Chowdhury
& A. K. Weiss), pp. 13-33. New York: Plenum Press.
SCHMELL, E., EARLES, B. J., BREAUX, C. & LENNARZ, W. J. (1977). Identification of a sperm
receptor on the surface of the eggs of the sea urchin Arbacia punctulata. J. Cell Biol. 72,
35-46.
SENTANDREU, R. & NORTHCOTE, D. H. (1968). The structure of a glycopeptide isolated from
the yeast cell wall. Biochem. J. 109, 419-432.
SNELL, W. J. (1976a). Mating in Chlamydomonas: a system for the study of specific cell
adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components
involved in adhesion. J. Cell Biol. 68, 48-69.
3
CEL 36
30
G. P. Bolwell, J. A. Callow, M. E. Callow and L. V. Evans
W. J. (19766). Mating in. Chlamydomonas: a system for the study of specific cell adhesion. II. A radioactive flagella-binding assay for quantitation of adhesion. J. Cell Biol. 68,
7O-79.
VAQUIER, V. D. & PAYNE, J. E. (1973). Methods for quantitating sea urchin sperm-egg binding.
Expl Cell Res. 8a, 227-235.
WreSE, L. (1974). Nature of sex-specific glycoprotein agglutinins in Chlamydomonas. Ann. N. Y.
Acad. Sci. 234, 383-395.
WIESE, L. & SHOEMAKER, D. W. (1970). On sexual agglutination and mating-type substances
(gamones) in isogamous heterothallic chlamydomonads. II. The effect of Concanavalin A
upon the mating-type reaction. Biol. Bull. mar. biol. Lab., Woods Hole 138, 88-95.
WIESE, R. L., GOODENOUGH, D. & GOODENOUGH, U. (1977). Membrane differentiation at sites
specialised for cell fusion. J. Cell Biol. 72, 144-160.
YOUNG, N. M. (1974). The effects of maleylation on the properties of concanavalin A. Biocltim.
biophys. Acta 336, 46-52.
SNELL,
{Received 17 August 1978)