Jf. Cell Set. 32, 45-S4 (1978) 45
Printed in Great Britain © Company of Biologists Limited
FERTILIZATION IN BROWN ALGAE. I. SEM
AND OTHER OBSERVATIONS ON
FUCUS SERRATUS
MAUREEN E. CALLOW, L. V. EVANS, G. P. BOLWELL AND
J. A. CALLOW
Department of Plant Sciences, The University, Leeds LS2 gjfT, England
SUMMARY
The cell wall secreted immediately following sperm entry into an egg can be visualized by
the fluorescent dye Calcofluor white. Cell wall secretion precedes nuclear fusion by 10-20 min.
SEM observations of the surface of unfertilized and fertilized eggs and sperm attachment to
eggs are described. These results are discussed in relation to fertilization in sea urchins and the
biochemical phenomena associated with egg-sperm recognition in Fucus.
INTRODUCTION
Eggs of Fucus spp. are bounded only by a plasma membrane (Pollock, 1970;
Matthews, Evans & Callow, 1976; Brawley, Wetherbee & Quatrano, 1976), but
following fertilization there is rapid secretion of a cell wall. This has been detected by
various methods such as treatment with zinc chloride (Pollock, 1970), plasmolysis and
birefringence (Quatrano & Stevens, 1976). However, these methods are relatively
insensitive since they depend on a substantial degree of structural and mechanical
integrity having developed in the new cell wall. The first formed wall component is
alginic acid which can be detected chemically within minutes of fertilization (Quatrano,
personal communication). Cellulose becomes detectable after 1 h and from 4-24 h the
composition of the wall is stable, consisting of alginic acid (60%), cellulose (20%)
and sulphated fucan (20%) (Quatrano & Stevens, 1976).
In Fucus, sperm are attracted to eggs by chemotaxis (Cook, Elvidge & Heilbron,
1947/48). The conjugated octatriene fucoserraten has been isolated from F. serratus
eggs (Miiller & Jaenicke, 1973; Jaenicke & Seferiadis, 1975), but the attraction is not
species specific (Miiller & Seferiadis, 1977). Although, there do appear to be biochemical
barriers to cross fertilization amongst Fucus spp. (Bolwell, Callow, Callow
& Evans, 1977, 1978).
The structure of F. serratus spermatozoids has been described in detail (Manton &
Clarke, 1950, 1951, 1955). The posterior flagellum is very elongated, greatly exceeding
the Flimmer-bearing anterior one in length. The anterior end of the cell is prolonged
into a flattened structure, the proboscis which possesses an interior framework of
microtubules derived from a modified flagellar root. Spermatozoids attach to the egg
surface by the anterior flagellum, leaving the posterior one free thus causing the egg
to rotate (Thuret, 1854; Thuret & Bornet, 1878; Strasburger, 1897; Levring, 1947).
4 CEL 32
46 M. E. Callow, L. V. Evans, G. P. BolweU and J. A. Callow
The present study is concerned with sperm attachment and the morphological
changes in the egg surface which occur following fertilization in F. serratus. The
timing of such changes is correlated with the timing of nuclear fusion. Since the term
fertilization in the literature is widely used to refer to sperm entry rather than nuclear
fusion per se, for uniformity fertilization will be used synonymously with sperm entry
in this paper.
MATERIALS AND METHODS
Mature plants of F. serratus were collected from the East Coast of Yorkshire, England.
Gametes were released as described by Callow, Coughlan & Evans (1978).
Calcofluor staining
To 5000 eggs, 2 cm* sperm suspension were added at a concentration of 2000 sperm per egg.
At various times after gamete mixing, sperm were inactivated by addition of 20 /*1 of 0-2 % I, in
2% KI. Seawater was pipetted off the eggs and a saturated solution of Calcofluor White ST
in seawater added (American Cyanamid Co.). After 10 min the eggs were washed with 2
changes of seawater. Staining and washing procedures were performed at o °C. Cells were
examined with a Reichert Diapan fluorescence microscope using exciter filters BG38 and
UGI and barrier filter GG13 + W2B. Photographs were taken using the Reichert photoautomatic
system on Kodak 35-mm Tri-X.
Scanning electron microscopy
Cells were fixed in 5 % glutaraldehyde in o-i M cacodylate buffer containing 0-25 M sucrose
at pH 7-0. Dehydration was carried out using an ethanol series with 30-min changes (30, 50,
7°> 85, .95, 100, ,and 100%). Cells were pipetted into perforated Beem capsules lined with
10-fim nylon mesh and critically point dried from liquid CO, in a Polaron E3000 critical point
dryer. The contents of the nylon mesh containers were shaken on to stubs covered with glue,
coated with gold and examined in a Cambridge Stereoscan 600 electron microscope.
Assessment of nuclear fusion
Cells were fixed in freshly mixed glacial acetic acid/absolute ethanol (1:3, v/v) overnight
and then transferred to 70 % ethanol for storage. Prior to staining the cells were washed in
distilled water then in a dilute solution of ferric chloride. The cells were squashed in acetocarmine
and treated as described by Evans (1966) until the nuclei were stained dark red. The
number of nuclei per cell was recorded, a minimum of 100 cells per sample being counted.
RESULTS
The difference in appearance between fertilized and unfertilized eggs after
staining with Calcofluor can be seen in Fig. 3A,B. Fertilization was detected within
minutes of adding sperm to eggs and was essentially complete after 10 min (Fig. 2).
Repeated attempts to prepare stained nuclei using Feulgen reagent (Evans, 1966)
for subsequent quantitation by microspectrophotometry all failed, presumably due to
the presence of interfering substances (Jensen, 1962) or the frequent dispersion of
DNA in egg nuclei (Ruthman, 1970). Thus, the method used to determine the timing
of nuclear fusion was indirect. Fig. 1 shows the distribution of cells with different
numbers of nuclei over a 60-min period after mixing eggs and sperm. After 10 min
Fertilization in brown algae 47
100 r
o
a?
40 h
20 h
10 20 30 40 50
Time after mixing eggs and sperm, min
Fig. i Distribution of cells with i nucleus (•), 2 nuclei (O), and more than 2 nuclei
(A) per cell, at various times after mixing egg and sperm.
100
80
60
40
20
10 20 30 40 50
Time after mixing eggs and sperm, min
60
Fig. 2. Distribution of cells stained with Calcofluor (O) and with fused egg and sperm
nuclei (•) at various times after mixing. Both sets of data were obtained from the
same batches of egg and sperm. Each point is based on a minimum count of ioo cells.
4-2
48 M. E. Callow, L. V. Evans, G. P. Bolwell and J. A. Callow
Fertilisation in brown algae 49
the increase in the percentage of cells with 2 nuclei (1 egg+1 sperm) corresponds well
with the percentage of cells stained by Calcofluor. After 10 min the percentage of
cells with 2 nuclei declines, with a concomitant increase in the percentage of cells
with one nucleus. This increase in the percentage of cells with a single nucleus has
been taken to represent those cells in which nuclear fusion has occurred. Fig. 1 also
shows that in approximately 5-10% of cells more than one sperm can enter an egg,
i.e. polyspermy may occur. The number of sperm nuclei in an egg varied from 2 to 8.
Unfertilized eggs were occasionally (1 in 400) found to contain 2 or 3 nuclei.
Fig. 2 shows that sperm entry triggered the secretion of a cell wall as evidenced by
Calcofluor staining, and this was essentially complete before nuclear fusion began.
Calcofluor-stained cells invariably gave rise to segmented zygotes if cultured.
Scanning electron microscopy
The difference in the surface topography of unfertilized and fertilized eggs is shown
in Fig. 4. The surface of the unfertilized egg is characteristically rough, due to protrusion
of the cytoplasmic vesicles lying beneath the plasma membrane (Fig. 5). On
sperm entry those vesicles which contain alginic acid presumably fuse with the
plasma membrane thus discharging their contents by exocytosis (Fig. 6). The secreted
cell wall is relatively smooth (Figs. 7-9) and its appearance does not change from
10 min to 16 h.
Variable numbers of sperm attach sufficiently firmly to the egg surface to be retained
in intact form throughout specimen preparation. Entry of a sperm into the
egg appears not to affect others which may be bound to the egg. The anterior
flagellum of each sperm attaches to the plasma membrane and following fertilization
the anterior flagellum of any attached sperm becomes encased in secreted wall
material. The unattached sperm body and posterior flagellum remain on the surface
of the secreted wall material (Figs. 8, 9). Agglutinated clumps of sperm are frequently
found attached to the surface of unfertilized and fertilized eggs (Fig. 9).
DISCUSSION
Egg-sperm recognition in Fucus as in mammalian (Nicolson, 1974) and sea-urchin
fertilization (Aketa, 1973) appears to be based on the association of surface-localized
complementary macromolecules (Bolwell et al. 1977, 1978). However, there are many
differences between the fertilization process in Fucus and, for example, sea urchins.
Fucus eggs are naked cells, without vitelline membranes or jelly coats (Lillie, 1914)
outside the plasma membrane. Although the presence of 2 layers external to the
Fig. 3. Cells of Fiicus serratus stained with Caleofluor white ST (A) with barrier
filters GG13+W2B and(B) with barrier filters GG13+ W2B +exciter filters BG38 +
UGi. In B only fertilized cells which are stained by Calcofluor fluoresce. X95.
Fig. 4. Group of cells taken 10 min after mixing eggs and sperm seen in the SEM.
Note the smooth surfaces of the fertilized cells and the rough surface of the
unfertilized egg (lower), x 700.
M. E. Callow, L. V. Evans, G. P. Bolwell andJ.A. Callow
Fig. 5. SEM photograph of the surface of an unfertilized egg showing the irregularity
due to the protrusion of cytoplasmic vesicles, x 3500.
Fertilization in brown algae 51
plasma membrane in Fucus eggs, an 'egg membrane' and an outer gelatinous coat,
have been described (Levring, 1952; Takamura, 1976) the present authors consider
these to represent remnants of oogonial mucilages (see McCully, 1968) since repeated
washing of eggs removes all traces of adhering mucilage.
In animals, sperm attachment and penetration is mediated through an acrosome
reaction (see Monroy, 1965). Although the proboscis of the Fucus sperm could have
some acrosomal function (see Manton, 1969) the initial attachment to the egg surface
is through the anterior flagellum. In a detailed cinemicrographic study of Ascophyllum,
Friedmann (1961) observed initial attachment to and subsequent penetration of the
egg membrane by the tip of the anterior flagellum. Since sperm also attached to glass
slides he postulated that a non-specific stickiness of the flagellar tip may establish the
first contact between sperm and egg surface. Pollock (1970) noted a swelling at the
tip of the anterior flagellum of Fucus and speculated on a possible acrosomal function.
Although no particular role could be ascribed to the proboscis, Friedmann noted it
was always pointed towards the egg surface. The observations reported here also show
that sperm attachment is mediated through the tip of the anterior flagellum. Since
current biochemical evidence is consistent with the presence of mannosyl and fucosyl
residues on the egg surface being recognized by appropriate carbohydrate binding
proteins on the sperm (Bolwell et al. 1978), and since attachment takes place by the
flagellar tip, it is possible that the carbohydrate-binding proteins are located in this
region of the Fucus sperm. The carbohydrate-containing residues on the egg may
also be localized in discrete regions since there are a finite number of sperm-binding
sites (Bolwell et al. 1978) and the sperm probes the egg surface with its anterior
flagellum prior to attachment (Friedmann, 1961). Localization of the recognition
sites on the egg surface is currently being investigated using labelled lectins.
Following fusion of the egg and anterior flagellar membranes it has been proposed
that the body and then the posterior flagellum are drawn into the egg (Friedmann,
1962). At this stage wall material is rapidly secreted. Following sperm entry in sea
urchins, cortical granules release material which is incorporated into the vitelline
membrane to form the fertilization membrane (Endo, 1952, K)6ia,b). The fertilization
membrane is rigid and prevents further entry of sperm into eggs (Eddy &
Shapiro, 1976). The secreted wall in Fucus also performs the same function. The
ultrastructure of nuclear fusion in Fucus has been described by Brawley et al. (1976)
and the results presented here clearly show that this takes place after cell wall formation.
Calcofluor white stains /Minked polysaccharides (Maeda & Ishida, 1967; Hughes
& McCully, 1975) hence its affinity for the wall components rapidly secreted after
fertilization, namely alginic acid (y5i-3-linked) and cellulose (/?i-4-linked).
The incidence of polyspermy recorded here appears high (5-10%). This may be
the result of a high sperm/egg ratio (2000 :1), although polyspermy in Fucus has been
observed previously (Yamanouchi, 1909). Whether the additional male nuclei degenerate
or take part in multipolar spindle formation (Yamanouchi, 1909) is not
known. Likewise the fate of nuclei in multinucleate eggs, also observed by Farmer &
Williams (1896) is unknown.
M. E. Callow, L. V. Evans, G. P. Bolwell and J. A. Callow
Fertilization in brown algae 53
We are grateful to the Science Research Council (G.P.B.) and International Paint Marine
Coatings (M. E. C.) for financial support and to Mr A. Nichells and Mr K. Wood for skilled
technical assistance.
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{Received 10 January 1978)