J. Cell Set. 9, 621-635 (1971)
Printed in Great Britain
621
THE ULTRASTRUCTURE OF FERTILIZATION
AND ZYGOTE FORMATION IN THE GREEN
ALGA ULVA MUTABJLIS F0YN
T. BRATEN
Electron Microscopical Unit for Biological Sciences,
University of Oslo, Blindern, Oslo 3, Norway
SUMMARY
Fertilization and zygote formation in the multicellular green alga Ulva mutabilis Foyn were
studied with the aid of transmission and scanning electron microscopes. The growth mutant
'slender' was used in this investigation. The initial contact between mating gametes is made
by theflagella,whereupon cytoplasmic contact is established within a few seconds. Fusion of
the cytoplasm of the 2 cells is completed in 5 min and the nuclei can be seen to fuse 30 min
after the onset of copulation. The settling of the young zygotes initiates a process during which
the 4flagellaare absorbed into the cell. Cell wall formation also starts when the zygotes settle.
Observations indicate disintegration of one of the 2 chloroplasts in the young zygote.
INTRODUCTION
Few studies have been made of the ultrastructure of the fertilization process in
algae. The first study of this kind was done by Manton & Friedmann (i960) on the
anisogamous green alga Prasiola stipitata. In spite of the limited techniques available
at that time, the work of Manton & Friedmann furnished a wealth of information
about this important phase of the algal life-cycle. Subsequent publications have dealt
with the fine structure of the fertilization of Chlamydomonas moewusii (Brown, Johnson
& Bold, 1968) and C. reinhardii (Friedmann, Colwin & Colwin, 1968). Crawley (1966)
describes the ultrastructure of the zygote of Acetabularia, but due to technical difficulties he was not able to observe the early stages of copulating gametes. No ultrastructural studies have been published on the fertilization process in Ulva, but
Levring (1955) gives a short account of the process in Ulva lactuca as seen in the light
microscope.
The details of the fertilization process in algae, as observed with the aid of the electron
microscope, show great variations even in closely related species. Thus the process in
Chlamydomonas reinhardii as described by Friedmann et al. (1968) differs considerably
from that of C. moewusii (Brown et al. 1968). In C. reinhardii the first cytoplasmic
contact is made via a fertilization tubule possessed by the plus gamete. This species thus
turns out to be anisogamous. In C. moewusii it was found that successful cytoplasmic
contact depended on a precisely timed formation of the so-called plasma papilla in
both sexes. The fate of the flagella after fertilization also differs in algae. In Chlamydomonas theflagellaare abscissed from the zygote (Brown et al. 1968), while in Prasiola
the male flagella are taken into the zygote (Manton & Friedmann, i960).
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T. Brdten
In the present investigation study of the behaviour of the flagella during the fertilization process with the aid of the scanning electron microscope was found to be most
rewarding.
MATERIAL AND METHODS
Observations reported in this paper were made on the mutant strain ' slender' of the green
alga Ulva mutabilh Foyn. No morphological differences can be found between the gametes of
this mutant and those of the wild type.
Algae were grown in a light-dark cycle of 17 h light and 7 h dark at 19 °C in Nordby's
modification of Provasoli's medium (Levlie, 1969). Almost synchronous release of gametes was
obtained by changing to fresh medium in a culture of mature gametophytes. Large numbers of
gametes could then be harvested on the side of the culture vessel exposed to the light. Copulation was achieved by mixing approximately equal numbers of plus and minus gametes, which
were then fixed at intervals ranging from a few seconds to several days after copulation. The
initial stages were fixed while still motile and kept in suspension throughout the preparation
procedure. Zygotes older than 10 min to be studied in the transmission electron microscope
were allowed to settle on agar before being fixed. Zygotes to be studied in the scanning electron
microscope were made to settle on platinum disks.
The following stock solution of fixative was used: 4% glutaraldehyde, 4% formaldehyde
(prepared from paraformaldehyde as described by Robertson, Bodenheimer & Stage, 1963) and
4% polyvinylpyrrolidone (PVP, Taab Laboratories, Reading, England; approx. mol. wt.
40000) in 0-2 M cacodylate buffer. This stock solution was diluted with an equal volume of
seawater before use. The total osmolarity of the fixative was approximately 1300 m-osmol,
which is slightly hypertonic to seawater. For transmission electron microscopy the material was
fixed for 3—5 h and then rinsed for 15 min in a solution composed of equal volumes of seawater
and 0-2 M cacodylate buffer containing 4% PVP. Postfixation was carried out overnight in
2 % OsO4 made up in 0-2 M cacodylate buffer/seawater 1:1. All solutions had a pH of 7-5 and
the fixation and rinsing were carried out at room temperature. This was also the case for the
subsequent dehydration in graded ethanols followed by propylene oxide before the final
embedding in Araldite (Fluka). Silvery sections were cut with a diamond or glass knife and
double-stained in uranyl acetate and lead citrate, using the method described by Venable &
Coggeshall (1965).
For scanning electron microscopy the same fixation procedures were performed except that
the initial fixation was extended to 24 h. After postfixation in OsO4 the specimens were rinsed
several times in 30% acetone and transferred to a desiccator, where they were placed over
100% acetone in a slight vacuum. The desiccator also contained CaCl, as a water absorbent
(Sitte, 1962). The specimens were left overnight in the desiccator, then air-dried and coated
with a thin film (approx. 30 nm) of gold/palladium.
RESULTS
Ulva mutabilis is isogamous and even at the ultrastructural level no morphological
differences can be found between the 2 sexes. The internal morphology of an Ulva
gamete as it appears in a median longitudinal section is shown in Fig. 2.
The mixing of plus and minus gametes results in the formation of clusters held
together by the tips of their flagella (Fig. 3). The clusters break up almost immediately
into mating pairs, still held together by the flagella (Fig. 4). Direct cytoplasmic contact
is, however, established between the cell bodies within a few seconds of the onset of
copulation. The flagella then separate from each other and the mating pair swims
away from the light source. The initial cytoplasmic contact between the 2 gametes
takes place at the anterior end of the gamete slightly posterior to the flagellar base
Fertilization and zygote formation in a green alga
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(Fig. 5). No specific organelle can be found to be associated with the first cytoplasmic
contact. The plasma membranes of the copulating gametes simply fuse at the point
of contact. The fusion of the 2 gametes is a rapid process and stages like the one shown
in Fig. 6 can be found only 30 s after the initial mixing of the 2 sexes. Increasingly
larger areas of the 2 gametes come into contact as the two fold together in a jack-knife
manner; 3 min after the onset of copulation the cytoplasm of the 2 gametes has fused
completely (Fig. 7). During the process of fusion the zygote remains motile with its
4 flagella cruciately arranged at the anterior end. Approximately 5 min after copulation, firmly settled zygotes can be found. The young zygote attaches itself to the substratum at the anterior end, where secretion of electron-dense particles can be seen
(Fig. 8). The settling down of the young zygote initiates a process in which the
Fig. 1. Schematic drawing of a median longitudinal section of a young zygote
illustrating the uptake of the flagellar core through the cell surface.
4flagellaare absorbed into the cell body. With the basal bodies of theflagellapointing
posteriorly, the flagella are taken up through the cell surface. The process starts at
the anterior end of the gamete and continues posteriorly as illustrated in the schematic drawing of Fig. 1. Only the core of the flagellum with its arrangement of microtubules is taken into the cell. The process of the uptake of theflagellain young zygotes
can easily be studied in the scanning electron microscope and a series of micrographs
of the process is shown in Figs. 9-12. It is clear from these photographs that because
of the length of the flagellum only the proximal part can be taken up directly through
the cell surface. The apical end of the flagellum is probably drawn into the cell by
some kind of contraction mechanism leaving the flagellar membrane behind (Fig. 12).
The whole process of flagellar uptake is accomplished about 15 min after copulation.
The zygote has at this stage become spherical and the arrangement of flagellar microtubules can easily be detected inside the cell (Fig. 17). It can be shown by serial
sectioning of young zygotes that the flagellar core remains intact in its entire length
within the cell, and suitably oriented sections show the flagellar core over a considerable distance (Fig. 14). The diameter of the flagellar core is the same (0-14 fim. as an
average of 20 measurements) whether measured on intactflagellaoutside the cell body
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T. Brdten
or after the core has been absorbed by the cell. Lying in the cytoplasm of the zygote,
the flagellar microtubules slowly depolymerize. After approximately 12 h they can no
longer be detected.
The settling of the young zygote also initiates the production of a cell wall. Newly
settled zygotes are seen surrounded by a thin coat of fibrous material (Fig. 15), while
free-swimming zygotes of the same age do not produce a cell wall. The wall increases
rapidly in thickness, as can be seen from Fig. 16. The primary wall is seen to be composed of a more electron-dense material than the secondary wall (Fig. 16, inset).
Nuclear fusion also takes place soon after copulation. Closely opposed nuclei can
be seen after 10 min and the fusion is completed in about 30 min. The fusion of the
2 nuclei starts with the disintegration of the outer nuclear membrane at the site of
contact between the 2 nuclei (Fig. 13) and is followed by the fusion of the inner
membranes.
The fate of the chloroplast is more difficult to follow because of the irregular shape
of this organelle. Many young zygotes show disintegrating chloroplasts and serial
sections of young zygotes frequently show one intact and one disintegrating chloroplast (Figs. 18, 19). This strongly suggests that the chloroplast from one of the gametes
is destroyed in the zygote.
DISCUSSION
The ultrastructure of the Ulva gamete is essentially the same as that described by
Evans & Christie (1970) for the zoospores of Enteromorpha. Small vesicles with an
electron-dense content similar to those described from the apical end of Enteromorpha
and believed to be the cementing substance used when the zygote (or zoospore) settles,
can also be found in Ulva. In addition, Ulva gametes and zoospores (Braten & Lovlie,
1968) show large vacuoles with a content of medium electron density. These vacuoles
do not seem to be present in Enteromorpha. Their function in Ulva is unknown, but
they can frequently be observed extruded through the cell surface. It is not clear,
however, whether this represents an artifact due to unbalanced osmotic conditions or
real secretion. Several of these vacuoles remain inside the cell and gradually become
more condensed as the zygote gets older. Similar vacuoles have been observed in the
gametes of Acetabularia (Crawley, 1966).
It is established through a series of investigations (Forster & Wiese, 1954, 1955;
Forster, Wiese & Braunitzer, 1956) that Chlamydomonas secretes substances, termed
gamones, which are responsible for the mating-type reaction. These substances were
shown to have an agglutinating effect and the flagella were shown to secrete the
gamones. In Chlamydomonas gamete copulation starts by cluster formation, in which
the gametes are held together by their flagella. Wiese (1965) gives evidence that the
gamones are the surface component responsible for this flagellar contact. In Ulva
copulation also starts by cluster formation, the only difference from Chlamydomonas
being that this stage is of very short duration. Gametes must be fixed immediately
after being mixed if this stage is to be preserved. In all, the extreme rapidity of the
copulation and zygote-forming processes is characteristic for Ulva, compared with
Fertilization and zygote formation in a green alga
625
Chlamydomonas. This could be an adjustment to the exposed habitat in which Viva
lives.
The first cytoplasmic contact between the mating cells in the species of algae
studied so far has been associated with a specialized structure either in one or in both
gametes (Friedmann et al. 1968). In Ulva no such structure has been observed and
the cytoplasmic contact is made through simple fusion of the surface membranes.
This always takes place at the anterior end of the gametes in a region seen to have a
high secretory activity. It is therefore probable that material secreted in this region
not only represents cementing substances (Evans & Christie, 1970) but also substances
which cause the cells to fuse.
The withdrawal of the flageUar microtubules into the cell body of the zygote was
first reported by Manton & Friedmann (i960). No such absorption takes place either
in Chlamydomonas (Brown et al. 1968) or in Acetatndaria as judged from Crawley's
(1966) micrographs. It is clear from the scanning electron-microscope observations
that the proximal part of the flagella is taken in through the cell surface, as flagella can
be seen to stick out from the cell at various distances between the anterior and the
posterior end. However, some kind of contraction mechanism must also be involved
in this process because of the length of the flagella compared with the size of the cell
body. The constant diameter of the flagellar core, whether in the intact flagellum or
after being absorbed by the zygote, illustrates the rigidity of the arrangement of
flagellar microtubules. One can only speculate at present on the advantage of absorbing
flagellar microtubules. The flagella will, however, furnish a great deal of microtubular
proteins which might be used, for instance, in the mitotic spindle apparatus during
subsequent cell divisions.
Observations with the electron microscope on the disintegration of chloroplasts
from one of the mating cells in zygotes of green algae have not been reported previously. Brown et al. (1968) report the fusion of the 2 chloroplasts in Chlamydomonas
moewusii, and this also seems to take place in the zygote of Acetabularia (Crzwley, 1966).
Cavalier-Smith (1970) has shown that the 2 chloroplasts fuse in the zygote of Chlamydomonas reinliardii, and in a recent publication (Bisalputra, Shields & Markham, 1971)
evidence is given for the continuity of both paternal and maternal chloroplasts in the
zygote of the brown alga Laminaria. The observations reported here fit in with the
observations of A. Fjeld (in preparation), who was able to show that a larger portion
of the zygotes of Ulva mutabilis survived when he irradiated the plus gametes before
copulation than when the minus gametes were irradiated. This suggests that the
genetic information contained in the plus gamete is eliminated in the zygote, i.e. the
minus gamete might furnish the chloroplast in the normal zygote. The question of the
fate of the chloroplasts in the zygote is at present under further investigation.
I wish to thank Mrs Edel Naavik Olsen and Miss Tove Wold for skilled technical assistance.
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REFERENCES
T., SHIELDS, C. M. & MARKHAM, J. W. (1971). In situ observations of the fine
structure of Laminaria gametophytes and embryos in culture. I. Methods and the ultrastructure of the zygote. J. Microscopic 10, 83—98.
BRATEN, T. & LevLiE, A. (1968). On the ultrastructure of vegetative and sporulating cells of
the multicellular green alga Ulva mutabilis Foyn. Nytt Mag. Bot. 15, 209-219.
BROWN, R. M., JOHNSON, C. & BOLD, H. C. (1968). Electron and phase-contrast microscopy of
sexual reproduction in Chlamydomonas moeiuusii. J. Phycol. 4, 100-120.
CAVALIER-SMITH, T. (1970). Electron microscopic evidence for chloroplast fusion in zygotes of
Chlamydomonas reinliardii. Nature, Lond. 228, 333-335.
CRAWLEY, J. C. W. (1966). Some observations on the fine structure of the gametes and zygotes
of Acetabularia. Planta 69, 365-376.
EVANS, L. V. & CHRISTIE, A. O. (1970). Studies on the ship-fouling alga Enteromorpha. I.
Aspects of the fine structure and biochemistry of swimming and newly settled zoospores.
Ann. Bot. 34, 451-466.
FORSTER, H. & WIESE, L. (1954). Gamonwirkung bei Chlamydomonas eugametos. Z. Naturf. B
9. 548-55OFORSTER, H. & WIESE, L. (1955). Gamonwirkung bei Chlamydomonas reinliardi. Z. Naturf. B
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H., WIESE, L. & BRAUNITZER, G. (1956). tfber das agglutinierend wirkende Gynogamon von Chlamydomonas eugametos. Z. Naturf. B 11, 315-317.
FRIEDMANN, I., COLWIN, A. L. & COLWIN, L. H. (1968). Fine-structural aspects of fertilization
in Chlamydomonas reinhardi. J. Cell Sci. 3, 115-128.
LEVRING, T. (1955). Some remarks on the structure of the gametes and reproduction of Ulva
lactuca. Bot. Notiser 108, 40-45.
LOVLIE, A. (1969). Cell size, nuclei acids, and synthetic efficiency in the wild type and a growth
mutant of the multicellular alga Ulva mutabilis Foyn. Devi Biol. 20, 349—367.
MANTON, I. & FRIEDMANN, I. (i960). Gametes, fertilization and zygote development in
Prasiola stipitata Suhr. II. Electron microscopy. Nova Hedwigia 1, 443-462.
FORSTER,
ROBERTSON, J. D., BODENHEIMER, T. S. & STAGE, D. E. (1963). The ultrastructure of Mauthner
cell synapses and nodes in goldfish brains. J. Cell Biol. 19, 159-199.
P. (1962). Einfaches Verfahren zur stufenlosen Gewebe-Entwasserung fur die elektronenmikroskopische Preparation. Naturwissensdiaften 49, 402-403.
VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron
microscopy. J. Cell Biol. 25, 407-408.
WIESE, L. (1965). On sexual agglutination and mating-type substances (Gamones) in isogamous
heterothallic Chlamydomonads. I. Evidence of the identity of the gamones with the surface
components responsible for sexual flagellar contact. J. Phycol. 1, 46—54.
SITTE,
(Received 5 May 1971)
Fig. 2. Median longitudinal section through the gamete of Ulva mutabilis. b, flagellar
base; c, chloroplast; g, Golgi apparatus; n, nucleus; p, pyrenoid; s, starch grain; v,
large vacuole. Arrows point to secretory granules believed to be cementing substance,
x 21000.
Fig. 3. Scanning electron micrograph of a cluster of mating gametesfixeda few seconds
after the mixing of plus and minus gametes. Arrows point to agglutinating flagellar
tips, x 3600.
Fig. 4. Mating gametes fixed just before cytoplasmic contact is established. The
gametes are held together by flagella (arrows), x 7150.
Fig. 5. Mating gametes showing the first cytoplasmic contact, m, mitochondrion; n,
nucleus; v, large vacuole. x 16000. Inset: scanning electron micrograph of mating
gametes just after cytoplasmic contact has been made, x 6200.
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Fig. 6. A more advanced stage than in Fig. 5. This young zygote was fixed 30 s after
copulation, c, chloroplast;/, flagellum; n, nucleus; v, large vacuole. x 18000. Inset:
scanning electron micrograph of the same situation. Note large vacuole extruding
through the cell surface (arrow), x 8350.
Fig. 7. Young zygote fixed 5 min after copulation. Cytoplasmic fusion is completed,
while the 2 nuclei (n) and the 2 chloroplasts (c) can still be distinguished, x 24000.
Inset: Same situation as seen in the scanning electron microscope. Note extruding
vacuole (arrow) and 4 flagella. x 8000.
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Fig. 8. Part of the anterior end of a 15-min-old zygote showing the secretion of
electron-dense particles (arrows). Note theflagellarmicrotubules (/) in the cytoplasmic
matrix, x 57 800.
Fig. 9. Scanning electron micrograph showing the uptake of flagella at an early stage.
Only the proximal part of one flagellum is absorbed (arrow), ant., anterior end of
zygote. X I I 600.
Fig. 10. A slightly more advanced stage offlagellaruptake than in Fig. 9. ant., anterior
end of zygote. x 16200.
Fig. 11. At this stage the 4flagellahave been absorbed along the entire length of the
zygote (arrows). Small spherical objects are contaminating microorganisms, ant.,
anterior end of zygote. x 11 600.
Fig. 12. This micrograph shows the posterior end (post.) of a zygote with the apical
parts of the flagellar cores being drawn into the cell. The flagellar sheaths are left
behind (arrows), x 12800.
Fertilization and zygote formation in a green alga
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Fig. 13. The fusion of the 2 nuclei at an early stage. This zygote was fixed 30 min
after copulation. The outer nuclear membrane can be seen to disintegrate at the place
of contact, x 40250.
Fig. 14. Zygote 30 min after copulation showingflagellarcore in transverse and longitudinal sections (arrows), x 24750.
Fig. 15. Part of 2 15-min-old zygotes showing the first sign of cell wall formation.
c.w., cell wall; p.m., plasma membrane, x 38500.
Fig. 16. Several 19-h-old zygotes showing thick cell wall. Note also the condensed
appearance of the large vacuoles (i>). x 8000. Inset: higher magnification of cell wall.
p.tv., primary wall; s.w., secondary wall, x 40500.
Fertilization and zygote formation in a green alga
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Fig. 17. Young zygote 20 min after copulation. Flagellar microtubules can be seen in
the cytoplasm (arrows) just beneath the cell surface, x 27000.
Fig. 18. Zygote 30 min old showing one intact chloroplast (c) and one disintegrating
chloroplast (d.c). n, nucleus, x 19500.
Fig. 19. Zygote of same age as in Fig. 18, but showing a more advanced stage of chloroplast disintegration, c, intact chloroplast; d.c, disintegrating chloroplast; n, nucleus,
x 19600.
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