J. Cell Sci. 13, 69-81 (1973)
Printed in Great Britain
69
ULTRASTRUCTURE OF MEIOSIS AND
CENTRIOLE BEHAVIOUR IN ULVA
MUTABILIS F0YN
T. BRATEN
Electron Microscopical Unit for Biological Sciences,
University of Oslo, Blindern, Oslo 3, Norway
AND 0. NORDBY
Department of Zoology, University of Oslo, Blindern, Oslo 3, Norway
SUMMARY
The present work deals with the ultrastructure of the meiotic processes leading to the
formation of zoospores. The formation of an exit pore on the outer surface of the sporangium
is the first visible sign that the cell will undergo meiosis and not just a somatic division. Prophase I nuclei differ from nuclei in mitotic prophase by having one or several invaginations
of the nuclear membrane. Electron-dense lines probably representing the lateral elements of
the 8ynaptinemal complex are observed during prophase I. The central element of the complex
has, however, never been observed.
The fate of the centrioles during meiosis is described in detail. The number of duplications
of the centrioles is found to be the same at meiotic and mitotic zooid formation. The number
of cell divisions is, however, different in the 2 cases.
INTRODUCTION
Among organisms suitable for the study of specific preparations for meiotic
or reductional division, the sea-lettuce Ulva and other members of the order Ulotrichales possess the outstanding property that closely similar meiotic and mitotic
zooid formation processes can be compared in division-synchronized diploid and
haploid plants. This fact makes it possible to separate processes concerned with the
differentiation of reproductive cells, from processes involved with the meiotic process
itself.
Ulva mutabilis Foyn has now for several years served as an object for genetical,
physiological and morphological studies in our laboratories (Lovlie, 1968). As part
of current morphological and biochemical studies of synchronized zooid formation
(meiotic and mitotic) in this organism (Nordby & Hoxmark, 1972), the present work
contains a description of some ultrastructural features of the meiotic zoosporogenesis.
Particular attention is paid to the replication of the centrioles, the number of
which is 4 per cell in the haploid zoospore and 2 per cell in the haploid gamete.
Several light-microscopical investigations exist on meiosis in green algae (e.g. Foyn,
1934; Ramanathan, 1939). To the author's knowledge no investigations have, however,
been made on the ultrastructure of meiosis, in these plants.
70
T. Brdten and 0. Nordby
MATERIAL AND METHODS
Diploid sporophytes of the fast-growing mutant 'Slender' (Sl/Sl) were used in this
investigation. The algae were grown at 18 °C in enriched seawater as described elsewhere
(Lovlie, 1969). After approximately 5 weeks' growth in a I7h/7h light/dark programme with
the light period running from 04.00 to 21.00 hours, the algae were harvested at 14.00 hours
and cut into small fragments. The thoroughly washed fragments were transferred to fresh
growth medium in Petri dishes and placed in the sporulation chamber. This chamber has a
temperature of 21 °C, and an illumination of 10-500 lux measured at dish centre. Here the
light period in the 17 h/7 h light/dark programme runs from 21.00 to 14.00 hours. Under
these conditions more than 90 % of the cells in the fragments will regularly carry out synchronous meiosis 40 h later (Nordby & Hoxmark, 1972).
Specimens for light microscopy were fixed in Bouin-Dubosque's solution (Lovlie, 1964)
and stained in Gomori's haematoxylin according to Melander & Wingstrand (1953).
Preparation for electron microscopy was as described in a previous paper (Braten, 1971).
RESULTS
General
Principally JJlva grows as a hollow tube, the wall of the tube being made up of a
single coherent layer of cells. As described by Lovlie & Braten (1970) and indicated
in Fig. 1, the somatic mitosis is initiated by a wandering of the cell nucleus towards
the inner surface of the cell. The nucleus divides while lying close to this wall, and
always with its spindle axis parallel to the inner surface. After completed division the
formed daughter nuclei approach each other and glide outwards along the newly
formed division furrow to take up their interphase position in the vicinity of the
cup-shaped chloroplast. A new cell wall is then laid down vertical to the surface
plane. A detailed light-microscopical description of the meiotic division is in preparation. The result is summarized as follows:
Meiotic formation of motile zoospores deviates in several respects from somatic
mitosis. The first sign of zooid formation is the appearance of a bulge on the outer
surface of the cell. This will later become the exit pore for the fully formed spores.
Shortly thereafter the cells round up within the cell wall envelopes, apparently
losing their turgor. The meiotic division is also initiated by a wandering of the
nucleus from its interphase position close to the chloroplast, to a position close to
the cell wall opposite the chloroplast. This cell wall is, however, not the inner surface
wall as in mitosis. In meiosis the nucleus takes up a position at the centre of the
wall shared with the neighbouring cell. The spindle axis lies parallel with the outer
and inner surface plane, but already during the anaphase the spindle and the forming
daughter cells rotate so (hat they end up lying on top of each other (Fig. 1). As
during mitosis the division furrow appears as a ring around the whole cell.
After the completion of the first division, essentially the same division and movement patterns are repeated 3 more times giving rise to 16 daughter cells within the
cell wall of each spore mother-cell.
In the usual light/dark regime used in these experiments meiotic prophase stages
appear some 40 h after transfer to the sporulation chamber. The 16 zoospores per
sporangium are formed within the next 12 h, and they will be released through the
exit pore at some time during the next light period (Fig. 12).
3
Metaphase I
Metaphase
Telophase I
Telophase
Release of 16
zoospores
Reconstltution of interphase cells
Fig. i. Schematic view of nuclear orientation and movement during somatic mitosis and meiotic zoospore formation. Left: A somatic
interphase cell seen in 2 projections, where abed designates cell walls against neighbour cells, 0 and i the outer and inner thallus
surface respectively. In the cell is shown the nucleus (black) and the chloroplast (hatched). To the right are shown stages in somatic
mitosis (upper half) and in meiosis (lower half) as seen in the same projections. Note differences in the position of the nucleus and
movement of daughter cells after meiosis.
Meiotic
prophaie
Mitotic
prophase
II I
I"
I
3
72
T. Brdten and 0. Nordby
Formation of the exit pore
The exit pore is always formed on the outer surface of the sporangia. Onset of
spore formation is the first visible sign that the cell will undergo meiosis and not
just a somatic division. Spore formation starts by an outward bulging of the outer
cell wall. At the same time a weakening of the wall at this place can be seen (Fig. 2).
On electron micrographs this weakening is seen as less densely packed and more
randomly oriented fibrils than in the rest of the cell wall. Membrane-bound vesicles
are frequently observed at the site of cell wall weakening.
The nuclear division
The marked difference in structure between the 'synizesis'-like and 'spiremal'
stage of meiotic prophase as seen in light-microscope preparations is not encountered
in the corresponding electron micrographs. It is however, a widespread notion that
the tight 'synizesis knot' is actually a fixation artifact of light microscopy (Rhoades,
1961).
The morphology of the nucleus at prophase I is strikingly different from what is
seen at mitosis. The nucleus has an irregular surface due to usually one extensive
invagination of the nuclear membrane (Fig. 4). Nuclei with large numbers of membrane imaginations have also been found (Fig. 3). Characteristically the centrioles
occupy a position at the opening of one of the invaginations of the nuclear membrane.
Microtubules are found in the cytoplasm of this invagination. One other characteristic
of the prophase I nucleus is the appearance of electron-dense lines often found to
be paired (Figs. 4, 5). Each line measures approximately 30 run in width and the 2
paired lines are separated by a space of 0-15-0-2 /tm. We think that these lines represent the lateral element of the synaptinemal complex (see Moses, 1968, for terminology). Unpaired lines may represent the so-called axial elements thought to be
early formation stages of the synaptinemal complex. We have, in spite of extensive
search, never observed complete synaptinemal complexes containing the central
element.
The metaphase picture is the same as seen during mitosis. The nuclear surface
has regained a smooth appearance except at the 2 poles where large fenestrae can be
seen (Fig. 7). The polar region is a homogeneous mass of cytoplasm devoid of
ribosomes and other cell organelles. Spindle microtubules are numerous and seem
to stop rather abruptly at the level of the openings in the nuclear envelope (Fig. 8).
Anaphase proceeds without any sign of cytokinesis (Fig. 6). The nucleus takes the
shape of a long cylinder open at both ends, but with the nuclear envelope intact over
the rest of the surface.
The behaviour of the centrioles during division and cytokinesis
When the centrioles are first observed during meiosis they lie in the narrow space
between the prophase nucleus and the cell wall (Fig. 4). As mentioned in the previous
section, when an invagination develops into the interior of the nucleus the centrioles
are found just outside this invagination. In this position the centrioles duplicate.
Ultrastructure of mewsis in Ulva
73
The 2 daughter pairs now move towards the spindle polar areas and are found to
lie close to this area during metaphase. The position of the centrioles at early anaphase
is to one side of the polar region between the nucleus and the cell wall (Fig. 8).
We have no pictures showing the movement of the centrioles during ana- and telophase. When reconstituted nuclei are found after telophase, however, these lie close
together. Between the nuclei all 4 centrioles may be found packed closely together.
Figs. 9 and 10 clearly show that the subsequent division furrow will cut right through
the centre of the centriole group, distributing 2 centrioles to each daughter cell.
This equatorial position with the 4 centrioles close together is interpreted as being
due to the fact that the 2 nuclei start to rotate and move together immediately after
reconstitution (0. Nordby, in preparation). The same pattern of centriole duplication
and movement is seen during the following 3 mitotic divisions. It is to be noted
here that a distinct interphase stage appears between each of these divisions, and
that the centrioles duplicate before any sign of nuclear division is apparent.
Subsequent to the last nuclear division, an additional centriole duplication takes
place, thus furnishing each future zoospore with 4 centrioles. These remain in rightangled pairs, and are seen to lie in a distinct bulge on the somewhat elongated cell.
These 4 centrioles then form the basal bodies for the 4 flagella which emerge during
the subsequent differentiation of the zoospores (Fig. 11). Usually the 16 zoospores
form a hollow sphere inside the sporangium with their flagella pointing towards each
other.
DISCUSSION
The irregularly shaped nuclei with one or several extensive invaginations of the
nuclear membrane are characteristic of meiotic prophase. Nuclei undergoing ordinary
mitosis show ovoid nuclei with a smooth surface (Lovlie & Braten, 1970). We have
not observed attachment of chromosomes to the invagination(s) of the nuclear membrane at prophase, as described in some organisms (e.g. Underbrink, Ting & Sparrow,
1967; Moens & Perkins, 1969).
We are puzzled by the fact that no complete synaptinemal complex has been
found, this, in spite of the extensive search through large numbers of prophase I
nuclei. One sees electron-dense parallel lines, but the central element of the complex
has never been observed. Moses (1968) states that the appearance of the central
element varies widely among species and even within an individual. Our failure to
observe it can hardly be due to extremely short duration of this particular stage.
It is possible that our method of preparation fails to stain the central element. The
low affinity of Ulva tissue for osmium is a general problem in preparation for electron
microscopy. This for instance leads to difficulties in identifying hetero- and euchromatic regions of chromosomes in ordinary vegetative tissue. Our measurements of
the size of the synaptinemal complex lie within those reported from other organisms
(Moses, 1968).
Two pairs of centrioles exist throughout meiosis except for early interphase.
The position of the centrioles at anaphase, somewhat to one side of the polar region,
74
T. Brdten and 0. Nordby
confirms observations made from vegetative cells that centrioles do not take direct
part in the formation of the spindle (Lovlie & Braten, 1970). Recent investigations
(Szollosi, Calarco & Donahue, 1972) have shown that the presence of centrioles is
not necessary for the formation of meiotic spindles in mouse oocytes.
The position of the 4 centrioles at the equator during cytokinesis strongly confirms the suggestion made by Johnson & Porter (1968) that the centrioles determine
the position of the cleavage plane. This is particularly evident in Fig. 9 where the
cleavage furrow can be seen to pass right between the 4 centrioles.
The interpretation that the flagella are formed at the 16-cell stage is somewhat
circumstantial, since we have no serial sections of this stage. However, since flagella
are never seen in dividing cells, this interpretation appears to be the most probableThe number of flagella is, apart from the difference in size, the only way of distinguishing between zoospores and gametes on a morphological basis. Interesting
in this connexion is the fact that each gametangium apparently produces 32 biflageUated gametes while each sporangium produces 16 quadrinagellated zoospores.
This means that after the last centriole duplication, the future gametes undergo
another division, leaving each cell with 2 centrioles to become the basal bodies of the
2 flagella. In the case of the zoospores no division takes place after the last centriole
duplication; thus each cell is given 4 centrioles to become the basal bodies of the 4
flagella. In other words: the number of duplications of the centrioles is the same
at meiotic and mitotic zooid formation. The number of cell divisions is, however,
different in the 2 cases.
We wish to thank Mrs Eva Jenssen for skilled technical assistance. Financial support from
Royal Norwegian Council for Scientific and Industrial Research is gratefully acknowledged.
REFERENCES
T. (1971). The ultrastructure of fertilization and zygote formation in the green alga
Ulva mutabilis Foyn. J. Cell Set. 9, 621-635.
FOYN, B. (1934). Lebenszyldus, Cytologie und Sexualitat der Chlorophycee Ulva lactuca L.
Arch. Protistenk. 83, 154-177.
JOHNSON, U. G. & PORTER, K. R. (1968). Fine structure of cell division in Chlamydoinonas
reinliardi. Basal bodies and microtubules. J. Cell Biol. 38, 403-425.
LOVLIE, A. (1964). Genetic control of division rate and morphogenesis in Ulva mutabilis Foyn.
C.r. Trav. Lab. Carhberg 34, 77-168.
LOVLIE, A. (1968). On the use of a multicellular alga {Ulva mutabilis Foyn) in the study of
general aspects of growth and differentiation. Nytt Mag. Zool. 16, 39—49.
LeVLiE, A. (1969). Cell size, nucleic acids and synthetic efficiency in the wild type and a
growth mutant of the multicellular alga Ulva mutabilis Foyn. Devi Biol. 20, 349-367.
LevuE, A. & BRATEN, T. (1970). On mitosis in the multicellular alga Ulva mutabilis Foyn.
J. Cell Set. 6, 109-129.
MELANDER, Y. & WINGSTRAND, K. G. (1953). Gomori's hematoxylin as a chromosome stain.
Stain Technol. 28, 217-223.
MOENS, P. B. & PERKINS, F. O. (1969). Chromosome number of a small protist: Accurate determination. Science, N.Y. 166, 1289-1291.
MOSES, M. J. (1968). Synaptinemal complex. A. Rev. Genet. 2, 363-412.
NORDBY, 0. & HOXMARK, R. C. (1972). Changes in cellular parameters during synchronous
meiosis in Ulva mutabilis Foyn. Expl Cell Res. 75, 321-328.
RAMANATHAN, K. R. (1939). The morphology, cytology and alternation of generations in Enteromorpha compressa (L) Grev. var. lingulata (J. AG.) Hauck. Ann. Bot. 3, 375-398.
BRATEN,
Ultrastructure of meiosis in Ulva
75
M. M. (1961). Meiosis. In The Cell, vol. 3 (ed. J. Brachet & A. E. Mirsky), pp. 1-76.
New York and London: Academic Press.
SZOLLOSI, D., CALARCO, P. & DONAHUE, R. P. (1972). Absence of centrioles in the first and
second meiotic spindles of mouse oocytes. J. Cell Set. 11, 521-541.
UNDERBRINK, A. G., TING, Y. C. & SPARROW, A. H. (1967). Note on the occurrence of a synaptinemal complex at meiotic prophase in Zea mays. Can. J. Genet. Cytol. 9, 606-609.
RHOADES,
(Received 6 December 1972)
76
T. Brdten and 0. Nordby
Fig. 2. Part of a cell in an eaily stage of meiosis, but showing an advanced state of
exit pore formation. Part of the chloroplast (cJit) can be seen close to the future exit
pore (ex), x IOIOO.
Fig. 3. Prophase I nucleus showing a highly irregular surface. X 14300.
Fig. 4. Part of a prophase I nucleus showing an extensive invagination of the nuclear
membrane. One centriole (ce) can be seen just outside this invagination. Arrows
indicate lines probably representing the synaptinenial complex, x 25 200.
Ultrastructure of meiosis in Ulva
•
1*\
78
T. Brdlen and 0. Nordby
Fig. 5. Prophase I nucleus showing typical dense lines thought to be the synaptinemal complex, x 9600
Fig. 6. The nucleus at anaphase. Note an intact nuclear envelope except at the poles
where large fenestrae can be seen. Electron-dense areas (chr) represent chromosomes,
x 10200.
Fig. 7. The nucleus at metaphase I. Chromosomes (chr) and spindle microtubules
can be seen, x 28900.
Ultrastructure of meiosis in Ulva
T. Brdten and 0. Nordby
? : ^ _
Fig. 8. The polar region of a nucleus at anaphase. Note that the spindle microtubules
are roughly parallel to each other and not converging towards the pole. Note also the
position of the 2 centrioles (arrows), w, cell wall, x 25500.
Fig. 9. Four centrioles found at one equator during cytokinesis. The division furrow
(arrows) can be seen to pass through the middle of the 4 centrioles. x 39600.
Ultraslructure of meiosis in Uha
81
Fig. 10. The position of the centrioles (arrows) just after cytokinesis, n, nucleus; w,
cell wall, x 24700.
Fig. 11. Part of a cell undergoing differentiation to become a zoospore. The centrioles
(arrows) now become the basal bodies for the forming flagella (/). x 22000.
Fig. 12. Scanning electron micrograph of the surface of the thallus after sporulation.
The exit pores (arrows) can clearly be seen. Note that almost every cell has sporulated.
x 800.
.&
CI5L
13